ESM
European Society
of Mycobacteriology
In co-organization with the Instituto Nacional de Saúde Dr. Ricardo Jorge
CONTENTS
• Welcome Message
• Introduction to the European Society of Mycobacteriology
• Congress Organization
• Program at a glance
• Programme of Guest Lectures, Oral Presentations and Symposia
• Programme of Poster Presentations
• Abstracts of Guest Lectures (GL)
• Abstracts of Oral Presentations (OP)
• Abstracts of Poster Presentations (PP)
• Author Index
European Society of Mycobacteriolog y | 30 th Annual Congress | July 2009 | Por to - Por tugal
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Welcome Message
Dear Colleagues,
It is with great pleasure that we warmly welcome you to the 30th Annual Congress of the
European Society of Mycobacteriology, held in the city of Porto in Portugal from July 5-8, 2009.
The aim of ESM2009 is to promote the exchange of distinguished experts from all around
the world who will have the opportunity to update information in the front-line of scientific
achievement, share experience and ideas, and actively participate in contribution to the field of
mycobacteriology.
We have worked hard to make your stay in Porto a pleasant, useful and memorable occasion.
On behalf of the ESM and the Local Organisers,
Suzana David
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ESM 2009
European Society
of Mycobacteriology
30th Annual Congress
July 5-8, 2009
Porto, Portugal
The European Society of Mycobacteriology (ESM, http://www.esmycobacteriology.eu), founded in 1980, is a non-profit
international scientific society dealing with different aspects of mycobacteriology and related diseases. It is considered
one of the most active international scientific societies in this area, being committed to:
- Encouraging the highest standards for research to facilitate the discovery of new knowledge;
- Coordinating and providing information and expertise to other organizations worldwide;
- Disseminating knowledge on all aspects of mycobacteriology and related diseases, through scientific
meetings and publications,
- Encouraging and providing the highest standards of training to interested health care providers;
- Establishing, reviewing and revising guidelines;
- Promoting high quality and cost effective diagnostic procedures;
- Advising, cooperating and participating with government and non-government agencies in matters of
common interest;
- Participating in activities whose aim is to prevent mycobacterial diseases worldwide.
The ESM meetings are held each year in a different country of Europe. The ESM2009 congress is hosted in the city of
Porto, in Portugal. International specialists will treat themes at the front-line of scientific achievement in the field of
mycobacteriology and related diseases. As in previous meetings, the scientific program of the conferences covers a wide
range of topics in both applied and fundamental research in areas of priority such as:
- Diagnostics of active and latent tuberculosis
- Diagnostics of atypical mycobacteria
- Molecular epidemiology of tuberculosis
- Antibiotic resistance, MDR, XDR
- Tuberculosis drug development
- Immunology of mycobacterial infections
- Molecular Biology of mycobacteria
- Taxonomy of the genus Mycobacterium
- Veterinarian and environmental Mycobacteriology
- Methods for diagnostics adapted to economically disfavoured settings
- Laboratory safety
- Short and long term programs and recommendations
European Society of Mycobacteriolog y | 30 th Annual Congress | July 2009 | Por to - Por tugal
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Congress Organization
EUROPEAN SOCIETY OF MYCOBACTERIOLOGY
http://www.esmycobacteriology.eu
Chairman of the ESM 2009 congress
Suzana David (Lisbon, Portugal)
Steering Committee
General Secretary
Enrico Tortoli (Firenze, Italy)
President
Todor Kantardjiev (Sofia, Bulgaria)
Treasurer
Malcolm Yates (East Dulwich Grove, U.K.)
Members
Maria-Jesus Garcia (Madrid, Spain)
Sven Hoffner (Solna, Sweden)
Stefan Niemann (Borstel, Germany)
Gabriela Pfyffer (Luzern, Switzerland)
Nalin Rastogi (Guadeloupe, France)
Veronique Vincent (Geneva, Switzerland)
HONORS COMMITTEE ESM 2009
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Ana Jorge
Minister of Health
Mariano Gago
Minister of Science, Technology and Higher
Education
Manuel Pizarro
Secretary of State for Health
Francisco Ramos
Secretary of State for Health
José Pereira Miguel
President of the National Health Institute
Dr. Ricardo Jorge
Jorge Sampaio
UN Secretary General’s Special Envoy to
Stop TB
Mario Raviglione
Director of the Stop TB Department,World
Health Organization
Maria do Céu Machado
High Comissioner for Health
Francisco George
Director-General of Health
Jorge Soares
Director of the Health and Human development Service Calouste Gulbenkian
Foundation
Jorge Torgal
Director do Institute of Hygiene and Tropical
Medicine
Rui Rio
Mayor of the city of Porto
Fernando Augusto Fiuza de Melo
Director of the Clemente Ferreira Institute,
São Paulo, Brasil
ESM 2009
COORGANIZERS
European Society of Mycobacteriology
http://www.esmycobacteriology.eu
Instituto Nacional de Saúde Dr. Ricardo Jorge, INSA
http://www.insa.pt
PARTNERS
Foundation for Science and Technology (FCT)
http://www.fct.mctes.pt
Fundação Calouste Gulbenkian
http://www.gulbenkian.pt
Luso-American Foundation (FLAD)
http://www.flad.pt
STOP-TB Working Group on New Diagnostics, Point of
Care sub group
http://www.stoptb.org
SPONSORS
The Organization expresses its thanks and appreciation to all those who generously contributed to the success of the 30th Annual Congress of ESM.
HAIN LifeSciences
http://www.hain-lifescience.com
Platinum Medal Sponsor
Beckton Dickinson & Quilaban
http://www.bd.com
http://www.quilaban.pt
Gold Medal Sponsor
BioMerieux
http://www.biomerieux.com
Gold Medal Sponsor
Microsens (logo)
http://www.microsens.com
Cepheid
http://www.cepheid.com
Genoscreen
http://www.genoscreen.com
European Society of Mycobacteriolog y | 30 th Annual Congress | July 2009 | Por to - Por tugal
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Program at a glance
Sunday, July 5th
09h00
REGISTRATION
11h00 – 12h00
SYMPOSIUM SPONSORED BY HAIN LIFESCIENCE GMBH:
RAPID MOLECULAR GENETIC DETECTION OF MDR- AND
XDR-TB
Chair: Michael Weizenegger
Speakers: Doris Hillemann and Christopher M Gilpin
12h00 – 13h30
SYMPOSIUM SPONSORED BY STOP-TB WORKING
GROUP ON NEW DIAGNOSTICS, POINT OF CARE SUB
GROUP: A SYMPOSIUM ON POINT-OF-CARE TESTS FOR
TUBERCULOSIS
Speakers: Catharina Boehme, Carol Nawina Nyirenda, Rosanna
Peeling, Gerd Michel, Ruth McNerney and Amy P Wong
13h30 – 15h30
Break for Lunch
15h30 – 16h30
SYMPOSIUM CO-SPONSORED BY BECTON, DICKINSON
AND COMPANY AND QUILABAN - QUÍMICA
LABORATORIAL ANALÍTICA LDA: SUCCEPTIBILITYTESTING
AND TREATMENT OF TB IN THE ERA OF MDR-TB AND
XDR-TB. DO WE USE THE RIGHT DRUGS AND TOOLS?
Chair: Francoise Portaels and Salman Siddiqi
Speakers: Stefan Winkler, Andre De Bock, and Virginia Crews
17h00 – 19h00
OPENING SESSION
Welcome Address
TB CONTROL PROGRAMS
Chair: Jaime Nina and Cristina Furtado
Guest Lecture-1: Miguel Villar
Guest Lecture-2: Fernando Fiuza de Melo
Monday, July 6th
19h15
Welcome Reception
09h00 – 13h15
SCIENTIFIC SESSION ON MOLECULAR EPIDEMIOLOGY
AND DRUG RESISTANCE SURVEILLANCE
Chair: Stefan Niemann and Cristina Gutierez
9h00 – 9h45
Guest Lecture-3: Sebastien Gagneux
9h45 – 11h00
Oral Presentations
11h00 – 11h30
Coffee and tea
Chair: Nalin Rastogi and Dr. Philip Supply
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11h30 – 12h15
Guest Lecture-4: Dick van Soolingen
12h15 – 13h015
Oral Presentations
13h15 – 14h00
Lunch
ESM 2009
Tuesday, July 7th
14h00 – 15h00
POSTER SESSION
15h00 – 16h45
SCIENTIFIC SESSION ON NON TUBERCULOUS
MYCOBACTERIA
Chair: Enrico Tortoli
15h00 – 15h45
Guest Lecture-5: Joseph O. Falkinham, III
15h45 – 16h45
Oral Presentations
16h45 – 17h15
Coffee and tea
17h15 – 19h15
SCIENTIFIC SESSION ON ISSUES IN THE MODERN TB
LABORATORY
Chair: Gabriela Pfyffer and Thomas Shinnick
17h15 – 18h00
Guest Lecture-6 : Dr. Jean-Pierre Zellweger
18h00 – 18h30
Guest Lecture-7 : Lee W. Riley
18h30 – 19h15
Oral Presentations
19h30
Visit and Dinner at the Port Wine Cellars
09h00 – 12h30
SCIENTIFIC SESSION ON VACCIN DEVELOPMENT
AND PATHOGENESIS
Chair: David Minnikin
09h00 – 09h45
Guest Lecture-8: Carlos Martin
09h45 – 10h30
Guest Lecture-9 : Lee W. Riley
10h30 – 11h00
Coffee and tea
11h00 – 12h30
Oral Presentations
12h30 – 13h15
Lunch
13h15 – 14h15
BUSINESS MEETING
14h15 – 18h15
SCIENTIFIC SESSION ON LABORATORY STRENGTHENING
Chair: Dr. Sven Hoffner and Dr. Mark Perkins
14h15 – 15h00
Guest Lecture-10: Thomas Shinnick
15h00 – 15h45
Guest Lecture-11: Afrânio Kritski
15h45 – 16h30
Guest Lecture-12 : Moisés Palaci
16h30 – 17h00
Coffee and tea
17h00 – 17h45
Oral Presentations
17h45 – 18h15
BEST POSTER
18h30
Cultural Evening Dinner
European Society of Mycobacteriolog y | 30 th Annual Congress | July 2009 | Por to - Por tugal
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Wednesday, July 8th
09h00 – 11h00
SCIENTIFIC SESSION ON DRUG DEVELOPMENT
Chair: Dr. Nalin Rastogi
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09h00-09h45
Guest Lecture-13: Clarice Queico Leite
09h45 – 10h30
Guest Lecture-14 : Moisés Palaci
10h30 – 11h00
Oral Presentations
11h00 – 11h30
Coffee and tea
11h30 – 12h20
SESSION ON PRACTICAL ASPECTS AND QUALITY
ASSURANCE IN MOLECULAR EPIDEMIOLOGY
Chair: Dr. Elvira Richter and Prof. Christophe Sola
11h30 – 12h00
Guest Lecture-15 : Philip Supply
12h00 – 12h20
Oral Presentation
12h20 – 12h30
Discussion
12h30 – 13h30
SYMPOSIUM SPONSORED BY INSTAND, SOCIETY FOR
RESEARCH PROMOTION OF QUALITY ASSURANCE
IN MEDICAL LABORATORIES, WHO COLLABORATING
CENTRE, DUESSELDORF, GERMANY: EXTERNAL QUALITY
ASSURANCE
Speakers: Elvira Richter, Girts Skenders, Akos Somoskövy
13h:30 – 13h45
Closing Remarks
13h45 – 14h15
CLOSING OF CONGRESS
14h15
Conference Closes
ESM 2009
Programme of Guest Lectures,
Oral Presentations and Symposia
Sunday, July 5th
9h00 – 11h00
REGISTRATION
11h00 – 12h00
SYMPOSIUM SPONSORED BY HAIN LIFESCIENCE GMBH
RAPID MOLECULAR GENETIC DETECTION OF MDR- AND XDR-TB
Chair: Dr. Michael Weizenegger
Dr. Doris Hillemann, National Reference Center for Mycobacteria (Borstel, Germany)
Rapid detection of XDR-TB with the Genotype MTBDRsl assay
Dr. Christopher M Gilpin, PhD MPH, International Organization for Migration (Geneva,
Switzerland)
Implementation of new diagnostic tools and algorithms for enhanced
case detection of drug resistant forms of tuberculosis
12h00-13h30
SYMPOSIUM SPONSORED BY STOP-TB WORKING GROUP ON NEW
DIAGNOSTICS, POINT OF CARE SUB GROUP
A SYMPOSIUM ON POINT-OF-CARE TESTS FOR TUBERCULOSIS
Dr. Catharina Boehme, M.D., Foundation for Innovative Diagnostics (FIND) (Geneva,
Switzerland)
What is a POC test?
Carol Nawina Nyirenda (Zambia)
Why do we need rapid tests: a patient’s perspective?
Prof. Rosanna Peeling, Ph.D., London School of Hygiene & Tropical Medicine
(London, UK)
Rapid tests for TB: what is wrong with them?
Dr. Gerd Michel, Ph.D., Foundation for Innovative Diagnostics (FIND) (Geneva,
Switzerland)
Biomarker discovery: are we making progress
Dr. Ruth McNerney, Ph.D.,
(London, UK)
London School of Hygiene & Tropical Medicine
Volatile markers for TB: myth or reality?
Dr. Amy P Wong, Ph.D., X PRIZE Foundation (California, U.S.A.)
Barriers to TB test development
Discussion: The way forward
Platform and floor
13h30 – 15h30
Break for Lunch
15h30 – 16h30
SYMPOSIUM CO-SPONSORED BY BECTON, DICKINSON AND COMPANY AND
QUILABAN - QUÍMICA LABORATORIAL ANALÍTICA LDA
SUCCEPTIBILITYTESTING AND TREATMENT OF TB IN THE ERA OF
MDR-TB AND XDR-TB. DO WE USE THE RIGHT DRUGS AND TOOLS?
Chair: Prof. Francoise Portaels and Dr. Salman Siddiqi
Prof. Stefan Winkler, University of Vienna (Vienna, Austria)
Antibiotic therapy for TB and new treatment options
European Society of Mycobacteriolog
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Dr. Andre De Bock (BD)
Extended Drug Susceptibility Testing of M. tuberculosis
Virginia Crews (BD)
The new BD MGIT TBc Identification Test
16h30 – 17h00
OPENING SESSION
WELCOME
Minister of Health (Portugal) (to confirm)
Minister of Science, Technology and Higher Education (Portugal) (to confirm)
17h00 – 17h45
Welcome from the President of the Instituto Nacional de Saúde Dr. Ricardo Jorge,
Prof. José Pereira Miguel (Portugal)
Welcome from the General Secretary, Dr. Enrico Tortoli (Italy)
Welcome address, Dr. Suzana David (Portugal)
TB CONTROL PROGRAMS
Chair: Prof. Jaime and Prof Cristina Furtado
17h45 – 18h20
Guest lecture: Dr. Miguel Villar, Consultant or Thuberculosis, Directorate-General
of Health (Lisboa, Portugal)
Tuberculosis in Portugal
18h20 – 19h00
Guest lecture: Dr. Fernando Fiuza de Mello, Director of the Clemente Ferreira
Institute (São Paulo, Brasil)
The Brazilian experience in the control of multi-drug
resistant tuberculosis
Welcome reception Welcome Reception
Monday, July 6th
SCIENTIFIC SESSION ON MOLECULAR EPIDEMIOLOGY AND DRUG
RESISTANCE SURVEILLANCE
9h00 – 9h45
Guest lecture: Dr. Sebastien Gagneux, Ph.D., National Institute for Medical Research
(London, UK)
GL-3
Evolutionary forces in Mycobacterium tuberculosis
Chair: Dr. Stefan Niemann and Dr. Cristina Gutierez
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9h45 – 10h00
Honisch C, Mosko M, Arnold C, Gharbia S, Feuerriegel S, Niemann S
Mass Spectrometry for molecular typing of the Mycobacterium
tuberculosis complex: One platform and multiple assay formats
OP-1
10h00 – 10h15
Borile C, Refrégier G, Labarre M, Franz S, Mézard M, Sola C
Clustering of spoligo-patterns: towards an automation of
Mycobacterium tuberculosis complex classification
OP-2
10h15 – 10h30
Sandoval A, Cubillos A, Reyes A, Correa N, Robledo J, Zambrano MM,
Del Portillo P
Identification of the insertion element IS6110 in phop promoter
in a high transmission Mycobacterium tuberculosis strain: A clue
to phenotypic variation
OP-3
10h30 – 10h45
Oelemann MC, Gomes HM, Willery E, Lima KVB, Possuelo L, Locht C, Goguet de
la Salmonière YOL, Gutierrez MC, Supply P, Suffys PN
OP-4
ESM 2009
Genomic interrogation of mycobacterium tuberculosis isolates
from Brazil
10h45 – 11h00
Mokrousov I,Valcheva V, Sovhozova N, Aldashev A, Rastogi N, Isakova J
Penitentiary population of Mycobacterium tuberculosis in
Kyrgyzstan: Exceptionally high prevalence of the Beijing genotype and its Russia-specific subtype
11h00 – 11h30
Coffee and Tea
11h30 – 12h15
Guest lecture: Dr. Dick van Soolingen, Ph.D., National Institute for Public Health and
the Environment (Bilthoven, Netherlands)
OP-5
GL-4
Advances in the molecular epidemiology of tuberculosis
Chair: Dr. Nalin Rastogi and Dr. Philip Supply
12h15 – 12h30
Millet J, Miyagi-Shiohira C,Yamane N, Mokrousov I, Rastogi N
The unique endemic nature of Beijing genotype strains in
Okinawa, Ryukyu Islands of Japan as revealed by newly described 15 and 24-loci MIRU-VNTR typing schemes
OP-6
12h30 – 12h45
Shamputa IC, Lee J, Allix-Béguec C, Cho E-J, Lee J-I, Min JH, Goldfeder LC, Kim JH,
Kang HS, Hwang SH, Eum SY , Lee H, Park SK, Supply P, Cho SN,Via LE,
Barry III CE
Mycobacterium tuberculosis genetic diversity in South Korea
OP-7
12h45 – 13h00
Feuerriegel S, Homolka S, Post E, Oberhauser B, George AG, Westman L, Dafae F,
Rüsch-Gerdes S, Niemann S
Correlation of molecular resistance mechanisms and phenotypic
resistance to first-line drugs in Mycobacterium tuberculosis
strains from Sierra Leone
OP-8
13h00 – 13h15
McNerney R, Mallard K
Lam and HIV: correlation or co-incidence?
OP-9
13h15 – 14h00
Lunch
14h00 – 15h00
POSTER SESSION
SCIENTIFIC SESSION ON NON TUBERCULOUS MYCOBACTERIA
Chair: Dr. Enrico Tortoli
15h00 – 15h45
Guest lecture: Prof. Joseph O. Falkinham, III, Ph.D.,Virginia Tech
(Virginia, U.S.A.)
GL-5
Surrounded by mycobacteria
15h45 – 16h00
Lyberopoulos P, Frangopoulos F, Kontos F, Zerva L, Malagari Ai, Papiris S OP-10
Mycobacterium celatum: An emerging pathogen in the immunocompetent. A case report
16h00 – 16h15
Radomski N, Thibault V, Karoui C, De Cruz K, Cochard T, Gutiérrez C, Supply P,
Biet F, Boschiroli ML
Mycobacterium avium subspecies strains from human
and animal origin
OP-11
16h15 – 16h30
Leão SC, Tortoli E,Viana-Niero C, Ueki SYM, Lima KVB, Lopes ML,Yubero J,
Menendez MC, Garcia MJ
The characterization of mycobacteria from an outbreak suggests a revision of the taxonomic status of members of the
Mycobacterium chelonae-abscessus group
OP-12
European Society of Mycobacteriolog y | 30 th Annual Congress | July 2009 | Por to - Por tugal
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16h30 – 16h45
Santos R, Marques M, Oliveira P, Carvalho F, Carvalho C, Monteiro G, Cabral J,
Frade R, Silva M, Fernandes P
The sunny side of mycobacteria
16h45 – 17h15
Coffee and Tea
OP-13
SCIENTIFIC SESSION ON ISSUES IN THE MODERN TB LABORATORY
Chair: Dr. Gabriela Pfyffer and Dr.Thomas Shinnick
Guest lecture: Dr. Jean-Pierre Zellweger, M.D., Swiss Lung Association (Bern, Switzerland)
17h15 – 18h00
The use of Interferon Gamma Release Assays as an aid in the control of tuberculosis
GL-6
18h00 – 18h30
Guest lecture: Prof. Lee W. Riley, M.D., Ph.D., University of California at Berkeley
(California, U.S.A.)
GL-7
A novel diagnostic test to differentiate latent TB infection and
active disease
18h30 – 18h45
Kaal E, Kolk A, Kuijper S, Janssen HG
Fast identification of Mycobacterium tuberculosis in sputum and
cultures based on thermally-assisted hydrolysis and methylation
by gas chromatography-mass spectrometry
OP-14
18h45 – 19h00
Ängeby K, Juréen P, Giske C, Chryssanthou E, Werngren J, Hoffner S, Kahlmeter
G, Sturegård E, Schön T
How Wild-type MIC distributions can be useful to determine
clinical breakpoints in Mycobacterium tuberculosis
OP-15
19h00 –19h15
Miotto P, Cirillo DM
Molecular techniques to monitor TB patients’ treatment: selective removal of DNA from dead bacteria in mixed populations by
use of ethidum monoazide
OP-16
Visit and Dinner at the Port Wine Cellars
Tuesday, July 7th
SCIENTIFIC SESSION ON VACCIN DEVELOPMENT AND PATHOGENESIS
Chair: Dr. David Minnikin
9h00 – 9h45
Guest lecture: Prof. Carlos Martin, M.D., Ph.D., University of Zaragoza
(Zaragoza, Spain)
GL-8
New live tuberculosis vaccines strategies
09h45 – 10h30
Guest lecture: Prof. Lee W. Riley, M.D., Ph.D., University of California at Berkeley
(California, U.S.A.)
GL-9
Regulation of Mycobacterium tuberculosis cell wall lipid composition and its effect on in vivo bacterial persistence
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10h30 – 11h00
Coffee and Tea
11h00 – 11h15
Brzostek A, Pawelczyk J, Rumijowska-Galewicz A, Dziadek B, Dziadek J
Mycobacterium tuberculosis is able to accumulate and utilize
cholesterol
OP-17
11h15 – 11h30
Rodríguez-Güell E, Alonso C, del Val-Romero B, Clivillé R, Secanella SP, Roura-Mir
C, Cañete C, Navarro A, de Gispert FX , Luquin M, Julián E
Mycolic acid-induced IFN-g production by CD1-restricted T cells
from tuberculous patients
OP-18
11h30 – 11h45
Farnia P, Ali Veleyati A, Masjedi MR, Ibrahim TA, Tabarsei P, Haroun RZ, Kuan HO,
Omar AR
A report on new adapted forms of extensively drug resistance
tubercle bacilli : Transmission Electron Microscopy analysis
OP-19
ESM 2009
11h45 – 12h00
Homolka S, Niemann S, Russell DG, Rohde KH
Growth profile of clinical isolates of Mycobacterium tuberculosis
complex in murine macropghages
OP-20
12h00 – 12h15
van Ingen J, van der Wel N, Dekhuijzen R, Boeree M, van Soolingen D
Presence of esat-6 and cfp-10 genes does not lead to phagolysosome translocation of Mycobacterium szulgai
OP-21
12h00 – 12h30
Fraga AG, Braga JE, Cruz A, Martins TG, Pereira DR, Meyers WM, Portaels F,
Castro AG, Pedrosa J
Development of an adaptive immune response in the draining
lymph node during Mycobacterium ulcerans infection
OP-22
12h30 – 13h15
Lunch
13h15 – 14h15
BUSINESS MEETING
SCIENTIFIC SESSION ON LABORATORY STRENGTHENING
Chair: Dr. Sven Hoffner and Dr. Mark Perkins
14h15 – 15h00
Guest lecture: Dr. Thomas M. Shinnick, Ph.D., Centers for
Disease Control and Prevention (Georgia, U.S.A.)
GL-10
CDC’s global TB laboratory activities
15h00 – 15h45
Guest lecture: Prof. Afranio Kritski, M.D., Ph.D., Universidade Federal do Rio de
Janeiro (Rio de Janeiro, Brazil)
GL-11
Development and validation of new TB diagnostic tests in Brazil:
experience of Rede-TB
15h45 – 16h30
Guest lecture: Prof. Moisés Palaci, Ph.D., Universidade Federal do Espírito Santo GL-12
(Vitória, Brazil)
Experience of a successful mycobacteriology laboratory network
in Espirito Santo- Brazil
16h30 – 17h00
Coffee and Tea
17h00 – 17h15
Portugal C, Cardoso N, Sancho L, Sousa G
Tuberculosis software in a general hospital, working instrument
OP-23
17h15 – 17h30
den Hertog A, Koeleman M, Ingham C, Fey F, Langerak E, Klatser P, Anthony R
Development of an automated culture system for M. tuberculosis with autofluorescence detection
OP-24
17h30 – 17h45
Morcillo N, Imperiale B, Di Giulio B
Second-line drug susceptibility testing of Mycobacterium tuberculosis by MGIT 960 system, the microplate colorimetric-based
method and the proportion method
OP-25
17h45 – 18h15
BEST POSTER
Cultural Evening Dinner
Wednesday,
July 8th
SCIENTIFIC SESSION ON DRUG DEVELOPMENT
Chair:Dr. Nalin Rastogi
9h00 – 9h45
Guest lecture: Prof. Clarice Queico Fujimaro Leite, M.Sc., Ph.D.; Universidade
Estadual Paulista (Araraquara, Brazil)
European Society of Mycobacteriolog y | 30 th Annual Congress | July 2009 | Por to - Por tugal
GL-13
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Screening of molecules with anti-TB activity, from the Brazilian
cerrado plants, and synthetic metallo- organic compounds
09h45 – 10h30
Guest lecture: Prof. Moisés Palaci, Ph.D., Universidade Federal do Espírito Santo
(Vitória, Brazil)
GL-14
Clinical trials of drugs and diagnostic tests: the challenges in
mycobacteriology
10h30 – 10h45
van Ingen J, Boeree M, Amaral L, Pando RH, van Soolingen D
Thioridazine shows promising activity in a murine model of
multidrug-resistant tuberculosis
OP-26
10h45 – 11h00
Rodrigues L, Sampaio D, Couto I, Machado D, Kern WV, Amaral L, Viveiros M OP-27
Contribution of efflux pump activity for macrolide resistance in
M. avium complex
11h00 – 11h30
Coffee and Tea
SESSION ON PRACTICAL ASPECTS AND QUALITY ASSURANCE IN
MOLECULAR EPIDEMIOLOGY
Chair: Dr. Elvira Richter and Prof. Christophe Sola
11h30 – 12h00
Allix-Béguec C, HubansC, Ferreira S, Supply P
New, easy-to-use tools for quality-controlled genotyping of M.
tuberculosis complex strains
GL-15
12h00 – 12h20
Abadia E, Zhang J, Refrégier G, Sola C
Membrane- based versus microbead- based spoligotyping:
Preliminary results on a quality- insurance study on 10 sites
worlwide
OP-28
12h20 – 12h30
Discussion
SYMPOSIUM SPONSORED BY INSTAND, SOCIETY FOR RESEARCH
PROMOTION OF QUALITY ASSURANCE IN MEDICAL LABORATORIES,
WHO COLLABORATING CENTRE, DUESSELDORF, GERMANY
12h30 – 13h30
EXTERNAL QUALITY ASSURANCE
PD Dr. Elvira Richter, NRL (Borstel, Germany)
EQA in a low incidence, high income country
Dr. Girts Skenders, State Agency of TB and Lung Disease (Latvia)
Organization and EQA of Latvian TB laboratory network
Dr. Akos Somoskövy, M.D., Ph.D., D.Sc., Foundation for Innovative Diagnostics
(FIND) (Geneva, Switzerland)
Quality Assurance for new techniques – what is necessary, what
is possible?
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13h30 -13h45
Closing remarks
13h45 – 14h15
CLOSING OF CONGRESS
14h15
Conference closes
ESM 2009
Programme of Poster
Presentations
Miotto P, Baldan R, Cirillo DM
Evaluation of the high-throughput repetitive-sequence-based PCR diversilab
system in M. tuberculosis molecular epidemiology studies
PP-1
Zhang J, Abadia E, Refrégier G, Ruimy R, Boschiroli ML, Guillard B, Sola C
68 spacers Mycobacterium tuberculosis complex spoligotyping : A study using a
microbead-based high throughput format
PP-2
Niemann S, Khechinashvili G, Gegia M, Mdivani N, Tang YW
Association between Beijing genotype and drug resistance among Mycobacterium
tuberculosis isolates circulating in the Republic of Georgia
PP-3
Baboolal S, Millet J, Akpaka PE, Ramoutar D, Rastogi N
Mycobacterium tuberculosis epidemiology and genetic diversity in the Twin Island
Republic of Trinidad and Tobago
PP-4
Mestre O, Luo T, Rauzier J, Golec M, Rastogi N, Rasolofo V, Tonjum T, Sola C, Matic I, Mei J, Gao Q,Vultos
TD, Gicquel B
Diversity and evolution of M. tuberculosis
PP-5
Sharaf-Eldin GS, Elmoula IF, Ali MS, Saaed NS, Ali AB, Mallard K, McNerney R, Algamdi S
Spoligotype patterns and drug resistant profile of Mycobacterium tuberculosis
in Sudan
PP-6
Valcheva V, Mokrousov I, Panaiotov S, Bachiiska E, Zozio T, Sola C, Markova N, Rastogi N
Controversial dissemination pattern of the Bulgaria-specific M. tuberculosis
spoligotype ST125_BGR
PP-7
Panaiotov S, Bachiyska E, Brankova N, Levterova V
Mycobacterium tuberculosis Beijing genotype and origins of the Bulgarians
PP-8
Al-Maniri AA, Singh JPN, Al-Rawas O, Al Busaidi S, Al Balushi L, Ahmed I, Al- Mahruqi S, Haile
M, Diwan V, Hoffner S
A Snapshot on biodiversity and clustering of Mycobacterium tuberculosis among
nationals and immigrants in Oman using spoligotyping
PP-9
David S, Ribeiro JN, Maio JN, João I, Amorim A, Pereira E
The extent of the Latin American-Mediterranean Mycobacterium tuberculosis
spoligotype family in Portugal
PP-10
Von Groll A, Martin A, Felix C, Prata P, Honscha G, Portaels F, Almeida da Silva P, Palomino JC
Fitness Study of the RDRio lineage and LAM family of Mycobacterium tuberculosis in
a study population in Rio Grande, Brazil
PP-11
Perdigão J, Silva C, Portugal I
Genomic characterization of Lisboa family strains by deletion analysis
PP-12
Obrovac M, Katalinic-Jankovic V, Grce M, Zmak L
Importance of molecular typing in suspected intra-familial transmission
of tuberculosis
PP-13
Oral Zeytinli U, Kayar Mb, Karacali A, Sahan Kipalev A,Yula E, Köksal F
Detection of clonal complexity in clinical M. tuberculosis Isolates by MIRU-VNTR in
Cukurova Region, Turkey
PP-14
Leite CQF, Santos ACB, Pandolfi JRC, Malaspina AC, Pavan FR, Mendes NH,Viana BHJ
Molecular epidemiology study of tuberculosis patients in a small city of São Paulo –
Brazil, from 2002 to 2006
PP-15
Leite CQF, Nogutia EN, Malaspina AC, Santos ACB, Hirata RDC, Hirata MH, Cardoso RF
Genotyping of Mycobacterium tuberculosis in northwest of Paraná State of Brazil
PP-16
European Society of Mycobacteriolog y | 30 th Annual Congress | July 2009 | Por to - Por tugal
15
16
Mello FAF, Albarral MIP, Mendes NH, Pandolfi JRC, Santos ACB, Almeida EA, Cardoso RF
Spoligotyping of Mycobacterium tuberculosis isolated from patients of Clemente
Ferreira ambulatory in São Paulo, SP – Brazil
PP-17
Tajeddin E, Farnia P, Kargar M, Noroozi J, Ahmadi M, Kazempour M, Hadadi M, Masjedi M,Velayati A
Comparison Of Mycobacterium Beijing Genotype with VNTR, Spoligotyping and
RFLP-IS6110
PP-18
Ritacco V, Reniero A, Beltrán M, López B, Kantor I, Barrera L
Multiply recurrent tuberculosis in a pacient living with HIV: Reinfection or
reactivation?
PP-19
Tavares Magalhães A,Alves A, BragaR,Valente I, DuarteR, Miranda A
Molecular epidemiology of tuberculosis in Vila Nova de Gaia, Portugal
PP-20
Alves A, Miranda A
Molecular study of recurrent tuberculosis cases
PP-21
Ehricht R, Slickers P, Monecke S
Genotyping of drug resistance in Mycobacterium tuberculosis using diagnostic
microarrays
PP-22
Al-Hajoj S,Varghese B, Herbawi M, Al-Omari R, Allix-Béguec C
Genotyping of mono and multi-drug resistance TB in Saudi Arabia
PP-23
Machado D,Viveiros M, Rodrigues L, Couto I , Amaral L
Early detection of MDRTB by molecular tools in the control of drug resistant
tuberculosis in Portugal: a case of success
PP-24
Vladimirov K, Zaitseva E, Ivanov A
Drug-resistance of Mycobacterium tuberculosis at penitentiary institutions of St.
Petersburg, Russian Federation
PP-25
Stoffels K, Fauville-Dufaux M
An increase of drug resistance since 2001 in multidrugresistant M. tuberculosis
isolates from Belgium
PP-26
Perdigão J, Ferreira A, Malaquias A, Macedo R, Brum L, Portugal I
Mutational analysis of genes associated with resistance to injectable second-line
drugs in Mycobacterium tuberculosis clinical isolates from Lisbon, Portugal
PP-27
Nuak J, Ferreira D, Carvalho T, Gomes MH, Sarmento A
Multidrug-resistant tuberculosis
PP-28
Tudó G, Rey E, Alcaide F, Coll P, Codina G, Martín-Casabona N, Montemayor M, Moure R, Salvadó M,
González-Martín J
Characterisation of streptomycin mutations in Mycobacterium tuberculosis clinical
isolates in the area of Barcelona
PP-29
Fattorini L, Pardini M, Cirillo D, Borroni E, Miotto P, Filippini P, Cassone A
Surveillance of Drug-Resistant Tuberculosis in Italy
PP-30
Sancho L; Portugal C; Tancredo L; Silva M; Dias A; Silva F, Sousa G
Tuberculosis resistance in a general hospital in Portugal – 9 years surveillance
PP-31
Yates M, Brown T, Drobniewski F
Does a mutation in the rpoB mean that the M. tuberculosis is resistant
to rifampicin?
PP-32
Zaldumbide MA, Mazarrasa CF, Martinez-Martinez L, Balbin JA
Genotypic detection of isoniazid and rifampin resistance in Mycobacterium
tuberculosis clinical isolates
PP-33
Chan CYR, Chan WCE, Au TKM, Lai WMR,Yew WW,Yip CW, Kam KM
Physiological fitness and transmission potential of multi-drug resistant
Mycobacterium tuberculosis clinical isolates in Hong Kong
PP-34
ESM 2009
Sousa AS, Pinheiro MD, Carvalho T, Gonçalves H
Mycobacterium tuberculosis: 1999-2008 antituberculosis drugs surveillance in clinical
isolates from patients in the largest hospital in the North of Portugal
PP-35
Von Groll A, Martin A, Jureen P, Hoffner S, Portaels F, Palomino JC, Almeida da Silva P
Fitness cost of Mycobacterium tuberculosis clinical isolates resistant to
fluoroquinolones
PP-36
Von Groll A, Martin A, Jureen P, Hoffner S, Portaels F, Almeida DA Silva P, Palomino JC
In vitro activity of ofloxacin, moxifloxacin and gatifloxacin against Mycobacterium
tuberculosis by the resazurin colorimetric method
PP-37
Paasch F, Martin A, Docx S, Fissette K, Portaels F, Palomino JC
Rapid detection of extensively drug-resistant Mycobacterium tuberculosis by the
resazurin microtiter assay plate
PP-38
Montoro E,Yzquierdo S, Lemus D, Echemendia M, Takiff H
Detection of embB gene codon 306 mutations in ethambutol susceptible and
resistant Mycobacterium tuberculosis strains
PP-39
Yew WW,Yan SW, Fung SL, Chau CH, Chan Chiu Y
Tolerance of moxifloxacin in routine clinical treatment of tuberculosis
PP-40
Perdigão J, Sabino A, Milho C, Macedo R, Brum L, Portugal I
Characterization of gidB gene in Mycobacterium tuberculosis isolates in Lisbon
Health Region: role in streptomycin resistance and epidemiological markers
PP-41
Gaile I, Skenders G, Leimane V, Jansone I, Bauskenieks M, Pole I, Baumanis V
Fluorquinolone resistant Mycobacterium tuberculosis isolates and their molecular
characteristics
PP-42
Samper S, Millan I, Lopez-Calleja AI, Gavin P, Lezcano MA
Design of a rapid method of identification of a highly transmitted strain based on
the localization of IS6110
PP-43
Gutierrez MC, Brosch R, Marceau M, Tap J, Bourdon E, Brisse Smangenot S, Salvignol G, Barbe V, Médigue
C, Supply P
Driving forces on the evolution of the progenitor of M. tuberculosis
PP-44
Ruiz P, Causse M, Zerolo FJ, Gutierrez J, Casal M
Resistance, MDR and XDR of M. tuberculosis in Spain in the last years
PP-45
Radomski N, Lucas F, Cambau E, Moulin L, Haenn S, Régis M
Detection of non tuberculous mycobacteria in surface waters: comparison of
culture methods
PP-46
Spicic S, Cvetnic Z, Pate M, Duvnjak S, Zdelar-Tuk M, Racic I
Typing of Mycobacterium avium subsp. avium from different sources using PvuII–
PstI–IS901 restriction fragment length polymorphism (RFLP) in Croatia
PP-47
Spicic S, Duvnjak S, Obrovac M, Zdelar-Tuk M, Katalinic-Jankovic V, Racic I, Cvetnic Z
Tuberculosis in pets and wild animals living in urban environment
PP-48
Spicic S, Cvetnic Z, Pate M, Katalinic-Jankovic V, Duvnjak S, Ocepek M, Zdelar-Tuk M, Krt B
IS1245-RFLP based genetic relatedness of the Mycobacterium avium subsp.
hominissuis strains isolated from humans, animals and environment in Croatia
PP-49
Lucas F, Radomski N, Cambau E, Moulin L, Haenn S, Moilleron R
Development of real-time PCR assay for quantification of mycobacteria in
surface waters
PP-50
Amorim A, Macedo R, Pereira E
Nontuberculous mycobacteria, isolated from patients with lung disease, from
Lisboa e Vale do Tejo region, during 2008
PP-51
European Society of Mycobacteriolog y | 30 th Annual Congress | July 2009 | Por to - Por tugal
17
Lima KVB, Lopes ML, Furlaneto IP, Lima EJC, Conceição EC, de Sousa MS, Costa ARF
Nontuberculous mycobacteria infections in the State of Pará, Amazon
Region, Brazil
PP-52
Jahromi NS, Seif S, Farnia P, Kazempour M, Kargar M, Nowroozi J, Kazempour M, Masjedi M,Velayati A
Evaluation of Hsp65, Tb ,Sp Regions in Identifying Mycobacterium Other Than
Tuberculosis (MOTT); using PCR-RFLP
PP-53
Svensson E, Ridell M, Åkerström M, Andersson E
Mycobacterium avium alveolitis after cleansing hotel spa whirlpools
PP-54
Couto I, Machado D,Viveiros M, Rodrigues L, Amaral L
Identification of nontuberculous mycobacteria in clinical samples using molecular
methods: A three-year study
PP-55
^
18
Pate M, Ferme D, Žolnir Dovc M, Ocepek M
Mycobacteria in animals in Slovenia – an overview of the last decade
PP-56
Leite SRA, Silva P, Sato DN, Santos ACB, Miyata M, Leite CQF
Isolation and identification of Rhodococcus and Nocardia genders in sputum
samples with tuberculosis suspect
PP-57
Neonakis IK, Kontos F, Gitti Z, Baritaki S, Bazigos S, Mihailelis E, Zerva L, Spandidos DA
PCR-RFLP of hsp65 for identification of Mycobacterium leprae directly from a
clinical sample
PP-58
Neonakis IK, Kontos F, Gitti Z, Baritaki S, Kosmadakis G, Baritaki M, Zerva L, Spandidos DA
A case-report of Mycobacterium thermoresistibile from Greece
PP-59
Portugal C, Sancho L, Dias A, Tancredo L, Silva M; Sardinha T, Sousa JG
Isolation and frequency of Mycobacterium sp in a general hospital during
a 9-year period
PP-60
Diogo J, Rodrigues A, Nascimento I, Sardinha E, Raposo A, Figueira R, Monge I, Silva K, Gil MJ,
Rodrigues S
Laboratory Microbiology contribution to Mycobacterium spp. Diagnosis in three
district councils of Setubal (Portugal), an area with high mycobacterial
infection prevalence
PP-61
Santos C, Mendes AC, Fernandes SJ, Ramos MH
Mycobacterium lentiflavum as a causative agent of adenopathy
PP-62
Watson C, Lockwood D
Teaching old bones new tricks; single nucleotide polymorphism analysis of
european archaeological M. leprae DNA
PP-63
Greib C, Lazaro E,Viallard JF, Pellegrin JL, Maugein J
Interpretation of positive M. tuberculosis antigen specificifnγ release assays in
tuberculosis diagnosis
PP-64
Wang S, Neo ZY, Mak KX, Quieng MD, Sing LH
Direct Identification of Mycobacterium tuberculosis Complex, Mycobacterium avium
Complex and Mycobacterium kansasii in Smear-positive Clinical Specimens
PP-65
Müllerova M
Rapid diagnosis and drug susceptibility testing of tuberculosis infection: MTDTest2 and Bactec MGIT 960 system
PP-66
Levina K, Dementieva A, Saluotsa M
First experience with genotype MTBDR assai for rapid evaluation of MDR cases
PP-67
Fajfar N, Zolnir - Dovc M
Drug resistant tuberculosis in Slovenia and evaluation of genotype MTBDRPLUS
test in clinical laboratory
PP-68
ESM 2009
Causse M, Gutierrez-Aroca JB, Casal M
Evaluation of a new real-time PCR kit for the diagnosis of tuberculosis
inrespiratory specimens
PP-69
Karabela S, Papaventsis D, Nikolaou S, Konstantinidou E, Sainti A, Ioannidis P, Kanavaki S
Quantiferon-TB Gold assay (QFT) and tuberculine skin test (TST) clinical
performance for the diagnosis of active tuberculosis
PP-70
Karabela S, Papaventsis D, Nikolaou S, Konstantinidou E, Sainti A, Ioannidis P, Kanavaki S
Clinical performance of Quantiferon-TB Gold assay (QFT) for the diagnosis of
latent tuberculosis in different patient groups
PP-71
Nikolaou S, Karabela S, Papaventsis D, Sainti A, Konstantinidou E, Ioannidis P, Kanavaki S
Tuberculosis diagnosis by Quantiferon TB Gold assay in areas with differences in
TB incidence
PP-72
Havelkova M, Bartu V, Kubin M
Quantiferon -TB Gold In-Tube test used in Prague patients listed in the National
Tuberculosis Register
PP-73
Cacho J, García-Cañas A, González Torralba A, Cano I, Pérez Meixeira A, Ramos Martos, SánchezConcheiro M
Practical experience of using a DNA amplification assay for rapid detection of
Mycobacterium tuberculosis complex in respiratory specimens
PP-74
Morgan K
Real-time polymerase chain reaction for the direct detection of Mycobacterium
tuberculosis in clinical specimens
PP-75
Kontos F, Zerva L
The utility of molecular testing in routine mycobacteriology diagnosis
PP-76
Salas S, Hernández J, Ojeda P, Awad C, de la Hoz F, Murcia M
Detection of Mycobacterium tuberculosis DNA in formalin-fixed, paraffin-embedded
tissue specimens by spoligotyping: application to histopathological diagnosis
PP-77
Cardoso S, Coelho R, Paulo C, Abreu C, Silva S, Gomes H, Sarmento A
Pott´s Disease: an ancient disease?
PP-78
Loureiro C, Matos G, Balacó I, Mota M, Nogueira C, Lemos S, Rocha G
Osseous tuberculosis at age of 9 months
PP-79
Secanella SP, Luquin M, Julián E
Differences in direct antitumoral capacity among the various Mycobacterium bovis
BCG substrains
PP-80
Anoosheh S, Farnia P, Noruzi J, Kargar M, Kazempour M, Seif S, Masjedi MR,Velayati AA
Role of TNF-a gene polymorphisms in host genetic susceptibility to pulmonary
tuberculosis
PP-81
Torrado E, Fraga AG, Logarinho E, Martins TG, Carmona JA, Gama JB, Carvalho MA, Proença F, Castro
AG, Pedrosa J
Mycolactone interferes with the protective IFN-γ-dependent activation of
macrophages during infection with Mycobacterium ulcerans
PP-82
Montoro E,Valdés I, Aguilar D, Orozco H, Hernández-Pando R
Virulence, immunogenicity and protection induced by ´Mycobacterium habana´
strains in a murine model of pulmonary tuberculosis
PP-83
Saraiva M, Sousa C, Carmona JA, Cruz A, Pedrosa J, Castro AG
Dendritic cells differentially express IL12-family cytokines after infection with
Mycobacterium tuberculosis or M. bovis BCG
PP-84
Simões MF, Jordão L, Teles JMM, Couto S, Moniz-Pereira J, Pimentel M
Analysis of M. smegmatis mutants resistant to Ms6 infection
PP-85
European Society of Mycobacteriolog y | 30 th Annual Congress | July 2009 | Por to - Por tugal
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20
Julián EG, Rodríguez-Güell E, del Val-Romero B, Clivillé R, Cañete C, Navarro A, de Gispert FX,
Luquin M, Alonso C
Humoral response in tuberculous patients against the mycolic acids of
Mycobacterium tuberculosis
PP-86
López AG
Structural, functional and bioinformatic characterization of TlyA protein from
Mycobacterium tuberculosis
PP-87
Ferreira C, Afonso A, Duarte R, Lyashchenko K, Silva A, Rodrigues F, Miranda A, Tavares M, Caldas C,
Valente F,Valente A,Vasconcelos O, Amado J, Correia-Neves M
Evaluation of the applicability of serodiagnosis for tuberculosis in Portugal
PP-88
Martin A, Munga Waweru P, Babu Okatch F, Amondi Ouma N, Bonte L, Palomino JC,Varaine F, Portaels F
Implementation of the thin layer agar (TLA) for the diagnosis of smear negative
pulmonary tuberculosis in a high HIV prevalence setting
PP-89
Ferro RS, Shikama M-L,Villela G, Sato DN, Giampaglia CS, Martins MC, Martin A, Palomino JC
Direct detection of rifampin resistance in Mycobacterium tuberculosis by the nitrate
reductase assay applied directly in sputum samples
PP-90
Ferro RS, Shikama M-L,Villela G, Sato DN, Giampaglia CS, Martins MC, Martin A, Palomino JC, Telles MAS
Direct detection of rifampin resistance in Mycobacterium tuberculosis by the nitrate
reductase assay applied directly in sputum samples
PP-91
De Haas P, Zenhorst R, Mwamba P, Muvwimi M, Mwanza W, Mbulo G, Kapata N, Ayles H
MTBDRPLUS assay is a useful tool to screen for multi-drug resistant tuberculosis
in a national survey
PP-92
de Haas P, Moyoyeta M, Samutela M, Mwanza W, Musunsa A, Mbulo G, Muvwimi M, Ayles H
Contribution of laboratory factors to high MGIT culture contamination rate
in Zambia
PP-93
Ahmed A, Qazi F, Khan AJ
Programmatic Community-based Management of MDR-TB: Experience in
Karachi, Pakistan
PP-94
Muchwa C, Akol J, Mumbowa F, Orikiriza P, Morgan K, Eisenach K, Joloba M, Etwom A, Mugyenyi P,
Mugerwa R
Evaluation of Capilia (TAUNS) for rapid identification of Mycobacterium tuberculosis
complex from cultures
PP-95
Muchwa C, Akol J, Orikiriza P, Morgan K, Mumbowa F, Eisenach K, Etwom A, Joloba M
Comparison of capilia (TAUNS) and IS6110 PCR for rapid identification of
Mycobacterium tuberculosis complex from cultures in Kampala, Uganda
PP-96
Bwanga F, Hoffner S, Haile M, Joloba ML
Direct testing for multi drug resistant tuberculosis with four assays evaluated at
Kampala, Uganda
PP-97
McNerney R, Turner C, Mallard K, O’Sullivan D
PEA production by mycobacteria and its application in a rapid drug
susceptibility test
PP-98
Balmoi F
Various strategies to decontaminate acid fast bacilli positive liquid cultures
from Bactec MGIT 960
PP-99
Orikiriza P
Low cost isolation of Mycobacterium tuberculosis (MTB) from blood
PP-100
Kayar B, Oral Zeytinli U, Karacali A, Soyal A, Nagiyev T, Köksal F
Comparison of Rapid Colorimetric Method, Proportion Method and BACTEC460
TB System for testing susceptibility of M. tuberculosis to rifampine and isoniaside
PP-101
ESM 2009
Crews V, Warns M, Pfeltz R, Beaty PS, Rosales J, Kopher K, Joshi S, Hoosen A, Said H
Evaluation of the MGIT TBc ID test vs two commercially available rapid
immunoassays for M. tuberculosis complex organism detection from liquid
and solid culture
PP-102
Montoro E, Milián Y, Lemus D, Echemendía M,Yzquierdo S, Martin A,Van der Stuyft P, Palomino JC
Nitrate reductase assay applied to direct detection of drug resistance in
Mycobacterium tuberculosis
PP-103
Montoro E, Lemus D, Madruga M, Mirabal N, Milián Y,Yzquierdo S, Echemendía M, Martín A,Van der
Stuyft P, Palomino JC
Use of nicotinamide in colorimetric methods for rapid detection of pyrazinamide
resistance in Mycobacterium tuberculosis
PP-104
Hepple P, Novoa-Cain J, Cheruiyot C, Richter E, Ritmeijer K
Implementation of liquid culture for tuberculosis diagnosis in a remote setting:
Lessons learned
PP-105
Ichijo T, Izumi Y,Yamaguchi N, Nasu M
Rapid detection of respiratory active mycobacteria by auramine O-CTC
double staining
PP-106
Rey E, Tudó G, González-Martín J
Synergistic activity of two antituberculous drug combinations against clinical
isolates of Mycobacterium tuberculosis resistant to isoniazid
PP-107
Stoffels K, Traore H,Van Hoof R, Fauville-Dufaux M
Tobramycin-clarithromycin combination on Mycobacterium tuberculosis
clinical isolates
PP-108
Au-Yeang CKW, Au TK, Chan EWC, Chan RCY
Prevalence of Efflux-Mediated Rifampicin Resistance in Mycobacterium tuberculosis
Clinical Isolates
PP-109
Leite CQF, Pavan FR, Maia PIS, Deflon VM, Sato DN, Azevedo AA, Poelhsitz GV, Leite SRA, Franzblau SG
Intra and extracellular activity of ruthenium complexes against Mycobacterium
tuberculosis and their cytotoxicity
PP-110
Leite S, Pavan F, Maia P, Deflon V, Batista A, Sato D, Franzblau S, Leite C
Anti-Mycobacterium tuberculosis activity of thiosemicarbazones, semicarbazones
and hydrazones
PP-111
Ramos J, Rodrigues L, Couto I, Amaral L,Viveiros M
Methods for assessment of ethidium bromide transport across Mycobacterium
smegmatis cell wall
PP-112
Martins M,Viveiros M, Couto I, Amaral L
The human macrophage as a model to select compounds active against
MDR/XDR-TB
PP-113
Cynamon M, Mookherjee S, Shoen C
In vitro activities of jpc 2067 alone and in combination with SMX against
nocardia species
PP-114
Nina J
Nosocomial TB in a laboratory setting
European Society of Mycobacteriolog y | 30 th Annual Congress | July 2009 | Por to - Por tugal
PP-115
21
Abstracts
of Guest
Lectures (GL)
European Society of Mycobacteriolog y | 30 th Annual Congress | July 2009 | Por to - Por tugal
23
GL-1
TUBERCULOSIS IN PORTUGAL
Miguel Villar
Consultant on Tuberculosis, Directorate-General of Health, Lisbon, Portugal
Tuberculosis is a global problem with an estimated number of 9 million cases per year, 83% of which are in Sub-Saharian
Africa and South-East Asia, where we find many of the high burden countries.
Concerning multidrug-resistance tuberculosis (MDR-TB), WHO estimates about half a million cases globally, per year,
including 50.000 extensively drug-resistant tuberculosis (XDR-TB).
In 2007, European Union (EU) had an incidence rate of 17/105, having Portugal one of the highest rates in the EU (27/105).
In the last 20 years, the incidence in Portugal has decreased consistently more than 7% per year in the last five years.
This reduction is mainly in the age group between 25 and 44 years old, leading to a shift to the right of the median age
both in the nationals and in the immigrants.
The foreign born cases have represented about 12% of the cases and the prevalence of HIV has been around 14%.
Most of the cases are pulmonary forms (74.1%) between 2003-2007, 67.5% of which are SS+ and 75.3% are culture positive.
Mixed multidrug-resistance tuberculosis, including XDR-TB, during the same period, represents 1.9% (154 cases) of the
TB cases at the start of treatment, varying from 1.3% (22 cases) in 2006 to 2.4% (38 cases) in 2004, with an average of 31
cases per year (1.9%). These proportions are representative as the coverage of drug sensibility tests (DST) is over 80%.
We will address the importance of the Micobacteriology Laboratory network, concerning case detection, definition of
confirmed cases, early diagnosis of MDR-TB and 1st and 2nd line DST.
As an important complement of the DOTS Strategy, the analysis of the outcomes will be discussed, concerning not only
the general population but also the different risc groups, having as a goal the 85% cure rate proposed by WHO.
We finish our presentation addressing the strategy for MDR/XDR-TB control in Portugal, namely the importance of the
reference network for MDR-TB with its national coordination.
European Society of Mycobacteriolog y | 30 th Annual Congress | July 2009 | Por to - Por tugal
25
GL-2
THE BRAZILIAN EXPERIENCE CONTROLING TB MULTI-DRUG RESISTANCE
Fernando Augusto Fiuza de Melo
• Médico, Diretor do Instituto Clemente Ferreira – Coordenadoria de Controle de Doenças da Secretaria de Estado
da Saúde de São Paulo – ICF/CCD/SP
• Doutorado em Medicina, área de pneumologia, pela Escola Paulista de Medicina da Universidade Federal de São
Paulo - EPM/UNIFESP
• Membro do Comitê de Assessoria Técnico-Científico do Programa Nacional de Controle da Tuberculose do
Ministério da Saúde - PNCT/MS
Correspondence:
Rua Santo Estácio, 248 – Cidade Vargas, CEP. 04319-010 - Brasil - São Paulo,SP
Tele-fax: 0055 11 3218 8653 - Telemóvel 0055 11 8469 4330 - e-mail: [email protected]
Brazil was the first developing country using the short duration regimen of rifampin (R) and isoniazid (H) combined in
one capsule for six months, plus pyrazinamide (Z) during the first two months after reorganizing the Brazilian National
Tuberculosis Control Program of (BNTBCP, Programa Nacional de Controle da Tuberculose, PNCT) in 1980. At that
moment Brazil adopted the anti-TB regimen named E-1 (2RHZ/4RH) for all forms of TB with no known previous treatment. A similar regimen was adopted for Meningitis TB, named E-2, adding corticoids in the intensive phase with the
recommendation to lengthen the continuation phase of treatment to 7 months (2RHZCort/7RH). For those TB cases of
relapsing (RC) or re-treatment after defaulting (RA) the anti-TB regimen adopted was E-1R with ethambutol (E) during
the intensive phase (2RHZE/4RH). The recommended regimen for failure (F-1) cases was named E-3 and included Z and
E, associated with streptomycin (S) and the ethionamida (Et) during at least 12 months (3SZEEt/9EEt)¹
The E-1 was evaluated under pragmatic clinical “non-study” conditions during decades 1980/90 and also during half of the
first decade of the new millennium and shown an efficacy of 94.6 and 93.9%, an effectiveness of 77.8 and 77.1%, a default
rate of 13.7 and 13.1%13%, a failure rate of 1.5 and 1.7%%, a serious adverse events rate of 3.1 and 3.3% and a mortality
rate of 3.9 and 4.8%, respectively. (²,³).The small worsening on mortality rate might be related to high rates of defaulting and
the HIV co-infection.The good quality of the National Aids Program Control and the increasing rate of TB treatment under
direct supervision in Brazil are possible reasons for Brazil rates of resistance were not too high. On the other hand, results
of E-3 were not nice with efficacy and effectiveness varying between 57,5% - 85,2 and 66,7 - 84.7%, respectively (4).
4% of 80,000 cases of TB notified in Brazil were resistant to R+H or were unable to be treated with these drugs for any
other reason and were defined as F-1 case. These patients were treated with E-3 (3SZEEt/9EEt) and are considered in
Brazil as a case of MDR-TB. Brazil estimates a rate between 0,3 and 0,4% of cases of F-1 not responding to E-3. These
cases are defined in Brazil as a Multiresistant TB (MRTB) and there is no well established anti-TB regimen for these patients in the BNTBCP5.
In 1995 an anti-TB regimen including amicacin (AM), ofloxacin (OFX), terizidona (TRZ), clofazimine (CFZ) and E was
evaluated in some TB Centers in Brazil with reasonable results (6).
In 2000 the Monitoring Program for MRTB was created, centralizing the notification of all cases of TBMR in Brazil and
creating a work group including health professionals involved with MRTB in order to define and organize a way to control
MRTB in Brazil. In 2007, Guidelines for MRTB were published presenting the knowledge about TBMR and establishing
rules for diagnosis, treatment, prevention and biosafety; providing orientation on epidemiologic surveillance, building human and material resources, and implementing a specific National Notification System for these patients. The current
alternative regimen for MRTB cases is defined by the sensitivity tests and administered under supervision, including: AM
(or S if sensible) for 12 months, OFX, TRZ and E for 18 months and Z (if sensible) for 6 months7. Metronidazole (MTZ)
replacing Z, especially in cases of intolerance or resistance, is used in some clinics.
TBMR rates were evaluated in Brazil between 2000 and 2005, showing the following results:
Between 323 and 334 cases had been annually notified from 2000 to 2004, with an increase in 2005 to 383 cases notified
among a total of 80.000 TB cases.
Increasing cure rates over time (40 to 62%), with some organized units showing better rates (75 to 85%).
26
ESM 2009
Default rates between 5 and 7%; failure rates between 10 e 15%; mortality rates had decreased from 33% to 11% over time.
Most of MRTB were post-primary cases (74 to 80%); 6 to 8% were the primary cases, especially contacts and risk groups;
11 to 20% were indeterminate.
TBMR rate among HIV co-infected patients was low, between 1.6 e 3%7,8.
Extensively multi-drug resistant (X-MDR) TB cases, presenting resistance to 2 first line drugs and 3 second line drugs,
have been observed since 20009, however for more accurate estimates, a national survey is necessary. In recent survey
at the Clemente Ferreira Institute, 34 cases resistant to fluoroquinolone were notified, and 16 cases were also resistant
to AM and 18 to S. The patients were treated with an alternative regimen indicated for MRTB, with 9 cure cases and 25
failure cases, including 17 deaths. An important finding was the occurrence of 3 primary X-MDR cases10.
References
Ministério da Saúde/Fundação Nacional de Saúde/Comitê Técnico-Científico de Assessoramento à Tuberculose/Comitê
Assessor para Co-infecção HIV-Tuberculose. Tuberculose: guia de vigilância epidemiológica, Brasília, 2002.
Ministério da Saúde/Fundação Nacional de Saúde/Centro de Referência Prof. Hélio Fraga-Rio de Janeiro, Documento
Básico da Reunião de Avaliação operacional e epidemiológica do PNCT na década de 80. Bol Pneumol Sanit 1993,
Numero Especial.
Ministério da Saúde/Secretaria de Vigilância em Saúde/Centro de Referência Prof. Hélio Fraga-Rio de Janeiro. Análise da
situação da tuberculose no Brasil nos anos 90 e início da década atual. Bol Pneumol Sanit 2005;13:133-179.
Campos HS, Melo FAF. Efetividade do esquema 3 (3sSZEEt/9EEt) no retratamento da tuberculose na rotina das unidades
de saúde. Bol Pneum Sanit 2000;8:7-14.
Melo FAF, Ide Neto J, Seiscento M, Pinto JA, Afiune JB: Tuberculose Multirresistente. J Pneumol 1993;19:73-82.
Dalcolmo MP, Fortes A, Melo FAF, Motta R, Ide Neto J, Cardoso N,Andrade M, Barreto AW, Gerhardt G. Estudo de efetividade de esquemas alternativos para o tratamento da tuberculose multirresistente no Brasil. J Pneumol 1999;25:70-77.
Ministério da Saúde/Secretaria de Vigilância em Saúde/Centro de Referência Prof. Hélio Fraga/Projeto MSH. Tuberculose
multirresistente: guia de vigilância epidemiológica;2005:89pg
Melo FAF, Afiune JB, Ide Neto J, Almeida EA, Spada DTA, Antel ANL, Cruz ML. Aspectos epidemiológicos da tuberculose
multirresistente em serviço de referência na cidade de São Paulo Rev da Soc Brasil Med Trop 2003;36:733-40.
Emergence of Mycobacterium tuberculosis with extensive resistance to second line drugs worldwide 2000 – 2004.
MMWR 2006;55(11)
Savioli MTG, Melo FAF, Morrone N e Rodrigues DS. Tuberculosis with extensive resistance to drugs in a TB reference
center in Sao Paulo, Brazil. Poster accepted for UICTER 2009;Cancum, Mexico.
European Society of Mycobacteriolog y | 30 th Annual Congress | July 2009 | Por to - Por tugal
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GL-3
EVOLUTIONARY FORCES IN Mycobacterium tuberculosis
Sebastien Gagneux
Division of Mycobacterial Research, MRC National Institute for Medical Research, The Ridgeway, Mill Hill, NW7 1AA,
London, United Kingdom; [email protected], phone:+4420 8816-2399, fax: + 4420 8816-2564
The Mycobacterium tuberculosis complex (MTBC) consists of genetically monomorphic organisms. Studying the genetic
population structure and evolution of monomorphic bacteria is hindered by the lack of DNA sequence variation; methods
such as multilocus sequence typing (MLST), which have been well established in other bacteria, are not applicable. Because
of this limitation, most current genotyping methods for MTBC are based on mobile or repetitive DNA elements (e.g.
IS6110 RFLP, spoligotyping, MIRU-VNTR). Mobile and repetitive DNA regions change relatively quickly, which makes them
ideal markers for molecular epidemiological analyses. However, because these markers can exhibit convergent evolution
leading to homoplasy (similar patterns emerging in unrelated strains), they are less robust to infer phylogenetic relationships. Furthermore, actual DNA sequence data is preferred for population genetic analyses. To get around this problem,
we sequenced 89 genes in 108 MTBC strains. We used these DNA sequence data to explore the evolutionary forces that
have shaped the genetic diversity in MTBC. Our findings show that MTBC is under greatly reduced selective constraint (i.e.
purifying selection is reduced in MTBC), and as a result, much of the genetic diversity in MTBC is likely to have functional
consequences. These findings have important implications for the development of new tools to control tuberculosis.
28
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GL-4
ADVANCES IN THE MOLECULAR EPIDEMIOLOGY OF TUBERCULOSIS
Dick van Soolingen1, Jakko van Ingen1, Philip Supply2, Anita Schürch1, Ida Parwati3, Reinout van Crevel4, Nguyen Van Hung5,
1- Frank Cobelens6, and Kristin Kremer1 National Tuberculosis Reference Laboratory, Nat. Inst. for Public Health and the
Environment (RIVM), Bilthoven, the Netherlands; [email protected]
2 - National Center for Scientific Research, Institut Pasteur, Lille, France
3 - Dept. of Clin. Path. Hasan Sadikin Hosp., Med. Fac. Padjadjaran Univ.,Bandung, Indonesia
4 - Dept. Int. Med., Radboud University Nijmegen Medical Center, Nijmegen, the Netherlands
5 - Nat. Hosp. of Tuberculosis and Respiratory Diseases. Hanoi,Vietnam
6 - Center for Poverty-related Comm. Dis., Acad. Medical Center, Amsterdam, the Netherlands
Although elimination of tuberculosis in Europe is not yet in sight, the ECDC held a meeting in Stockholm in April 2009
to re-define the indicators of a successful TB control on this continent. Molecular epidemiology was identified as a key
component to detect the level of active transmission. In 2009, a new ECDC project has been initiated to re-activate
the molecular surveillance of (MDR/XDR) TB in Europe, with strong focus on a high coverage of MDR/XDR cases in
Central and Eastern Europe. In the previous project transmission of MDR/XDR-TB in Europe was largely caused by
Mycobacterium tuberculosis Beijing genotype strains.
In a recent (2009) resistance survey in Vietnam, a significant correlation between resistance and Beijing strains was observed. Moreover, in previous studies in Ho Chi Minh City treatment failures and relapses were more frequently found
in patients infected by Beijing strains. However, in a recent, larger study in Indonesia patients infected with Beijing genotype strains also more often had a positive sputum culture after six months treatment (RR:1.95; CI 95%:1.25-3.02), but
this was not correlated with differences in drug resistance. Therefore, this suggests that M. tuberculosis Beijing genotype
strains have a higher capacity to withstand tuberculosis treatment, even in the absence of drug-resistance.
The new European network on molecular epidemiology will implement 24-loci VNTR typing as a standard. Although the
utility of VNTR typing has been shown in multiple studies, a broad and nation wide comparison of IS6110 RFLP typing
and VNTR typing is still missing. In the Netherlands 4400 M. tuberculosis isolates from the period of 2004-2008 have been
subjected to IS6110 RFLP as well as VNTR typing and a concordance of 81% has been observed. Moreover,VNTR typing
showed a higher degree of concordance with findings in the conventional contact tracing than RFLP typing.
To come to the highest resolution of DNA typing, two isolates from the Harlingen tuberculosis outbreak, that have been
isolated with an interval of 12.5 years and which were separated by four person-to-person transmissions were subjected
to whole genome sequencing. Four single nucleotide polymorphisms (SNPs) and one tandem repeat polymorphism
(TRP) and a IS6110 transposition were identified. Typing of all 104 isolates in the IS6110 RFLP cluster with the six DNA
polymorphisms endorsed the separate line of transmission established by contact tracing.These findings suggest that the
microevolution of M. tuberculosis can be used to resolve separate transmission chains in large outbreak clusters.
European Society of Mycobacteriolog y | 30 th Annual Congress | July 2009 | Por to - Por tugal
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GL-5
SURROUNDED BY MYCOBACTERIA
Joseph O. Falkinham, III
Department of Biological Sciences
Virginia Tech
Blacksburg,Virginia 24061-0406
Phone 1-540-231-5931
FAX
1-540-231-9307
E-mail [email protected]
Humans, animals, and plants are surrounded by mycobacteria. The environmental opportunistic mycobacteria (also called
nontuberculous or atypical mycobacteria) include over 100 species; many of which cause disease. Infections include cervical lymphadenitis in children and pulmonary disease and skin infections in adults. Evidence that the environment was
the source of human disease was gained from the identity of DNA fingerprints of mycobacterial isolates from patients
and either their household water or potting soils. Recently, a number of reports have documented a dramatic increase
in pulmonary disease caused by these mycobacteria amongst elderly and slender men and women who lack all of the
classic predisposing risk factors (e.g., smoking, exposure to dusts). Although slowly growing with generation times of
one-half to one day, the environmental opportunistic mycobacteria survive, grow, and persist in a number of habitats
that are shared with humans and animals. The environmental opportunistic mycobacteria are oligotrophs; able to grow
in water containing greater than 50 µg AOC/L. Survival and persistence in the environment is due, in part, to the thick,
impermeable, hydrophobic, lipid-rich envelope of mycobacterial cells. Although the hydrophobic wall reduces the rate of
transfer or hydrophilic nutrients, it promotes attachment to surfaces where mycobacteria form biofilms. Hydrophobicity
also contributes to disinfectant- (e.g., chlorine and biocides) and antibiotic-resistance. For example, mycobacteria entering a water treatment system on particulates survive disinfection and grow during travel in the distribution system in the
absence of competitors. Hydrophobicity also promotes the aerosolization of mycobacteria from water to air in environments such as showers and hot tubs in the home and occupations where aerosols are generated.
30
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GL-6
The use of Interferon Gamma Release Assays as
an aid in the control of tuberculosis
Jean-Pierre Zellweger
Swiss Lung Association, Berne, Switzerland
Interferon Gamma Release Assays (IGRAs) are in vitro tests detecting the presence of latent tuberculosis infection (LTBI)
in asymptomatic persons who may have been infected by M. tbc in a recent or remote past and who may benefit from a
preventive treatment to decrease the risk of later reactivation of tuberculosis.
Basically, the IGRA tests rely on the same immunological phenomenon as the tuberculin skin tests, but they do it in a
much more specific way, because the tests are not influenced by a prior vaccination with BGC or by an infection with
most of the non-tuberculous mycobacteria present in the environment. Therefore, the indications and the use of the
IGRA tests are fundamentally the same as for the tuberculin skin tests :
• Detection of LTBI in persons in contact with an index case of tuberculosis
• Detection of LTBI in persons with a high risk of tuberculsois, if infected (immunosuppressed patients, patients
receiveing or due to receive immunosuppressive therapy, small children)
• Surveillance of exposed health care workers (as the test can be repeated without risk of inducing a booster effect)
• Aid to the diagnosis of tuberculosis in cases where a bacteriological examination is not feasible or not reliable
(severe extrapulmonary TB, TB in children)
In spite of their superiority, the IGRAs are not totally devoid of problems in practice and the best use of them is still
a matter of debate. Some Guidelines recommend their use only for the confirmation of positive TST among contacts
(the so-called two-step testing procedure) whereas others recommend the routine replacement of the TST by IGRAs.
Performing only one test is easier, and avoids a possible influence of a prior TST on the IGRA response.
The predictive value of the new IGRAs seems to be superior to the predictive value of TST, reinforcing their usefulness,
if the preventive treatment is corectly precribed and followed. One intriguing phenomenon is the possible reversion of
a positive IGRA after conversion, possibly indicating that some infected contacts may be able to eradicate the mycobacteria without treatment.
European Society of Mycobacteriolog y | 30 th Annual Congress | July 2009 | Por to - Por tugal
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GL-7
A NOVEL DIAGNOSTIC TEST TO DIFFERENTIATE
LATENT TB INFECTION AND ACTIVE DISEASE
Lee W. Riley
MD, School of Public Health, University of California, Berkeley
It is well recognized that the treatment of latent TB infection (LTBI) is a highly effective TB prevention strategy, which is
still not widely practiced in most parts of the world. Most TB-endemic countries rely on BCG vaccine to prevent TB.
LTBI treatment requires contact investigation, which is not done in most “BCG countries”. One reason for this reluctance to practice contact investigation is the lack of a reliable test that can distinguish LTBI from TB. Thus, a test that can
unequivocally distinguish LTBI from TB could alter the current national prevention programs in TB-endemic countries.
We have identified a set of M. tuberculosis cell wall proteins that are expressed when the bacilli replicate in vivo, but not
when they are in a nonreplicative state. Their continued expression is associated with disease progression in infected
mice, and mouse T cells are sensitized as these proteins are continually expressed in vivo. Exploiting this observation, we
developed a bioassay that is able to distinguish LTBI from active disease in a mouse model. The assay is based on IFNγ
induction by T cells exposed to a set of synthetic peptides based on the cell wall protein called Mcep1A. The Cornell
mouse model was used to study the response of spleen cells exposed ex vivo to these peptides. Cells from untreated
mice expressed 7-9-fold higher levels of IFNγ than those from treated mice at 24 and 32 weeks of infection, as measured by ELISA. Blood cells from healthy tuberculin skin-test positive, QuantiFERON-negative (n=3) and TST-negative,
QuantiFERON-negative (n=3) human volunteers showed no response to the peptides. These peptides are currently
under evaluation in newly diagnosed TB patients. If the assay can show a response in these TB patients at levels similar
to those observed in diseased mice, this assay can be converted into an immunochromatographic (“dip stick”) format.
Such a test then can be used to readily differentiate those with LTBI and active disease, and could then be incorporated
as part of National TB Control Programs.
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GL-8
NEW LIVE TUBERCULOSIS VACCINE STRATEGIES
Jesus Gonzalo Asensio, Ainhoa Arbues and Carlos Martín
Department of Microbiology, University of Zaragoza. Spain
http://genmico.unizar.es
BCG, the current vaccine against tuberculosis (TB), has been used for more than 80 years but is ineffective at providing
protection against adult pulmonary TB. New tuberculosis vaccine candidates and TB vaccination strategies, conferring
better protection against pulmonary tuberculosis than the current vaccine BCG, are needed.
In the recent decade, a global pipeline of novel TB candidates has emerged. Pioneering strategies for the development
of more effective vaccines today have lead to the discovery of subunit vaccines, which have proved ineffective at providing better protection that BCG in various animal models. Different heterologous prime BCG and boost with subunit
strategies are in clinical trials with the aim to improve efficacy of BCG. More recently, clinical trials with recombinant
BCG vaccines have started with the aim to find candidates to be used as prime, preventive vaccines. Another innovative
strategy, live attenuated Mycbacterium tuberculosis vaccines, in late preclinical investigation, are promising new preventive
vaccine candidates to replace BCG.
Based upon the observation that phoP is an essential gene for M. tuberculosis virulence, we rationally attenuated the
tubercle bacillus by inactivating phoP (Perez et al, Mol Micro 2001). The mutant was shown to be strongly attenuated in
cellular and animal models. Moreover, the phoP mutant resulted more attenuated than BCG Pasteur in immunocompromised SCID mice and this vaccine candidate protected guinea pigs and non human primates against tuberculosis infection (Martin et al Vaccine 2006,Verreck et al PLoS ONE 2009).
Both, the attenuated phenotype and the protective immunity conferred against tuberculosis infection can be accounted
for by the mechanism of action of PhoP, which has been recently shown to be crucial for intricate virulence network of
M. tuberculosis (Gonzalo Asensio et al PLoS ONE 2008). This observation was used to construct a new generation of live
vaccines based on phoP inactivation carrying a second additional mutation which affects the synthesis of a new family of
lipids associated to M. tuberculosis virulence.
It is estimated that at least 20 vaccine candidates should enter phase I safety trials with around half going forward for
immunological evaluation in phase II trials and leading to four phase III efficacy trials with the goal to reach an effective
licensed vaccine in 5-7 years (Young and Dye, Cell 2006). The discovery and use of a new TB vaccine better than BCG is
key to reach the 2050 objective of TB eradication.
European Society of Mycobacteriolog y | 30 th Annual Congress | July 2009 | Por to - Por tugal
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GL-9
REGULATION OF Mycobacterium tuberculosis CELL WALL LIPID
COMPOSITION AND ITS EFFECT ON IN VIVO BACTERIAL PERSISTENCE
Lee W. Riley
School of Public Health, University of California, Berkeley
The hallmark of M. tuberculosis is its ability to survive for many years in an infected host to establish latent tuberculosis
infection (LTBI). We propose a new model of latent infection that is based on the idea that this organism may simply have
readapted its “housekeeping” metabolic function to a new environment for its long-term survival. We propose that M.
tuberculosis, which evolutionarily most likely originated in soil, has readapted a soil-survival strategy its ancestral species
possessed to the granuloma environment in the human host. Granuloma cells constantly turn over every few days to
weeks, and after they die, they undergo replacement by new cells that migrate into the granuloma. Hence, M. tuberculosis
needs to readapt to this constantly changing environment, and we provide evidence that this adaptation is mediated by
M. tuberculosis remodeling its cell envelope in response to signals produced by dead granuloma cells. This remodeling
is mediated by a family of operons called mce (mce1,2,3,4). Disruption of the operons results in profound changes in
lipid profile of the cell wall. The mce1 operon mutant causes free mycolic acids (MA) to accumulate on its surface, and
other operon mutants (mce2,3,4) show evidence of lipid profile changes in the cell wall. These operon products serve as
energy-dependent lipid importers. Lipid products released from dead host granuloma cells that turnover may be used as
carbon sources for the resident M. tuberculosis. Thus, the “housekeeping” lipid metabolic function of M. tuberculosis may
have been readapted in the granuloma environment as a way for this organism to survive, similar to the way its ancestral
saprophytic organism survived in soil by scavenging dead organic materials as carbon sources. Further elucidation of the
interaction between M. tuberculosis cell wall and granuloma cell turnover may contribute to a new understanding of the
mechanism of LTBI.
34
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GL-10
CDC’S GLOBAL TB LABORATORY ACTIVITIES
Thomas M. Shinnick,
Associate Director of Global Laboratory Activities, Division of TB Elimination, Centers for Disease
Control and Prevention, 1600 Clifton Road, MS-G35, Atlanta Georgia 30333 USA. email: [email protected]; FAX: 1-404639-1287; Tel: 1-404-639-1474
A key bottleneck in health service delivery is weak laboratory capacity. This is particularly true for drug-resistant
TB — less than 5% of MDR TB cases are currently being detected globally. To meet the 2015 targets of the Stop TB
Global Plan, 60 million culture tests and 5 million drug susceptibility tests will be needed annually.This will require establishing at least 2,000 new culture laboratories and training of more than 20,000 laboratorians. At least US$ 1 billion will
be needed annually for building TB laboratory infrastructure and recurring costs. However, the benefit to cost ratio of
such investments is estimated to be 9:1 in populations with a high prevalence of HIV infection. Meeting the 2015 goals
could save countries in sub-Saharan Africa alone as much as US$ 52 billion annually.
While training of bench-level technicians relies on TB-specific expertise, laboratory capacity building relies more on
cross-cutting expertise in infrastructure, biosafety, human resource development, supply chain management, logistics,
quality assurance programs, management principles, information systems, data management, and accreditation processes.
As such, TB laboratory strengthening efforts can build on lessons-learned from the building of laboratory networks for
polio, measles, SARS, influenza, HIV/AIDS, and other diseases.
The goal of our TB laboratory strengthening efforts is the creation of a network of laboratories that can provide reliable, high quality testing and which is based on quality laboratory management principles and integrated across disease
programs, especially HIV and TB. A systems approach is used to optimize laboratory testing and information exchange.
The approach involves understanding the structure, performance, and cost of the network; developing referral processes
to ensure prompt flow of specimens and information; and using quality-improvement principles to continually evaluate
and improve the performance of the network. While TB laboratory strengthening plays the central role in our efforts,
the overriding goal is to ensure that a broader health systems approach is used to maximize the impact and sustainability
of the investments.
European Society of Mycobacteriolog y | 30 th Annual Congress | July 2009 | Por to - Por tugal
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GL-11
DEVELOPMENT AND VALIDATION OF NEW TB
DIAGNOSTIC TESTS IN BRAZIL: EXPERIENCE OF REDE-TB
Brazilian Tuberculosis Research Network: A Multi Disciplinary Collaborative Research Project
Introduction
In Brazil, which has an estimated 124,000 cases of TB per year, there has been a significant gap in communication and
understanding between TB programmatic experts, academics, the community, and nongovernmental organizations. In recognition of this gap, a National TB Research Network was established in 2002 to bring these constituencies together to
promote an integrated, multi-disciplinary and multi-institutional strategy for TB control in Brazil. In the last years, RedeTB has established a solid relationship among the National Tuberculosis Control Program and Oswaldo Cruz Foundation
that has helped to foster Brazilian leadership and competency in the development and evaluation under field conditions
of new diagnostics for TB.
Objectives
To develop new diagnostics, vaccines and drugs for the prevention and cure of TB, including MDR-TB, and develop
improved, therapeutic alternatives with the renewal of drugs, new formulations, drug associations, use of drugs already
available in the market, and immunotherapy.
To perform pre-clinical and clinical studies of new diagnostic tests against TB using adequate ethical standards and to
capacitate clinical sites for explanatory and pragmatic trials.
To carried out pragmatic clinical trials and cost-effective analysis of alternative interventions for TB control that include
diagnostic methods and health service strategies
To improve case detection by changing health behaviour and mobilizing communities.
To build a successful research partnership model that has every potential to stimulate changes in national standards and
practice in ways that serve country needs.
Conclusions
Through this approach, it is expected that locally algorithms based on clinical features, antibiotic response, and chest radiography will be validated, and clear guidelines issued about which patients might also benefit from mycobacteria culture
or new phenotypic / molecular diagnostic techniques. Additionally, the incorporation of new tests into clinical practice
will better planned and regulated, and national policy makers with better decision-making models will be provided.
36
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GL-12
EXPERIENCE OF A SUCCESSFUL MYCOBACTERIOLOGY
LABORATORY NETWORK IN ESPIRITO SANTO- BRAZIL
Moisés Palaci
Universidade Federal do Espírito Santo,Vitória, Brazil
The State of Espírito Santo occupies an area of approximately 6,750 square miles on the coast of Brazil. The economy
is mainly based on the production of steel, harborage activity, agriculture, a large number of small industries, and tourism. The population of the State is approximately 3.2 million, with the majority living in metropolitan Vitória, the capital,
which is located on an island and connected to the mainland by several bridges. The annual incidence of TB on the island
of Vitória is approximately 70 cases per 100,000 inhabitants. Each year approximately 1,500 new cases of TB (65% smear
positive) are reported for the State of Espírito Santo with 60% occurring in the City of Vitória and its 5 neighboring cities.
The Núcleo de Doenças Infecciosas has organized a local network of mycobacteriology laboratories in the Epírito State,
Brazil, Five local laboratories have been integrated into this network. This network was established by NDI researchers
and is committed to keep a partnership with the City Department of Health of each location and their laboratories. The
first phase of these partnerships consisted in reforming and restructuring the laboratories to enable them to accomplish
the technical requirements, data processing, and biosafety regulations involved in these projects. To this extent, basic
equipment and computers were installed to allow for the maintenance of mycobacterial culture and sharing of data over
an Internet database. The second phase consisted of training laboratory staff to properly and safely complete the necessary bench work and data processing procedures. Therefore, a considerable efforts and time has been spent for setting
up and to keep this system working. With the establishment of this network, NDI has gained earlier access to TB patients
and better conditions to conduct several clinical trials, including IND trials. In addition, capacitating local TB laboratories
in the metropolitan region of Vitória to perform mycobacterial cultures, allowed us to increase the detection rate of TB
cases at about 24%.
European Society of Mycobacteriolog y | 30 th Annual Congress | July 2009 | Por to - Por tugal
37
GL-13
SCREENING OF MOLECULES WITH ANTI-TB ACTIVITY, FROM THE BRAZILIAN
CERRADO PLANTS, AND SYNTHETIC METALLO-ORGANIC COMPOUNDS
Clarice Leite
Universidade Estadual Paulista, Faculdade de Ciências Farmacêuticas
Worldwide, tuberculosis (TB) remains the most frequent and important infectious disease causing morbidity and death.
Among all countries in the Americas, Brazil reports the second-highest TB mortality and morbidity, comprising a prevalence of 62/100.000. The global resurgence of TB and the rapid emergence of MDR-TB, underscore the importance of
the development of new antituberculous drugs.
Plants have provided many drugs in the past, and they remain a rich source of novel compounds. Plant extracts are among
the most attractive sources for developing new drugs and have been shown to produce promising results in the treatment of several diseases. Our research group deals in projects that integrate the chemical and anti-TB activity of plants
that compose the bioma of the Brazilian Cerrado, a savannah like vegetation. Many of those plants are commonly used
as natural remedies by people living in these areas to treat many illnesses. To perform the phytochemical step we used
chromatographic techniques, and to determine the structure of the isolated compounds we used mainly spectrometric
methods. To evaluate the activity of the extracts, enriched fractions and pure substances against M. tuberculosis we use
the resazurin microtiter assay (REMA) and M. tuberculosis H37Rv ATCC 27294 strain. In total were studied 77 extracts
from 39 plants, distributed into 20 families. From all extracts assayed 23% showed promising activity, bellow or equal to
125 µg/mL.The triterpene bassic acid from B. fagifolia showed strong antitubercular activity with MIC values of 2.5 µg/mL
comparable to MICs of some first-line tuberculosis drugs.The results indicated that plants of "cerrado" present fractions
and compounds with promising anti tuberculosis activity.
By the way, the use of natural compounds from plants is problematic, due to difficulty in obtaining pure substances and
their low availability.
Within the pipeline of new synthetic compounds with potential effectiveness in the treatment of TB, there are 7 novel
compounds, which are in various stages of clinical development. Inside this group however, there are complexes that
associate metals to organic compounds. Using the thiosemicarbazones, semicarbazones and hidrazones derivates as ligands, we proposed the complexation with Vanadium, to obtain organo-metallic compounds. We determined the anti-M.
tuberculosis activity of these compounds using REMA and the study of the citotoxicity of the ligands and complexes was
performed using murine macrophage cell line J774. We analyzed 37 compounds (14 free ligands and 23 vanadium complexes) and from of this, 17 (46%) presented promising MIC values varying between 0.97 and 7.80 μg/mL. The vanadium
complexes of hydrazones, semicarbazones and tiossemicarbazones derivates showed high antiTB activity, most of the
time this activity was increased from 2 to 10 times when compared with the free ligands. However due to high citotoxicity of hydrazones, semicarbazones and tiossemicarbazones derivates, the increase in the activity of the complexes didn’t
compensate the citotoxicity of the ligands.
38
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GL-14
CLINICAL TRIALS OF DRUGS AND DIAGNOSTIC TESTS:
THE CHALLENGES IN MYCOBACTERIOLOGY
Moisés Palaci
Universidade Federal do Espírito Santo,Vitória, Brazil
Tuberculosis (TB) therapy has three major microbiologic goals: (1) initial killing of actively multiplying organisms in order
to achieve early control of the disease and reduce infectivity (early bactericidal activity [EBA]); (2) eliminating slowly
growing mycobacteria in order to minimize relapses (sterilizing activity); and (3) preventing the emergence of drug
resistance (1). Currently, two month sputum culture conversion on solid medium is the best established predictor of
treatment outcome. Early bactericidal activity (EBA), determined by the serial decline in sputum M. tuberculosis colony
counts (CFU), is a commonly used tool for comparing new drugs to current anti-TB drugs and dose finding. EBA has been
measured as the rate of decrease in colony counts of mycobacteria in quantitative sputum cultures obtained during the
first days of therapy. Measurement of EBA is intended to be a rapid means of assessing the relative potency of new drugs
during early treatment. The development of new drugs for TB treatment has been hampered by the lack of an early surrogate marker that reflects long term non-relapsing cure. Measurement of EBA by quantitative culture however is time
consuming and labor intensive. The ideal marker would measure events early during treatment and be accurate regardless of the drug action or regimen being tested. In this lecture we will explore the potential role and the main limitation
of surrogate markers for TB treatment.
European Society of Mycobacteriolog y | 30 th Annual Congress | July 2009 | Por to - Por tugal
39
GL-15
NEW, EASY-TO-USE TOOLS FOR QUALITY-CONTROLLED
GENOTYPING OF M. tuberculosis COMPLEX STRAINS
Caroline Allix-Béguec1,2,3, Christine Hubans3, Stéphanie Ferreira3, and Philip Supply1,2,3
1 - INSERM U629
2 - Institut Pasteur de Lille, Lille
3 - Genoscreen, Lille, France, France
Mycobacterial interspersed repetitive unit-variable number of tandem repeat (MIRU-VNTR) typing has become a major
method for fast and high-resolution genotyping of Mycobacterium tuberculosis complex isolates. A system based on 24
loci has been proposed for international standardization. Several population-based studies have been published, showing
similar predictive value of this method compared to the previous gold standard IS6110 RFLP for studying tuberculosis
transmission in Western European settings. As a result, this method is being internationally adopted, often in combination with spoligotyping, as the new standard method for TB molecular epidemiology. New, easy-to-use tools and options
have recently become available, which facilitate quality-controlled use of this technique and interpretation of the results
obtained. MIRU-VNTR typing services are already used by international Reference Centers and laboratories, for outsourcing their genotyping (including of M. bovis strains) and/or for QA/QC evaluation. Quality-controlled MIRU-VNTR
calibration, validation and typing kits, as well as on-site trainings greatly facilitate standardized set up and efficient use of
MIRU-VNTR typing in user’s laboratory. Bioinformatic tools, including MIRU-VNTR Data Manager, have been developed
for further automating and streamlining the genotyping process, as well as ensuring direct compatibility with MIRUVNTRPlus Database for data interpretation. We hope that the availability of these tools for easier and more efficient
real-time genotyping will contribute to improve molecular-guided TB control and surveillance.
40
ESM 2009
Abstracts
of ORAL
PRESENTATIONS (OP)
European Society of Mycobacteriolog y | 30 th Annual Congress | July 2009 | Por to - Por tugal
41
OP-1
Mass Spectrometry for Molecular Typing of the Mycobacterium
tuberculosis Complex: One Platform and Multiple Assay Formats
C. Honisch1, M. Mosko1, C. Arnold2, S. Gharbia2, S. Feuerriegel3, S. Niemann3
1 - SEQUENOM, Inc., San Diego
2 - Health Protection Agency, London
3 - Molecular Mycobacteriology, NRC for Mycobacteria, Forschungszentrum Borstel, Borstel
Objectives
The analysis of nucleic acids by mass spectrometry has evolved to a user friendly technology for characterizing DNA,
and RNA via SNP genotyping and comparative sequencing in clinical research, agricultural applications, molecular medicine and non-invasive prenatal diagnostics research. Recently, the technology has become a versatile tool for microbial
detection and identification utilizing comparative sequence analysis. An example is the successful application to 16S based
typing of mycobacteria. Here, we adopted the technology to perform high throughput spoligotyping and detection of
resistance conferring SNPs.
Methods
Assays were designed in silico for spoligotyping analysis and detection of key resistance mutations. Both assays were evaluated by using well characterized reference collections.
Results
For MassARRAY 43 spacer oligonucleotide probes were designed and grouped into two multiplexed assays (TypePLEXTM).
Over 200 characterized strains from different reference centers representing the major M. tuberculosis complex lineages
were analyzed by the MassARRAY spoligotyping assays. Results were in concordance with classical spoligotyping data.
For detection of resistance mutations, assay were developed based on the MassCLEAVETM system. Resistance regions
are amplified by PCR with a tagged primer system followed by in vitro transcription of both DNA strands. Subsequent
endonuclease digests of the RNA transcripts at the bases cytosine and uracil result in four mixtures of RNA cleavage
products. Resistance is identified by correlating acquired spectra with theoretical peak patterns predicted for in silico
cleavages of sequences contained in a reference database. The first assays have been successfully evaluated in a set of
reference strains, further analyses are in progress.
Conclusion
Mass spectrometry specific assay formats for genotyping and comparative sequence analysis generate highly accurate
qualitative and quantitative data and provide a toolbox for molecular typing of microbes and viruses. Existing typing
schemes can be translated onto the mass spectrometry platform and new typing schemes can easily be developed.
European Society of Mycobacteriolog y | 30 th Annual Congress | July 2009 | Por to - Por tugal
43
OP-2
Clustering of spoligo-patterns: towards an automation
of Mycobacterium tuberculosis complex classification
Borile C 1, Refrégier G 2, Labarre M 1, Franz S 1, Mézard M 1, Sola C 2
1 - LPTMS, bât. 100, Université Paris-Sud, Centre scientifique d’Orsay, 15 rue Georges Clémenceau, 91405 Orsay cedex
2 - IGEPE bât. 400, Université Paris-Sud, Centre scientifique d’Orsay, rue Gregor Mendel, 91405 Orsay cedex
Spoligotyping is a typing method detecting the presence or absence of specific regions called spacers. These spacers
are grouped on what is called the DR locus (Direct Repeat locus) that belongs to the CRISPR locus family (Clustered
Regularly Interspaced Palindromic Repeats). The DR locus in Mycobacterium tuberculosis complex is believed to evolve
solely by deletion.
Using the spoligotyping technique, specific families of Mycobacterium tuberculosis complex strains have been recognized,
showing that the DR locus is phylogenetically informative. Specific signatures are recognized by experts so that each
spoligo-pattern is easily assigned to a specific family. Amazingly however, when using all available softwares for clustering
the data, the strains of a specific family are not always clustered in the same groupe using various methods.
We implemented a new algorithm to cluster these data. Until now, the single publicly available software to achieve this
task, SpotClust, provided only sub-optimal results. Our system is based on an evolutionary model taking into account
that spacers can be deleted as a large group. This is not the case when using the Jaccard distance in the commonly used
Bionumerics software, that mimicks a one-by-one spacer deletion process.
We will present data showing under what conditions this algorithm gives significantly better results than the commonly
used one.This studies leads toward an automation of Mycobacterium tuberculosis complex classification, an otherwise time
consuming and sometimes debatable topic.
44
ESM 2009
OP-3
IDENTIFICATION OF THE INSERTION ELEMENT IS6110 IN PHOP
PROMOTER IN A HIGH TRANSMISSION Mycobacterium
tuberculosis STRAIN: A CLUE TO PHENOTYPIC VARIATION
Andrea Sandoval1,2, Andrés Cubillos1,2, Alejandro Reyes3, Nidia Correa2,4, Jaime Robledo2,4, Maria Mercedes Zambrano1,
and Patricia Del Portillo1,2
1 - Corporación CorpoGen, Bogotá, Colombia.
2 - Centro Colombiano de Investigación en Tuberculosis CCITB, Bogotá, Colombia.
3 - Center for Genome Sciences, Washington University School of Medicine, St. Louis, Missouri, USA
4 - Corporación para Investigaciones Biológicas CIB, Medellín, Colombia
Aim
The insertion element IS6110 can mediate genetic diversity in Mycobacterium tuberculosis (MTB) strains due to its capacity to move and cause rearrangements, deletions and insertions. Transposition of these elements can therefore affect
gene expression and alter the phenotype of MTB. In this study we analyzed the insertion sites of IS6110 in two clinical
MTB Haarlem genotype strains that present differences in transmissibility as demonstrated in a cohort of patients and
household contacts from Colombia.
Methods
Restriction Fragment Length Polymorphism (IS6110-RFLP) was performed and revealed genomic differences between
the two strains. DNA was isolated, digested with XmaI and ligated to specific adapters designed for this purpose. Ligation
Mediated PCR (LM-PCR) was carried out to amplify the regions flanking IS6110 insertion sites. A library containing the
amplified products was constructed and sequenced clones were mapped against the annotated sequenced genomes. PCR
was used to confirm the sites of insertion.
Results
Twelve different insertions were identified; nine were common to both strains (Rv2336, Rv1754c, Rv0963c, Rv0403c,
Rv1358, Rv2813/DR, Rv2254c, Rv0795 and PPE34), two were specific for the high transmission strain (DR region and
transcriptional regulator phoP), and one was found just in the low transmission strain (PPE46).
Conclusions
LM-PCR allowed us to identify IS6110 flanking regions and localized the genomic differences between two strains with
contrasting transmission pattern. Most of the insertions occur in conserved hypothetical proteins, followed by proteins
involved in cell wall synthesis and cell processes, regulatory proteins and members of the PE/PPE family. The DR region,
considered a hotspot for insertions, had three insertions, two common to both strains and one in the high transmission
strain. Since the phoP gene regulates several functions implicated in virulence, it is possible that the IS6110 insertion in
the phoP promoter could enhance transmission by acting as a portable promoter and inducing gene expression, as has
been reported before in M. bovis.
Acknowledgment
Consorcio Colombiano de Investigación en Tuberculosis, CCITB. 4312004
European Society of Mycobacteriolog y | 30 th Annual Congress | July 2009 | Por to - Por tugal
45
OP-4
GENOMIC INTERROGATION OF Mycobacterium
tuberculosis ISOLATES FROM BRAZIL
Oelemann, Maranibia C 1, Gomes, Harrison M 1, Willery, Eve 2, Lima, Karla Valéria B 3, Possuelo, Lia 4, Locht, Camille 5,
Goguet de la Salmonière,Yves-Olivier L 6, Gutierrez, Maria Cristina 6, Supply, Philip 7, Suffys, Philip N 1
1 - Laboratory of Molecular Biology Applied to Mycobacteria, Oswaldo Cruz Institute, Rio de Janeiro, Brazil
2 - Laboratory of Molecular Mechanisms of Pathogenesis of Respiratory Pathogens, INSERM U629 and Institut Pasteur
de Lille, France
3 - Evandro Chagas Institute, Belém, Brazil
4 - Center of Scientific and Technological Development, Porto Alegre, Brazil
5 - Laboratory of Molecular Mechanisms of Pathogenesis of Respiratory Pathogens, INSERM U629 and Institut Pasteur
de Lille, France
6 - Department of Infection and Epidemiology, Institut Pasteur, Paris, France
7 - Laboratory of Molecular Mechanisms of Pathogenesis of Respiratory Pathogens, INSERM U629 and Institut Pasteur
de Lille, France
1 - Laboratory of Molecular Biology Applied to Mycobacteria, Oswaldo Cruz Institute, Rio de Janeiro, Brazil
Background
The Latin-American Mediterranean (LAM) spoligotype“clade” is among the six major M.tuberculosis spoligotype families and is
particularly prevalent in SouthAmerica.In certain regions,there is a dominance of geographically specific and genetically homogeneous strain lineages.Here,we have studied the genetic diversity and the consistency of the LAM and other families,by analyzing
Mtb isolates from three Brazilian regions including Rio de Janeiro (South East), Belém (North), and Rio Grande do Sul (South).
Methods and Findings
A PCR-based standardized genotyping system, based on amplification of 15 to 24 mycobacterial interspersed repetitive unit-variable number of tandem repeat (MIRU-VNTR) loci combined with spoligotyping, has been shown to
be proficient for molecular-guided evaluation of TB transmission. We tested the applicability of this system for molecular epidemiological analysis of 369 M. tuberculosis isolates from three regions of Brazil. Deligotyping, targeting multiple large sequence polymorphisms (LSPs), and MIRU-VNTRplus identification database were additionally
used to confirm phylogenetic identification. The high congruence between the different typing results showed the
countrywide supremacy of the Latin-American-Mediterranean (LAM) lineage, comprised of three main branches.
Nevertheless, by distinguishing 321 genotypes among the 369 isolates, combined MIRU-VNTR typing and spoligotyping demonstrated the presence of multiple distinct clones. Noteworthy, 27 of the 32 clusters identified were exclusively composed of patient isolates from a same city, consistent with expected patterns of local TB transmission.
Conclusions
Notwithstanding the challenges, high-capacity mycobacterial genotyping may now become a usable tool to guide
TB control efforts, at least on sentinel sites or targeted risk-populations in high TB burden countries. The interrogation of large sequence polymorphisms (LSPs) revealed that certain lineages or clones present genomic features that could change the phenotype and play a role in the clinical properties of specific Mtb strains. This is the
first countrywide report of an in-depth analysis on the genomic diversity of M. tuberculosis isolates from Brazil.
Acknowledgments
Fiocruz, CAPES, CNPq, FAPERJ, ICOHRTA, NIH, INSERM and Institut Pasteur.
46
ESM 2009
OP-5
PENITENTIARY POPULATION OF Mycobacterium tuberculosis
IN KYRGYZSTAN: EXCEPTIONALLY HIGH PREVALENCE OF THE
BEIJING GENOTYPE AND ITS RUSSIA-SPECIFIC SUBTYPE
Igor Mokrousov 1, 2,Violeta Valcheva 1, 3, Nurmira Sovhozova 4, Almaz Aldashev 4, Nalin Rastogi 1, Jainagul Isakova 4
1 - Institut Pasteur de Guadeloupe, France
2 - St. Petersburg Pasteur Institute, St. Petersburg, Russia
3 - Institute of Microbiology, Sofia, Bulgaria
4 - Institute of Molecular Biology and Medicine, Bishkek, Kyrgyz Republic
Objective
To identify genotypes and drug resistance properties of M. tuberculosis isolates from Kyrgyzstan’s prison inmates, a
population with high risk for TB; to compare in regional and global context.
Methods
56 M. tuberculosis DNA samples from sputum of HIV-negative Kyrgyz prison inmates, 2008, were typed by spoligotyping,
VNTR (12 MIRU and 3 hypervariable [HV] loci), IS6110-inverse-PCR, LAM-PCR. rpoB and katG mutations were detected
using TB-Biochip kit.
Results
Beijing genotype was detected in 42 of 56 samples. 12-locus MIRU-VNTR typing showed 8 of 56 samples to be mixed
cases; 7 of them contained a Beijing strain. MIRU analysis demonstrated a high homogeneity of the studied collection
(HGI=0.66) while 28 of 56 strains had a profile 223325153533 corresponding to Beijing/M2 subtype highly prevalent
in different Russian settings (Mokrousov, 2004, 2008). Four Beijing strains belonged to types M33 and M70 specific for
East Asia. Regarding non-Beijing variants, a comparison of their spoligoprofiles with SITVIT2 database (Institut Pasteur
de Guadeloupe) revealed a presence of minor global and Eurasia (Europe/Russia) specific types SIT262/Haarlem, SIT73,
SIT254/LAM found in ex-USSR and Europe but very rare in East Asia and global type SIT42/LAM that is also prevalent in
different parts in Eurasia. Three hypervariable loci, QUB-3232, VNTR-3820 and VNTR-4120, permitted to subdivide 28
Beijing strains with MIRU12 profile 223325153533 into 11 subtypes shared by 1 to 9 strains. RIF and INH resistance was
detected in 28% and 55% samples. 13 of 15 MDR strains belonged to Beijing genotype. Comparison of the rate of drug
resistance mutations in different Beijing subclusters revealed no statistically significant difference.
Conclusions
The penitentiary population of M. tuberculosis in Kyrgyzstan shows a strong affinity to the north-west Eurasia, especially,
Russia, and a weak relatedness to East Asia. Beijing genotype constituted 75% of the entire collection while half of the
studied strains belonged to the Beijing/M2 MIRU-defined subtype that is the major Beijing variant in Russia. MDR-TB was
detected in 27% samples that is similar to the Kyrgyzstan’s civilian population. IS6110-inverse PCR and HV-VNTR loci
were shown to be useful for detection of and subtyping within the Beijing genotype, directly in sputum-extracted DNA.
European Society of Mycobacteriolog y | 30 th Annual Congress | July 2009 | Por to - Por tugal
47
OP-6
THE UNIQUE ENDEMIC NATURE OF BEIJING GENOTYPE
STRAINS IN OKINAWA, RYUKYU ISLANDS OF JAPAN AS REVEALED
BY NEWLY DESCRIBED 15 AND 24-LOCI MIRU-VNTR TYPING SCHEMES
Julie Millet, 1 Chika Miyagi-Shiohira, 2 Nobuhisa Yamane, 2 Igor Mokrousov, 1,3 Nalin Rastogi 1
1 - Unité de la Tuberculose et des Mycobactéries, Institut Pasteur de Guadeloupe, Abymes, Guadeloupe
2 - Department of Laboratory Medicine, Graduate School and Faculty of Medicine, University of the Ryukyus, Okinawa, Japan
3 - St. Petersburg Pasteur Institute, St. Petersburg, Russia
Tuberculosis (TB) in the eastern Asiatic countries is mainly caused by strains of Mycobacterium tuberculosis belonging to
the Beijing lineage which was identified back in early nineties using IS6110-RFLP and spoligotyping. Associated with multiple drug-resistance (MDRTB), this highly homogeneous genogroup is characterized by little molecular diversity. Several
studies have recently emphasized the utility of using minisatellites in conjunction with IS6110-RFLP or spoligotyping for a
better discrimination of Beijing strains. In a recent study, we genotyped Beijing TB strains collected in Okinawa (Ryukyu
Islands, Japan), by using a discriminative selection of 8 MIRU loci (chosen from the classical 12-loci MIRUs), and 7 QUB
markers. This typing scheme excluded 4 MIRU loci (MIRU2, 4, 20, and 24), that were found to have a too low discriminatory power within an in-house Beijing database containing 694 strains (Millet et al., J. Clin. Microbiol. 2007, 45:3606–3615).
In the present study we evaluated the full “classical” 12-loci typing as compared to the newly described 15-loci and 24loci MIRU-VNTR typing schemes, which include 9 and 12 new MIRU loci respectively.We compared the results obtained
in Okinawa (an insular setting; n=72) with those recently published for Osaka (n=174 strains) and Kobe (n=175) (Wada
et al., FEMS Microbiol Lett. 2009, 291:35-43). A higher discriminatory power of 15-loci versus 12-loci format was seen
through percentage of clustered isolates; clustering for 12-loci format in Osaka, Kobe, and Okinawa was 78.3, 81.7, and
68.1% respectively, as compared to 55.7, 48.0, and 37.1% using 15-loci format. Corresponding discriminatory index (HGI)
in Osaka, Kobe, Okinawa were 0.901, 0.936, and 0.944 respectively for 12-loci format, as compared to 0.989, 0.989, and
0.992 for 15-loci format. A finer comparison of 15-loci patterns in the 3 settings revealed that contrary to Kobe and
Osaka which shared together a high number of similar patterns (25/114 and 25/107 respectively), Okinawa shared a
single pattern out of 55 with Kobe, and none with Osaka. The full 24-loci format results further reduced the clustering
observed (from 37.1% to 20%, HGI 0.996). The results analyzed by drawing a minimum spanning tree underlined the
unique endemic nature of the Beijing genotype strains in the insular setting of Okinawa, and suggest a local evolution of
M. tuberculosis Beijing genotype in this island starting from a common pool in mainland Japan.
48
ESM 2009
op-7
Mycobacterium tuberculosis genetic diversity in South Korea
Isdore Chola Shamputa1, Jongseok Lee2, Caroline Allix-Béguec3, Eun-Jin Cho2, Ji-im Lee2, Jin Hong Min4,
Lisa C. Goldfeder1, Jin Hee Kim4, Hyung Seok Kang4, Soo Hee Hwang4, Seok Yong Eum2 ,Hyeyoung
Lee5, Seung Kyu Park2,4, Philip Supply3,6, Sang Nae Cho7, Laura E.Via1, Clifton E. Barry III1
1 - Tuberculosis Research Section, National Institute of Allergy and Infectious Diseases, National Institutes of Health,
Bethesda, Maryland
2 - International Tuberculosis Research Center, Masan South Korea
3 - Genoscreen, Lille, France
4 - Masan National Tuberculosis Hospital, Masan, South Korea
5 - Department of Biomedical Laboratory Sciences,Yonsei University, Wonju, South Korea
6 - Centre National de la Recherche Scientifique, Institut Pasteur de Lille/Institut de Biologie de Lille, Lille France
7 - Department of Microbiology,Yonsei University College of Medicine, South Korea
South Korea has recorded a nine fold decrease in the incidence of TB in the last four decades, however challenges of TB
control remain significant as the Republic of Korea is among the 30 countries with the highest numbers of estimated
MDR-TB cases. Genotypic analysis of M. tuberculosis has greatly contributed to the control of TB by providing information
on transmission dynamics, assessing clonal distribution and expansion of the tubercle bacilli, in investigating of outbreaks
and pseudo-outbreaks, and in identifying laboratory cross contamination. However, there is limited information on the
molecular epidemiology of TB in South Korea.
Genetic diversity of 208 M. tuberculosis isolates from subjects enrolled in a prospective observational cohort study at
National Masan Tuberculosis Hospital in South Korea was determined using spoligotyping, IS6110-RFLP and standardised
MIRU-VNTR typing based on 24 loci. MIRU-VNTR analysis was performed independently and blindly from spoligotyping
and IS6110-RFLP results.
Analysis of MIRU-VNTR typing results use in conjunction with MIRU-VNTRplus database predicted that 202 (97.1%)
isolates belonged to the Beijing genotype. This prediction was fully confirmed by spoligotyping. Congruence analysis indicated the prevalence of 3 branches among Beijing strains respectively named Korea, Masan and China. Preliminary analysis did not show differential distribution of resistant, MDR or XDR strains among the 3 branches. MDR or XDR isolates
were detected in at least 4 clusters concordantly identified by the 3 genotyping methods. Using MIRU-VNTR typing and
spoligotyping, 23 clusters of 66 isolates were detected indicating a relatively large diversity of circulating strains despite
the prevalence of the Beijing lineage.
Standardised MIRU-VNTR typing appeared efficient as a first line discriminatory method for this country with high
prevalence of Beijing strains. However, preliminary analyses suggest that clustered cases may not be epidemiologically
linked and rather correspond to endemic strains circulating in South Korea.
European Society of Mycobacteriolog y | 30 th Annual Congress | July 2009 | Por to - Por tugal
49
OP-8
CORRELATION OF MOLECULAR RESISTANCE MECHANISMS
AND PHENOTYPIC RESISTANCE TO FIRST-LINE DRUGS IN
Mycobacterium tuberculosis STRAINS FROM SIERRA LEONE
Silke Feuerriegel1, Susanne Homolka1, Erik Post2, Barbara Oberhauser2, Abu Garawani George3, Lars Westman3, Foday
Dafae4, Sabine Rüsch-Gerdes1, Stefan Niemann1
1 - Research Center Borstel, National Reference Center for Mycobacteria, Parkallee 18, 23845 Borstel, Germany
2 - German Leprosy and TB Relief Association, Würzburg, Germany
3 - National Leprosy/TB Reference Laboratory, Freetown, Sierra Leone
4 - National Program Manager for Tuberculosis and Leprosy, Freetown, Sierra Leone
Background
Resistance to first-line drugs (INH, RMP, SM, EMB and PZA) displays a serious problem for the treatment of Mycobacterium
tuberculosis infections. Resulting MDR-tuberculosis (resistance to at least INH and RMP) implies an enormous threat for
tuberculosis control worldwide. It is therefore of great importance to analyze the genetic basis of clinical resistance,
especially in high incidence settings and to correlate molecular resistance data with phenotypic resistance data.
Methods
A total of 97 M. tuberculosis strains from previously treated patients in Sierra Leone which displayed resistance to INH
(n=32), RMP (n=16), SM (n=39), EMB (n=15) and PZA (n=10), respectively, were sequenced concerning the predominant resistance determining regions (katG, rpoB, rrs, rpsL embB and pncA). Strains resistant to INH with no mutation in
katG were also sequenced in the promoter regions of inhA and ahpC. From all strains analyzed 11 showed resistance to
INH and RMP and were therefore MDR-TB. Drug susceptibility testing was done by using the proportion method on
Löwenstein-Jensen medium.
Results
Among INH resistant strains the most common mutation detected is katG315 (65.6%). From all 32 resistant strains
3 had mutations in the promoter regions of inhA and ahpC. Among RMP resistant strains 50% displayed mutations at
rpoB531. Sensitivity and specificity of the DNA sequencing of katG and rpoB for detection of INH and RMP resistance
were about 90%. Concerning SM resistance none of the resistant strains showed any mutation in rrs, but 46.2 % had rpsL
mutations, either at codon 43 or 88. Among EMB resistant strains 46.7% showed mutations at embB306 and 13.3% at
codon 332 and 497 respectively. Strains resistant to PZA displayed a number of different mutations throughout the pncA
gene. Specificities of sequencing of rpsL, embB and pncA for detection of the respective resistance phenotypes were high
(96-100%), whereas sensitivities were lower (46% for sequencing of rpsL, 60% for embB, 70% for pncA).
Conclusion
There is a close correlation between data from molecular and phenotypic resistance testing for the determination of
INH and RMP resistance in strains from Sierra Leone. Sensitivities of sequencing of resistance determining genes for SM,
EMB and PZA resistance were low due to so far unknown resistance determining regions. Thus it is of great importance
to gather information on further mechanisms leading to drug resistant MTB strains in different settings.
50
ESM 2009
OP-9
LAM AND HIV: CORRELATION OR CO-INCIDENCE?
McNerney, Ruth, Mallard, Kim
London School of Hygiene & Tropical Medicine
The deadly synergy between TB and HIV presents a serious challenge to heath and development.Three decades after the
onset of the AIDS pandemic the region with the highest prevalence of HIV infection is sub-Saharan Africa, followed by
the Caribbean and Latin America. Molecular typing methods permit differentiation of M. tuberculosis into strain families
or genotypes. We have undertaken analysis of genotyping data to compare the prevalence of spoligotype lineage with
that of HIV infection. Data was taken from the peer reviewed literature, surveys testing only drug resistant strains or
those not fully describing the genotype population were excluded.The Latin-American-Mediterranean (LAM) lineage are
identified as typically lacking spoligotype spacers corresponding to oligonucleotides 21 to 24 and 33 to 36. They have
been observed in many geographic locations and include the F11 family reported in South Africa. Our analysis suggests
that LAM strains are more frequently found in regions with a high prevalence of HIV. Conversely, they are rare in settings where HIV has yet to emerge as a major threat to public health. Interestingly LAM genotypes appear less frequent
in African countries such as Uganda, Tanzania and Cameroon which have lower HIV prevalence’s than countries such as
Malawi, Zimbabwe and South Africa where HIV rates are in excess of 10%. Why tuberculosis of this genotype should be
linked to the HIV epidemic remains a matter of speculation. Although socioeconomic and political factors play a significant role they do not explain the uneven distribution of HIV in sub Saharan Africa. It is now evident that there is diversity
in the interaction of M. tuberculosis with its host and strains of differing genotype appear to elicit subtle but significant
differences in immune response. We present the hypothesis that strains of tuberculosis belonging to the LAM genotype
are associated with enhanced transmission of TB in immunosuppressed populations.
European Society of Mycobacteriolog y | 30 th Annual Congress | July 2009 | Por to - Por tugal
51
OP-10
Mycobacterium celatum: AN EMERGING
PATHOGEN IN THE IMMUNOCOMPETENT. A CASE REPORT
Lyberopoulos Panagiotis 1, Frangopoulos, Fragiskos1, Kontos, Fanourios 2, Zerva Loukia 2, Malagari Aikaterini 3, Papiris Spyridon 1
1 - 2nd Department of Pulmonary Medicine, Attikon University Hospital, Athens, Greece
2 - Department of Clinical Microbiology, “Attikon” University Hospital, Athens, Greece
3 - 2nd Department of Radiology, Attikon University Hospital, Athens, Greece
Mycobacterium celatum is a pathogen for immunocompromised patients but there is little evidence of its pathogenicity
among immunocompetent individuals. We report the isolation of M. celatum from a middle-aged immunocompetent woman with bronchiectasis that was initially considered a non-significant finding, but eventually proved extremely dangerous.
A 55-year-old Caucasian female never smoker, was referred to our hospital complaining of productive cough over the
preceding two weeks accompanied by general malaise. She lived in an urban area and had no history of alcoholism, use
of steroids or immunosuppressive drugs. A high resolution computed tomography (HRCT) of the chest showed illdefined peribronchial densities contained within the right upper lobe with air-bronchograms and branching pattern with
a few opacities of tree-in bud formation. There was no lymphadenopathy or pleural effusion.
Smears of five sputum specimens (before any treatment) were negative for acid-fast bacilli. These specimens were processed with the NALC/NaOH method and cultured in the BACTEC MGIT 960 system (Becton Dickinson, USA) and
on Löwenstein-Jensen medium (BioMerieux, France). Two mycobacterial strains were recovered from these cultures
and both strains were identified as M. celatum with the use of the Genotype Mycobacterium CM and AS assays (HainLifescience). PCR restriction analysis (PRA) of a 439-bp fragment of the hsp65 gene and sequencing of the 16S rRNA
gene confirmed that they were M. celatum type 1.
From the five aforementioned sputum specimens Pseudomonas aeruginosa and Serratia liquefaciens were also isolated and
were both sensitive to ciprofloxacin. Ciprofloxacin (1gr/day, per-os) plus low dose azithromycin (500mg twice a week)
were administered. Two months later, a control chest HRCT scan showed a considerable improvement; no pathogens
were isolated from consecutive sputum samples.
Eight months later the patient complained for general malaise, night sweats and cough with blood tinged sputum. Imaging
studies revealed a lung apical cavity.Two bronchial washing and two sputum cultures were smear positives and M. celatum
was recovered from all.
In conclusion, when the American Thoracic Society criteria for the diagnosis of NTM infection are met and M. celatum
is identified, it should be considered as the pathogen causing the pulmonary infection even in patients with apparently
normal cellular immunity.
52
ESM 2009
OP-11
Mycobacterium avium SUBSPECIES STRAINS
FROM HUMAN AND ANIMAL ORIGIN
Radomski, Nicolas 1, Thibault, Virginie 2, Karoui, Claudine 1, De Cruz, Krystel 1, Cochard, Thierry 1, Gutiérrez, Cristina 3,
Supply, Philip 3, Biet, Frank 2, Boschiroli, María Laura 1
1 - AFSSA-LERPAZ, Maisons Alfort
2 - INRA-UR1282, Nouzilly
3 - INSERM U629-Institut Pasteur, Lille
The Mycobacterium avium sbsp. avium and Mycobacterium avium sbsp. hominissuis are pathogenic emergent bacterial species belonging to the Mycobacterium avium complex (MAC). These two subspecies can infect and lead to disease to numerous animal species: birds, pigs, cattle, deer, sheep, goats, horses, cats, dogs, etc. Moreover, Mycobacterium avium subspecies have been isolated in HIV infected patients and in immuno-competent patients with pulmonary pathologies. MAC is
an ubiquitous bacterial group that can be found in water, in the environment, or even in food. A molecular typing study
was undertaken with the primary goal of improving the taxonomic and epidemiological knowledge of MAC. Different
strains of Mycobacterium avium sbsp. avium, Mycobacterium avium sbsp. hominissuis, and also of Mycobacterium avium sbsp.
silvaticum, isolated from animals, humans, and the environment, were typed by two methods: the Restriction Fragments
Length Polymorphism on insertion sequences IS1311 (RFLP1311), which is a standard method to characterise MAC, and
the Variable Number of Tandem Repeats-Mycobacterial Interspersed Repetitive Units (VNTR-MIRUs) characterisation,
which has been recently developed on Mycobacterium avium sbsp. paratuberculosis. Our results demonstrate that the
discrimination power of both methods is comparable (DI of more than 0.92). Therefore, VNTR-MIRUs seems a much
better typing method since it is a PCR based method that requires little genetic material for being performed, which is
easy to standardize in any laboratory and because the deduced numerical patterns do not require special softwares for
being compared in an inter-laboratories fashion.
European Society of Mycobacteriolog y | 30 th Annual Congress | July 2009 | Por to - Por tugal
53
OP-12
THE CHARACTERIZATION OF MYCOBACTERIA FROM AN
OUTBREAK SUGGESTS A REVISION OF THE TAXONOMIC STATUS OF
MEMBERS OF THE Mycobacterium chelonae-abscessus GROUP
Sylvia Cardoso Leão1, Enrico Tortoli2, Cristina Viana-Niero1, Suely Yoko Mizuka Ueki3, Karla Valeria Batista Lima4, Maria
Luiza Lopes4, Jesus Yubero5 María Carmen Menendez5, Maria Jesus Garcia5
1 - Universidade Federal de São Paulo, São Paulo, Brazil
2 - Centro Regionale di Riferimento per la Diagnostica dei Micobatteri, Ospedale di Careggi, Firenze, Italy
3 - Instituto Adolfo Lutz, São Paulo, Brazil
4 - Instituto Evandro Chagas, Belém, Brazil
5 - Universidad Autonoma de Madrid, Madrid, Spain
An outbreak of infections by rapidly growing mycobacteria (RGM) related to invasive procedures has been ongoing in
Brazil since 2004. Isolates from patients submitted to laparoscopic or plastic surgery and to mesotherapy, a cosmetic
procedure, were previously identified by molecular methods as Mycobacterium massiliense and M. bolletii, respectively. The
similarity of rpoB and hsp65 sequences from the clinical isolates and the corresponding sequences from both M. massiliense and M. bolletii type strains were above the accepted limit for interspecies variability, leading to conflicting results.
Therefore, an extensive characterization was carried out with six Brazilian clinical isolates from this outbreak study and
type strains from the members of the M. abscessus-M. chelonae group – M. abscessus, M. chelonae, M. immunogenum, M.
massiliense and M. bolletii. Phenotypic identification was performed by biochemical tests, high performance liquid chromatography (HPLC) and drug susceptibility testing. Molecular identification included PCR-restriction enzyme pattern
analysis (PRA) of the hsp65 gene, as well as rpoB and hsp65 gene sequencing and analysis of the corresponding phylogenetic trees. DNA-DNA hybridization (DDH) and restriction fragment length polymorphism (RFLP) of the 16S rRNA
gene were used as the gold standards for RGM speciation. The clinical isolates and the M. abscessus, M. massiliense and
M. bolletii type strains could not be separated by phenotypic tests and were grouped in the phylogenetic trees obtained.
Also, DDH results confirmed >70% relatedness, and indistinguishable RFLP 16S rRNA patterns were obtained. On the
contrary, separation from M. chelonae and M. immunogenum was supported by results from PRA-hsp65, rpoB and hsp65
phylogenetic trees, DDH and RFLP-16S. Taken together, these results led to the proposition that M. abscesus, M. massiliense and M. bolletii represent a single species, that of M. abscessus. Two subspecies are also proposed, M. abscessus subsp.
abscessus and M. abscessus subsp. massiliense which can be distinguished by two different PRA-hsp65 patterns, differing in
a single Hae III band, and by differences in rpoB (3.4%) and hsp65 (1.3%) sequences.
54
ESM 2009
OP-13
THE SUNNY SIDE OF MYCOBACTERIA
Santos, Ricardo 1, Marques, Marco 2, Oliveira, Pedro 2, Carvalho, Filipe 2, Carvalho, Carla 2, Monteiro, Gabriel 2, Cabral,
Joaquim 2, Frade, Raquel 2, Silva, Maria 3, Fernandes, Pedro 4
1 - Laboratório de Análises
2 - IBB-CEBQ-IST
3 - Faculdade de Farmacia, Univ. Coimbra
4 - IBB-CEBQ-IST
Mycobacteria are typically seen as cause of morbidity and mortality worldwide. There is however a Jekyll side to these
acid-fast bugs. Taking advantage of some key metabolic pathways, as well as of the particular nature of the hydrophobic
cell wall, that enables operation in aggressive, non-conventional environments, non-pathogenic mycobacteria can be
used for the production of compounds with application in the pharmaceutical, food and environmental areas. The present work aims to illustrate this concept, by providing examples of the application of non-pathogenic mycobacteria for
the production of steroid and siderophore molecules, and for the pinpoint modification of carbocycles. The assessment
of the required biocatalytic activity and the early stages of process characterization have been carried out in miniaturized bioreactors, allowing for a high level of parallelization, hence providing a high throughput platform for bioprocess
development. Going into detail, Mycobacterium sp. NRRL B-3805 was shown to effectively yield androstenedione (AD),
a key intermediate in the production of therapeutic steroids, from phytosterols, recovered from residues of the paper
industry, while operating in phthalate or liquid silicone environments, an approach that enhances process productivity.
Furthermore, this particular strain was shown to also yield the AD out of several polyhydroxylated steroids. Relying on
the high-throughput methodology, a library of non-pathogenic Mycobacterium spp. siderophore producers was developed in-house. Scaling-up the production process to bench bioreactor using the most promising strains is envisaged.The
same high-throughput methodology has been recently used to screen non-pathogenic mycobacteria for specific catalytic
activity, namely hydroxylation, on carbocyclic molecules, with promising results.
European Society of Mycobacteriolog y | 30 th Annual Congress | July 2009 | Por to - Por tugal
55
OP-14
FAST IDENTIFICATION OF Mycobacterium tuberculosis IN SPUTUM
AND CULTURES BASED ON THERMALLY-ASSISTED HYDROLYSIS AND
METHYLATION BY GAS CHROMATOGRAPHY-MASS SPECTROMETRY
Erwin Kaal 1,2*, Arend Kolk 3, Sjoukje Kuijper 3, Hans-Gerd Janssen 1,4
1 - Polymer-Analysis group, Van ’t Hoff Institute for Molecular Sciences, University of Amsterdam, Nieuwe Achtergracht
166, 1018 WV Amsterdam, The Netherlands.
2 - ATAS GL International, P.O. Box 17, 5500 AA Veldhoven, The Netherlands.
3 - KIT Biomedical Research, Royal Tropical Institute, Amsterdam, The Netherlands.
4 - Unilever Research and Development,Advanced Measurement and Imaging, P.O. Box 114, 3130 AC Vlaardingen,The Netherlands.
A fast gas chromatography-mass spectrometry (GC-MS) method with minimum sample preparation is described for
early diagnosis of tuberculosis (TB). The automated procedure is based on the injection of sputum samples which are
then methylated inside the GC-injector using thermally assisted hydrolysis and methylation (THM). The THM-GC-MS
procedure was optimized for the injection of sputum samples. For the identification of Mycobacterium tuberculosis the
known marker tuberculostearic acid (TBSA) and other potentional markers were evaluated. Hexacosanoic acid in combination with TBSA was found to be specific for the presence of M. tuberculosis. For validation of the method several
sputum samples with different viscosities spiked with bacterial cultures were analyzed. The detection limit was better
than 1 x 104 bacteria/ml. 17 methyl octadecanoic acid was used as standard.The detection limit for this compound was 20
pg/ml. Finally, 18 stored sputum samples collected in Vietnam from patients suspected to suffer from TB were re-analyzed
in Amsterdam by microscopy after decontamination/concentration and using the new THM-GC-MS method. No false
positives were found by THM-GC-MS and all patients who were diagnosed with TB were also found positive using our
newly developed THM-GC-MS method. These results show that the new fast and sensitive THM-GC-MS method holds
great potential for the diagnosis of TB.
This work was partially supported by the Foundation of New Diagnostics (FIND) and the Optimus Foundation.
56
ESM 2009
OP-15
HOW WILD-TYPE MIC DISTRIBUTIONS CAN BE USEFUL TO
DETERMINE CLINICAL BREAKPOINTS IN Mycobacterium tuberculosis
Ängeby, Kristian 1, Juréen, Pontus 2, Giske, Christian 1, Chryssanthou, Erja 1, Werngren, Jim 2, Hoffner, Sven 2, Kahlmeter,
Gunnar 3, Sturegård, Erik 4, Schön, Thomas 5
1 - Department of Clinical Microbiology, Karolinska University Laboratory and Karolinska Institute, Sweden
2 - Department of Bacteriology, Swedish Institute for Infectious Disease Control, Sweden
3 - Department of Clinical Microbiology,Växjö Hospital, Sweden
4 - Department of Clinical Microbiology, Malmö University Hospital, Sweden
5 - Department of Clinical Microbiology, Kalmar County Hospital, Sweden
The increasing prevalence of multidrug resistant (MDR) and extensively drug resistant (XDR) tuberculosis (TB) underscores the need for accurate drug susceptibility testing (DST) .It is unfortunate that the current critical antibiotic
concentrations (breakpoints) are based mostly on empiry and only to a limited extent on scientific evidence. This is
especially true for second line drugs.
For most other bacterial pathogens, wild-type minimal inhibitory concentration (MIC) distributions has been successfully
applied as one of other tools (such as pharmacokinetic and pharmacodynamic data) to determine clinical breakpoints for
DST. A microorganism is defined as wild-type by the absence of acquired and mutational resistance mechanisms to the
drug in question. In modern breakpoint determination, breakpoints that divide divide wild type distributions of MICs are
avoided. An isolate with a MIC above the wild-type distribution is highly likely to harbor resistance mechanisms and is
considered clinically resistant until there is evidence to the contrary.
At present, wild-type MIC-distribution data for M. tuberculosis is scarce, presumably in part because of the complexity of
DST. In order to establish wild-type MIC distributions for M. tuberculosis we used a 96-stick replicator method, which
allowed us to efficiently determine the MICs of 95 clinical isolates simultaneously for four first line drugs (isoniazid,
rifampicin, ethambutol and streptomycin) and 15 second line drugs (including four quinolones, four injectables, ethionamide, prothionamide, cykloserine, PAS, thioacetazone). Our findings clearly demonstrate how wild-type MIC distributions
among other tools can be used to define clinical breakpoints also in M. tuberculosis.
European Society of Mycobacteriolog y | 30 th Annual Congress | July 2009 | Por to - Por tugal
57
OP-16
MOLECULAR TECHNIQUES TO MONITOR TB PATIENTS’ TREATMENT:
SELECTIVE REMOVAL OF DNA FROM DEAD BACTERIA IN MIXED
POPULATIONS BY USE OF ETHIDUM MONOAZIDE
Miotto Paolo, Cirillo Daniela M.
Emerging Bacterial Pathogens Unit, San Raffaele Scientific Institute, Milan – ITALY
Drug-resistant tuberculosis (TB) represents a public health problem worldwide and is considered a real threat for TB
control programs. Modern nucleic acid amplification techniques (NATs) that exploit nucleic acids signals from clinical
samples represent useful tools to allow rapid detection of resistant M. tuberculosis strains. Identification of resistant bacteria in clinical samples is fundamental in order to prevent inadequate treatment and further development of resistant
strains. However, NATs can not be used for patients’ follow-up because DNA-derived signals can originate from nonviable
bacterial cells and, therefore, generate data that could be misinterpreted. A method developed for microbial food pathogens and already tested on environmental samples is here tested to distinguish between live and dead mycobacteria.
Ethidium monoazide bromide (EMA) is membrane impermeant that intercalates into both extracellular DNA and DNA
in nonviable cells and is excluded from viable bacteria. Exposure to a light source renders EMA-DNA incapable of contributing to PCR.
Liquid culture of M. tuberculosis H37Rv was heat inactivated by incubation at 95 °C for 30 min and then treated with 6
μM, 9 μM, 12 μM, respectively, EMA concentrations. EMA treatment consists of 20 min of incubation at +4 °C in the dark
followed by 30 min of exposure to a 500 Watt light. Controls used in the study were: inactivated bacteria untreated with
EMA, live bacteria untreated with EMA, and live bacteria treated with the same 3 EMA concentrations. After EMA treatment, samples and controls were processed for DNA extraction by thermal lysis (95 °C for 30 min) and directly used in
PCR. Amplicons were analyzed by capillary electrophoresis (Agilent Technologies).
EMA treatment at all three concentrations tested suppressed PCR amplification from heat inactivated cultures without
affecting amplification from live bacteria, proving that EMA could be used also for mycobacterial samples.
Preliminary studies were also conducted on mixed live/dead populations using two strains with a different MIRU-VNTR
genotype. Performing MIRU typing on mixed samples after EMA treatment, only the live strain could be detected.
Comparison of treated and untreated samples highlighted the significant contribution that nonviable bacteria can make
to DNA-based diagnostic analysis. EMA approach could be a simple and effective means to monitor patients’ treatment
allowing the use of NATs during follow-up.
58
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OP-17
Mycobacterium tuberculosis IS ABLE TO
ACCUMULATE AND UTILIZE CHOLESTEROL
Brzostek Anna 1,2, Pawelczyk, Jakub 2, Rumijowska-Galewicz, Anna 2, Dziadek, Bozena 3, Dziadek, Jaroslaw 2
1 - Laboratory of Mycobacterium Genetics and Physiology PAS, Institute for Medical Biology
2 - PAS, Institute for Medical Biology
3 - University of Lodz, Department of Immunoparasitology
Mycobacterium tuberculosis, is the causative agent of tuberculosis that infects one third of the human population. Tubercle
bacilli are able to persist in a dormant state, from which they may reactivate disease state. The presence of lipid metabolism genes in the genome of M. tuberculosis suggests that lipids, including steroids, are important carbon and energy
sources for this pathogen. One potential nutrient that is available in the mammalian host is cholesterol, a major sterol
of the plasma membrane. Cholesterol is essential for the uptake of mycobacteria by macrophages, and it has been found
to accumulate at the site of M. tuberculosis entry. Moreover cholesterol can be utilized by fast-growing, non-pathogenic
mycobacteria, but pathogenic mycobacteria might not be able to use cholesterol. Here, we show for the first time that M.
tuberculosis grown in media containing carbon source other than cholesterol is able to accumulate cholesterol in the free
lipid zone of its cell wall. This cholesterol accumulation decreases the permeability of the cell wall for the primary antituberculosis drug, rifampin, and partially masks the mycobacterial surface antigens. Furthermore, M. tuberculosis was able
to grow on mineral media supplemented with cholesterol as the sole carbon source. Targeted disruption of the Rv3537
(kstD) gene inhibited growth due to inactivation of the cholesterol degradation pathway, as evidenced by accumulation of
the intermediate, 9-hydroxy-4-androstene-3,17-dione. Our findings that M. tuberculosis is able to accumulate cholesterol
in the presence of alternative nutrients and use it when cholesterol is the sole carbon source in vitro may facilitate future
studies into the pathophysiology of this pathogen.
The work was supported partially by grants ICGEB (CRP/POL07-01) and State Committee for Scientific Research (N302
035 31/3172).
European Society of Mycobacteriolog y | 30 th Annual Congress | July 2009 | Por to - Por tugal
59
OP-18
MYCOLIC ACID-INDUCED IFN-γ PRODUCTION BY
CD1-RESTRICTED T CELLS FROM TUBERCULOUS PATIENTS
Rodríguez-Güell, E 1, Alonso, C 2, del Val-Romero, B 2, Clivillé, R 2, Secanella,SP 1, Roura-Mir,C 3, Cañete, C 4, Navarro, A 4,
de Gispert, FX 4, Luquin, M 1, Julián,E 1
1. Dept. Genètica i Microbiologia, Facultat de Biociències, Universitat Autònoma de Barcelona, Bellaterra (Barcelona)
2. Dept. Microbiologia, Consorci Sanitari Integral, L’Hospitalet de Llobregat (Barcelona)
3. Dept. Fisiologia i Immunologia, Facultat de Biociències, Universitat Autònoma de Barcelona, Bellaterra (Barcelona)
4. Unitat de Respiratori, Consorci Sanitari Integral, L’Hospitalet de Llobregat (Barcelona)
Introduction
The cell wall of Mycobacterium tuberculosis (MTB) has a large number of structurally diverse lipids. Mycolic acids (MAs) merit
special interest because their immunogenicity has been demonstrated in CD1-restricted T cell clones; specifically, they are
presented by CD1b molecules. To date, the recognition of MAs in tuberculous patients (TB) has not been studied.
The purpose of the study was to determine whether MAs are recognized by the immune system of TB patients and,
in the event of this being so, to study the evolution of this response throughout anti-TB treatment.
Methods
Immature dendritic cells (iDCs) isolated from 31 TB patients at the time of diagnosis and throughout anti-TB treatment,
20 PPD-positive and 20 PPD-negative donors were analysed by cytometry for CD1b surface expression. Subsequently,
the samples were irradiated and cultured with autologous lymphocytes in the presence of phytohemaglutinin, MTB and
purified MAs as stimuli. After 48 hours, IL-10 and IFN-γ were quantified by enzyme-linked immunosorbent assay.
Results
No significant differences among the surface expression of CD1b in iDCs from healthy donors or TB patients were observed at any time during the disease. iDCs from all the individuals were therefore able to present MAs.
Median levels of stimuli-induced IL-10 in samples obtained at the outset of anti-TB treatment were lower than those
obtained at the end; however, no significant differences were observed. Nor were any differences observed among the
IL-10 levels obtained from the different groups of individuals.
In TB patients, MA-induced IFN-γ median values obtained at the end of prophylaxis were significantly higher, statistically,
than those elicited at the beginning. Grouping the samples of the 31 TB patients in terms of collection time, IFN-γ median
levels followed an upward trend throughout anti-TB treatment, reaching maximum levels at the point of disease cure.
Furthermore, in PPD-negative donors IFN-γ median values were lower than those from PPD-positive donors, significant
differences only being established when MTB was used as antigen.
Conclusions
The specific cellular immune response against MAs in TB patients described here for the first time suggests a potential
immunoprotective role for MAs.
60
ESM 2009
OP-19
A REPORT ON NEW ADAPTED FORMS OF EXTENSIVELY DRUG RESISTANCE
TUBERCLE BACILLI : TRANSMISSION ELECTRON MICROSCOPY ANALYSIS
Parissa Farnia(PhD)1. Ali Akbar Veleyati (MD)1, Mohammal Reza Masjedi (MD)1, 2 Tengku Azmi Ibrahim (PhD)2, Payam
Tabarsei (MD), Rafiuz Zaman Haroun (MSC)2, Ho Oi Kuan (MSC))2, Abdul Rahman Omar (PhD)1
1 - Mycobacteriology Research Centre, National Research Institute of Tuberculosis and Lung Disease (NRITLD), WHO
Collaborating Centre, Shahid Beheshti University (Medical Campus), Darabad, Tehran ,19556, P.O: 19575/154, Iran.
E-mail: [email protected]
2 - Microscopic unit, Institute of Bioscience , University Putra Malaysia ,43400 UPM ,Serdang, Selangor Darul Ehsan , Malaysia
Background
Extensively drug resistance tuberculosis bacilli (XDR-TB), is caused by a strain of M. tuberculosis(MTB) that are resistant to
isoniazid and rifampin (which defines MDR tuberculosis) in addition to any fluroquinolone and at least one of the three
following injectable drugs: caperomycin, kanamycin and amikacin.Viewed under transmission electron microscopy (TEM),
spore like structure was observed inside the XDR-TB bacilli.
Methods
The susceptibility testing against first and second line drugs was performed on isolated M. tuberculosis strains. Subsequently,
a homogenous thick suspension of 107 to 108 cells at exponential phase was prepared and observed under Transmission
Electron Microscopy (TEM). Five isolates from each group (susceptible, MDR, and XDR TB) were used in this study.
Results
Viewed under the TEM, the XDRTB bacilli at exponential phase had three types of cell division 1) 80-70% of bacilli were
looks as norm one with symmetrical or asymmetrical cell division 2) 5-7% with extra ordinary thick cell wall ( 21 to 26
nm ) which was similar to stationary or dormant phase bacilli. But surprisingly the stationary phase XDR-TB bacilli were
at dividing process 3) 15-20% bacilli had spore formation inside them These spores were different from buds or a polar
division that formed during cell branching of MTB. No spore’s and stationary phase bacilli were detected in susceptible
or multidrug resistant strains of MTB.
Discussion
Dividing phenomena in XDR-TB bacilli with stationary phase appearance will generate new adapted types of M. tuberculosis which can resist the effect of all available anti tuberculosis drugs. At the same time, it is possible that under certain
conditions, the XDR-TB bacilli produce spore to overcome the hostile environment. Spore formation in XDR-TB bacilli
should take very seriously from epidemiological point of view.With these new forms of adaptation that occurs in XDRTB
bacilli , how should we treat and control the diseases.
European Society of Mycobacteriolog y | 30 th Annual Congress | July 2009 | Por to - Por tugal
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OP-20
GROWTH PROFILE OF CLINICAL ISOLATES OF Mycobacterium
tuberculosis COMPLEX IN MURINE MACROPGHAGES
Susanne Homolka1, Stefan Niemann1, David G. Russell2 and Kyle H. Rohde2
1 - Molecular Mycobacteriology, National Reference Center of Mycobacteria, Borstel Germany
2 - Department of Microbiology/Immunology, Cornell University,Vet. Med. Center, Ithaca, USA
Background
Mycobacterium tuberculosis and other members of the Mycobacterium tuberculosis complex (MTC) remain a major cause of
morbidity and mortality worldwide. Several studies based on spoligotyping, IS6110 fingerprinting and MIRU-VNTR typing
demonstrated that the global population structure of MTC is defined by phylogeographical lineages and genotypes that are
also associated with pathogenic differences. However, the influence of strain genomic variation on the outcome of infection
and the clinical presentation are not completely understood. In our study, we investigated growth differences of 15 strains
of five different genotypes in comparison to the CDC1551 reference strains in liquid culture and in macrophages.
Methods
Murine bone marrow derived macrophages were infected with liquid culture of different clinical isolates (MOI 3:1;
2x106CFU/ml). Survival of strains in resting and activated macrophages was determined at different time points up to 11
days post-infection. Additionally, growth kinetics of all strains in 7H9 liquid media were analyzed.
Results
Growth analyses of clinical isolates in liquid culture based on OD measurements showed no significant differences in
comparison to CDC1551. However, we observed both strain- and genotype-specific survival and growth profiles within
resting and activated macrophages.
Conclusions
The genomic diversity of clinical isolates influences the complex interaction of the pathogen with host phagocytes.
Further analyses to correlate intracellular gene expression profiles with the outcome of infection are in progress.
62
ESM 2009
OP-21
PRESENCE OF ESAT-6 AND CFP-10 GENES DOES NOT LEAD TO
PHAGOLYSOSOME TRANSLOCATION OF Mycobacterium szulgai
Jakko van Ingen1,2, Nicole van der Wel3, Richard Dekhuijzen2, Martin Boeree2, Dick van Soolingen1
1 - National Mycobacteria Reference Laboratory, National Institute for Public Health and the Environment, Bilthoven,
the Netherlands
2 - Department of Pulmonary diseases, Radboud University Nijmegen Medical Center, Nijmegen, the Netherlands
3 - Department of Cell Biology, Netherlands Cancer Institute, Amsterdam, the Netherlands
Background
Bacterial virulence factors in nontuberculous mycobacteria are mostly unknown. In Mycobacterium tuberculosis complex
bacteria, the esat-6 and cfp-10 genes are important virulence factors, which facilitate translocation from the phagolysosome to the cytosol of macrophages. Their presence and role among nontuberculous mycobacteria is largely unknown.
Methods
We assessed the presence of esat-6 and cfp-10 genes in all 5 M. kansasii subtypes based on 16S-23S internal transcribed
spacer sequencing (n=15), M. szulgai (4), M. marinum (4), M. avium (2), M. conspicuum (4), M. genavense (1), M. bohemicum
(2), M. interjectum (2), M. flavescens (5), M. xenopi (2), M. malmoense (2), “M. riyadhense” (1) and M. tuberculosis H37Rv by
PCR. We used Esa-12 CATGACAGAGCAGCAGTG and Esa-303 5’-GCCCTATGCGAACATCCC-3’ primers for esat-6
and opBR78 5’-GTAGCCCGGGATGGCAGAGATGAAGACCGATGCC-3’ and opBR103 5’-TCAGAAGCCCATTTGCGAGGACAGC-3’ primers for cfp-10. For PCR negatives, we performed Southern blotting with probes based on the
esat-6 gene of M. tuberculosis H37Rv.
One NTM species with esat-6 and cfp-10 genes was selected for macrophage infection. By cryo-immunogold electron microscopy we tested whether these genes effect translocation from the phagolysosome to the cytosol of macrophages.
Results
We were able to amplify esat-6 and cfp-10 genes in M. tuberculosis H37Rv, all M. kansasii subtypes, M. szulgai, M. marinum
and “M. riyadhense”. All esat-6 and cfp-10 sequences among these nontuberculous mycobacteria were species-specific;
multisequence alignment revealed an average of 90% homology with the M. tuberculosis sequences. The remaining species were repeatedly negative by both PCR and Soutern blotting. Mycobacterium szulgai was subsequently used for a
macrophage (THP-1) infection experiment, with an M. tuberculosis positive control. Seventy-two hours after infection, no
cytosolic M. szulgai bacteria were found, while 53% of the M. tuberculosis bacteria had translocated to the cytosol.
Conclusions
While some nontuberculous mycobacteria harbor esat-6 and cfp-10 genes, their products, at least for M. szulgai, do not
effect translocation of the bacteria from the phagolysosome to the cytosol in macrophages. ESAT-6 and CFP-10 protein
structure or secretion could be critical factors. While the presence of esat-6 and cfp-10 genes adds an interesting phylogenetic marker, the pathogenesis of NTM infections remains unexplained.
European Society of Mycobacteriolog y | 30 th Annual Congress | July 2009 | Por to - Por tugal
63
OP-22
DEVELOPMENT OF AN ADAPTIVE IMMUNE RESPONSE IN THE DRAINNG
LYMPH NODE DURING Mycobacterium ulcerans INFECTION
Alexandra G. Fraga; Joana E. Braga; Andrea Cruz; Teresa G. Martins; Daniela R. Pereira; Wayne M. Meyers; Françoise
Portaels; António G. Castro; Jorge Pedrosa
1 - Life and Health Sciences Research Institute (ICVS). School of Health Sciences, University of Minho. Braga, Portugal
2 - Mycobacteriology Unit, Department of Microbiology, Institute of Tropical Medicine, Antwerp, Belgium
3 - Armed Forces Institute of Pathology, Washington, D. C., USA
Buruli ulcer is a neglected tropical disease caused by infection with Mycobacterium ulcerans. The necrotic cutaneous lesions of BU patients are associated with the cytotoxic properties of the exotoxin mycolactone. Previous results from
our group show that the protective role of IFN-γ is impaired during infection with the highly virulent strain 98-912. To
further characterize the development of an adaptive immune response during progressive infection with a highly virulent
M. ulcerans strain, we decided to study the T cell dynamics in the draining lymph node (DLN) of mice infected with M.
ulcerans 98-912, as compared to the low virulent, mycolactone-deficient strain 5114.
Subcutaneous infection of mouse footpads with M. ulcerans 5114 induced a modest increase in the number of cells in the
DLN, prevalent throughout the experimental infection. Surprisingly, infection with strain 98-912 led to a 26-fold increase
in the number of cells in the DLN, followed by a significant drop to basal levels. However, this increase in the number of
cells did not correlate with a protective response in the infected footpad.
Following this observation, we explored whether the induction of an adaptive immune response occurs during infection
with strain 98-912. M. ulcerans infection elicited antigen-specific T cells producing IFN-γ in the DLN, with the response
being more prominent in M. ulcerans 98-912 infected mice. Additionally, infection with M. ulcerans followed by adoptive
transfer of OVA-transgenic T cells did not impair the accumulation, proliferation or activation of the OVA-specific T cells
in the DLN upon challenge with OVA. Moreover, this T cell accumulation and activation was significantly increased in
mice infected with strain 98-912. Interestingly, analysis of the DLN at later time points revealed the presence of viable
bacilli that correlated with the dramatic decrease in the number of cells. This decrease was due to apoptosis, as demonstrated by the higher number of activated-caspase 3 positive cells. Supporting our observations in the mouse model,
presence of bacilli and tissue destruction were also observed in DLN of BU patients.
In summary, the failure of the host to generate a protective response against infection with this highly virulent strain of
M. ulcerans is not associated with an impaired development of specific T cells in the DLN, but instead to their destruction
in the infection foci and, later, to the destruction of the DLN itself.
64
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OP-23
TUBERCULOSIS SOFTWARE IN A GENERAL HOSPITAL,
WORKING INSTRUMENT
Portugal Clara 1; Nuno Cardoso 2, Luísa Sancho 1; Germano Sousa 1
1 - Laboratory of Microbiology, Department of Clinical Pathology
2 - Planning and Management Control Director
Hospital Fernando Fonseca (HFF) - Amadora, Portugal
[email protected]
Background
Our Hospital is located in a Lisbon’s surroundings, its construction was completed in 1995 and, in this same year, it
started the operation.
The HFF was designed to house a population much smaller than that now covers, 750 000.
Our population has a low socioeconomic conditions and a high level of immigrants from Africa and in Eastern Europe,
which may be the main reason for concentrating a large number of tuberculosis’s cases. Portuguese Health Authorities
reported that 66% of TB cases in Portugal were concentrated in Lisbon’s surroundings.
In a few years after the opening of the Hospital, all laboratory’s data relating to tuberculosis became too numerous to be
recorded and cross in the general program.
Purpose
Given the need to gather and cross all the tuberculosis’s data, in 2000 was developed a software (MYCOHFF) that we
are going to present.
Methods
MYCOHFF results from the interconnection of 3 Excel files; MYCOYEAR related to the year in question, MYCOZIEHL
that brings only patient’s data with positive Ziehl-Nielseen (since 1997) and MYCOCULT that joins only the patient’s
data with positive cultural examinations (liquid and/or solid) (since 2000).
For each specimen submitted for mycobacterial culture, we record the name of the patient, process number, analysis
number, product’s type, the origin patient’s Service and product inoculation’s date. Thus takes place an observation’s
weekly list. For each week, we note the medium (liquid and Lowenstein-Jensen) growth or not, the contamination and/
or degradation, during 6 or 8 weeks (sometimes more).
The year’s records are placed in alphabetical order (patient’s name).
The patient’s analysis with positive culture (liquid and/or solid) automatically appears in red.
If the patient had already a positive culture, all the negative analysis appears in purple. If the patient had a positive ZiehlNielseen, then all the analysis will appear in yellow, becomes purple if it has other positive culture or red if this analysis
is positive.
On patient’s first isolation, we perform the Identification and Susceptibility Test.This date is noted as well as the final date
with the definitive results, which are presented to the clinician.
Results
• Warning Signs: When the patient’s registration happens, we know immediately, with different colors (program
alerts), if the patient has Ziehl-Nielseen positive, culture positive, and when.
• Search: Any search is fast because we are talking about an application that is made in Excel software.
• Weekly observation’s list: The weekly sheets are easily created by filtering the inoculation’s date field.
• Statistical indicators and graphics: With only a few records, statistical indicators are automatically calculated and
graphical analyses are available at any time.
European Society of Mycobacteriolog y | 30 th Annual Congress | July 2009 | Por to - Por tugal
65
Statistical indicators for each year are concerning to different parameters. 1- Concerning the analysis/patient relationship: Nº of patients, nº of analysis, total positive patients, total positive analysis, analysis/patient ratio, patients positive
rate, analysis positive rate; 2- Concerning the Ziehl-Nielseen (Z) and liquid/solid medium relationship: Nº of positive
direct tests (Z+), Nº of positive cultural tests (C+), true Z positive’s (Z+/C+) rate, false Z negative’s (Z-/C+) rate, false
Z positive’s (Z+/C-) rate, real Z negative’s (Z-/C-) rate, Ziehl-Nielseen’s positivity rate per analysis and per patient;
3- Concerning the identification and TSA: Nº of Mycobacterium sp. isolated, total identified strains, total strains with
the TSA and identification still in study, Nº and rate of Mycobacterium tuberculosis complex (CMT), CMT without resistance, with one resistance, MDR (Multi Drug Resistant - TB), XDR (Extensively Drug Resistant – TB), Nontuberculous
mycobacteria, average response time beginning with the isolation culture (identification plus TSA).
The statistics graphs show: 1 - Analysis requested / positive analysis by organic product; pleural biopsy, pleural fluid,
sputum, bronchial secretions, bronchoalveolar lavage, gastric fluid (respiratory specimens), urine, pus, mieloculture,
cerebrospinal fluid, biopsy , ascites fluid, fluids, blood; and their positivity, 2 - The isolated Mycobacterium sp.’s strains,
including the differentiation in CMT, zero resistance, one resistance, MDR, XDR, 3 - Compare the tuberculosis’s incidence rate in two populations, MDR and non-MDR, for the age range of patients, sex and HIV.
Conclusions
For us, MYCOHFF software is an essential tool for the process of gathering and managing tuberculosis data.
Quick reports of any patient are possible. In a few seconds, we can make the patient laboratory story on tuberculosis.
Each year a statistical report is communicated to all our hospital’s departments. Monthly, or when requested, for the CDP
(centre that follows the patients in the community), is sent a list identifying new positive cases and results (preliminary
and/or final).
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ESM 2009
OP-24
Development of an automated culture system for
M. tuberculosis with autofluorescence detection
den Hertog, Alice 1, Koeleman, Marc 1, Ingham, Colin 2, Fey, Frank 3, Langerak, Edwin 3, Klatser, Paul 1, Anthony, Richard 1
KIT Biomedical Research, Amsterdam, The Netherlands
Microdish BV, Wageningen, The Netherlands
CCM, Nuenen, The Netherlands
Due to the unavailability of rapid sensitive diagnostic methods for tuberculosis (TB), culture remains one of the most
important diagnostic tools - even though it is technically demanding and slow. The time required for culture leads to a
delay in TB diagnosis and treatment with effective drugs. A standardized method for more rapid drug sensitivity testing
(DST) of TB would be valuable.
We are developing a system in which mycobacterial (micro-)colonies are detected by their autofluorescence based on
the MODS (microscopic observation of drug susceptibility) method, which is much more rapid than the traditional culture methods, but is laborious.
By using microscopy with low-power magnification, colony growth could be determined much earlier than
when viewed by eye. Studying Mycobacterium smegmatis initially as a model organism, we have made sequential brightfield and fluorescence microscopy photographs of colonies growing on movable solid supports on media. These images were used to develop an image analysis protocol to determine the growth rate.
We found that the time to detection of microcolonies was less than half that required for visual detection. Furthermore,
as soon as the colonies could be visualized, further growth of colonies could be detected by the image analysis protocol
within 1-2 division times, from which the growth rate could be determined. As the bacteria were inoculated on movable
solid supports, exposing the microcolonies to selective media after their first detection could make DST of many individual colonies possible after only a few additional division times, allowing the rapid determination of the proportion of
antibiotic resistant and sensitive mycobacterial colonies present in a sample.
After confirmation of the recently described autofluorescence of mycobacteria, this method was used for the detection
and enumeration of viable mycobacteria. The specificity of the autofluorescence may be used to confirm the presence
of mycobacteria.
The use of (semispecific-) autofluorescence in combination with growth rate will increase the feasibility of developing
an automated detection system based on this method, which is very challenging for light microscopy methods (such as
MODS). Additionally, the possibility of performing quick DST without re-inoculation may lead to a reduce time to correct treatment.
European Society of Mycobacteriolog y | 30 th Annual Congress | July 2009 | Por to - Por tugal
67
OP-25
SECOND-LINE DRUG SUSCEPTIBILITY TESTING OF Mycobacterium
tuberculosis BY MGIT 960 SYSTEM, THE MICROPLATE
COLORIMETRIC-BASED METHOD AND THE PROPORTION METHOD
Nora Morcillo1, Belén Imperiale,1, 2, Beatriz Di Giulio3
1 - Reference Laboratory of Tuberculosis Control Program of Buenos Aires Province, Dr. Cetrángolo Hospital Buenos
Aires, Argentina.
2 - National Council of Scientific and Technological Research, Buenos Aires City.
3 - P.V. de Cordero Hospital, San Fernando, Buenos Aires, Argentina.
The accurate treatment of tuberculosis (TB) cases due to multidrug-resistant and extensively drug-resistant Mycobacterium
tuberculosis emphasizes the necessity of new tools for rapid detection of these strains in clinical laboratories. Minimal
inhibitory concentrations (MICs) by MGIT960 and the colorimetric microplate method using dyes as MTT or resarzurin
(CMM) were determined for the following drugs (µg/ml): amikacin (AMK): 2.0, 4.0, 8.0; kanamycin (KM), capreomycin
(CPM), ethionamide (ETH): 2.5, 5.0, 10.0; cycloserine (CS): 15.0; ofloxacin (OFX) and linezolide (LZ): 0.5, 1.0, 2.0; and
moxifloxacin (MOX) 0.25, 0.5, 1.0. MICs were performed on 94 clinical isolates.The proportion method on Middlebrook
7H11 (PM) was used as gold standard. Inoculated MGITs were incubated in the instrument for no longer than 21 days.
A strain tested by MGIT960 was considered resistant if a positive signal flagged from the drug-containing tube within 5
days of the positive control tube. Microplates of the CMM were incubated for an average of 8 days. Statistical methods
were applied to define drug-resistant strains on the basis of the comparison between results obtained by MGIT960 and
CMM with the PM. The following critical concentrations were identified (µg/ml): AMK: 4.0; CPM, ETH and KM: 5.0; CS:
30.0; LZ: 1.0; MOX: 0.5; OFX: 2.0. Accuracy of MGIT960 and M-MTT was 100% for AMK, CPM, OFX, MOX and LZ. In this
study tubes incubation and positivity detection was manually obtained from the MGIT960 instrument which actually can
be adapted to automatically detect both susceptible and resistant strains to each one of the second-line drugs. Results
were obtained in less than 10 days for both MGIT960 and CMM. On the other hand CMM, as a complete homemade
method, was cheaper but more laborious than MGIT960. Our results showed that both methods could be promissory
implemented as a rapid diagnosis tools to detect MDR and XDR-TB cases in clinical practice.
68
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OP-26
THIORIDAZINE SHOWS PROMISING ACTIVITY IN A
MURINE MODEL OF MULTIDRUG-RESISTANT TUBERCULOSIS
Jakko van Ingen1,2, Martin Boeree1, Leonard Amaral3, Rogelio Hernandez Pando4, Dick van Soolingen2
1 - Department of Pulmonary Diseases, Radboud University Nijmegen Medical Center, Nijmegen, the Netherlands
2 - National Mycobacteria Reference Laboratory, National Institute for Public Health and the Environment, Bilthoven,
the Netherlands
3 - Mycobacteriology Unit, Institute of Hygiene and Tropical Medicine, Universidade Nova de Lisboa, Lisbon, Portugal
4 - Experimental Pathology Section, Department of Pathology, National Institute of Medical Sciences and Nutrition
Salvador Zubiràn, Mexico City, Mexico
Background
Multidrug-resistant tuberculosis (MDR-TB) is a threat to TB control efforts worldwide for which very few active drugs
are currently available. The phenothiazines are antipsychotic agents that have potential as anti-tuberculosis drugs, at least
in vitro. Within this pharmacological class thioridazine is the most efficacious and causes the mildest side-effects when
used as an antipsychotic agent.Thioridazine is active against mycobacteria by targeting the type II NADH dehydrogenase,
succinate dehydrogenase, the binding of calcium to proteins and disruption of aerobic respiration under micro-aerobic
conditions. We tested its in vivo activity in a murine model.
Methods
We infected 3 groups of 40 Balb/c mice with pansusceptibe M. tuberculosis H37Rv. After 60 days, 20 mice in each group
started 2 months of thioridazine monotherapy at 16, 32 and 70 mg/kg dosages; the remaining 20 served as controls. This
experiment was repeated using a clinical multidrug-resistant M. tuberculosis (MDR-TB) isolate and two months daily oral
administration of 32 and 70 mg/kg. In a third experiment, 3 groups of 20 mice were infected with M. tuberculosis H37Rv;
one group received rifampicin, isoniazid and pyrazinamide, another received these 3 drugs and thioridazine, one untreated group served as controls. In all experiments, groups of five mice per group were sacrificed after 2, 4 and 8 weeks
of treatment; lung tissue was homogenized for quantitative cultures. The bacillary load of the lungs was determined by
colony forming units (CFU) quantification; histological damage was observed by microscopic examination.
Results
In both the M. tuberculosis �����������������������������������������������������������������������������������������
H37Rv and MDR-TB infection, monotherapy with 32 and 70mg/kg thioridazine lead to significant reductions in CFU counts from the lung tissue homogenates and in the extent of histological damage, at all time
points. ������������������������������������������������������������������������������������������������������������
Moreover, when 32 mg/kg of thioridazine was added to a regimen containing rifampicin, isoniazid and pyrazinamide for susceptible tuberculosis, a significant synergistic effect was achieved.
Conclusions
Thioridazine shows promising activity in our murine model. It has a potential effect in the treatment of susceptible
and MDR-TB, where few active drugs are available. The low price of this out of patent drug enables extended use in
resource–poor settings where the burden of multidrug-resistance is most grave.
European Society of Mycobacteriolog y | 30 th Annual Congress | July 2009 | Por to - Por tugal
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OP-27
CONTRIBUTION OF EFFLUX PUMP ACTIVITY FOR
MACROLIDE RESISTANCE IN M. avium COMPLEX
Liliana Rodrigues1,2*, Daniela Sampaio1, Isabel Couto1,3, Diana Machado1,Winfried V. Kern4,5, Leonard Amaral1,2,5 and Miguel Viveiros1,5
1 - Unit of Mycobacteriology, Instituto de Higiene e Medicina Tropical, Universidade Nova de Lisboa (IHMT/UNL), Lisbon, Portugal
2 - UPMM, IHMT/UNL, Lisbon, Portugal
3 - Centro de Recursos Microbiológicos (CREM), Faculdade de Ciências e Tecnologia, UNL, Caparica, Portugal
4 - Center for Infectious Diseases and Travel Medicine, University Hospital, Freiburg, Germany
5 - COST ACTION BM0701 (ATENS)
Mycobacterium avium complex (MAC), comprising M. avium and M. intracellulare, is clinically important since it can cause
severe infections in AIDS patients and other immunocompromised individuals. Therapy of MAC infections is problematic
due to the intrinsic resistance of these bacteria to many of the available antimicrobial drugs. The use of the macrolides
clarithromycin and azithromycin has improved the outcome of MAC infections, but therapeutic failure is still a major
problem. We have recently shown that efflux pumps of MAC play an important role on this resistance phenotype. In fact,
increased activity of efflux pumps is known to contribute to a multidrug resistance phenotype by extruding a wide variety
of chemically and structurally unrelated compounds from the cell, preventing them from reaching their cellular targets.
Thus, the characterization of such efflux pumps is crucial for the design of new antimycobacterial therapeutic strategies.
In this work, we have studied the efflux pump activity in MAC clinical strains by a fluorometric method that detects efflux
activity on a real-time basis, and evaluated the contribution of active efflux to the resistance to macrolides.
The results to be presented show that resistance to clarithromycin was significantly reduced in the presence of efflux
pump inhibitors (EPIs) such as the calcium-channel inhibitors thioridazine or chlorpromazine and the calcium ion influx
inhibitor verapamil. The same EPIs were effective in decreasing the efflux of ethidium bromide (a common efflux pump
substrate) from MAC cells, as shown by fluorometric analysis. Moreover, the retention of [14C]-Erythromycin by the same
inhibitors demonstrated that active efflux contributes to MAC resistance to macrolides.
In conclusion, this study demonstrates that efflux pumps play an important role in MAC resistance to antibiotics, particularly to macrolides, and opens the possibility to explore the usefulness of these EPIs, already used in clinical practice
for other purposes, such as thioridazine, as adjuvants to enhance the effectiveness of the therapeutic regimens against
MAC infections.
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OP-28
MEMBRANE- BASED VERSUS MICROBEAD- BASED SPOLIGOTYPING:
PRELIMINARY RESULTS ON A QUALITYINSURANCE STUDY ON 10 SITES WORLWIDE
Abadia E1, Zhang J1, Refregier G1, Sola C1.
1 - Institut de Génétique et Microbiologie, UMR8621, CNRS-Université Paris-Sud (Universud), Equipe IGEPE, bât. 400. France.
Spoligotyping have been developed 12 years ago and have been used worlwide for molecular epidemiological or molecular evolutionary studies. This technique is based on a reverse line- blot hibridization method. Due to it’s wide acceptance (458 references in Pubmed on 2009 April 27th) it can be considered as a basic technique in genotyping strains of
Mycobacterium tuberculosis complex together with MLVA (Multi locus variable analysis) typing. However, spoligotyping
has not been the focus of standarization nor of quality insurance studies. The transfer from the membrane- based to a
microbead- based format, through the development of the multiplex Luminex plataform was done in 2004 in the CDC
in Atlanta. We implemented this technique in our team in 2008.
We wanted to evaluate membrane- based spoligotyping results obtained on 10 sites (around 1000 DNA samples from
clinical isolates) comparing them with those from the Luminex microbead- based results obtained on the same samples.
The DNA samples were selected to fulfill the following goals: (1) Retrospectively assess the quality of spoligotyping
results, that have been produced in various laboratories worlwide during a decade (2) Solve inconsistencies, resolve discrepancies, improve quality of hard- to- interprete membrane- based spoligotyping results (3) Study if DNA extraction
procedure (quality, quantity) may have produced errors in spoligotyping patterns production (4) assess the economical
interest of the new Luminex- based technique and it’s contribution (or not) to and increased quality of services in molecular epidemiological studies.
We will present all results available until today. Our current experience and preliminary results suggest that the Luminex
platform it’s very versatile to produce high quality spoligotypnig results with both, a higher throughput and sensitivity
than the membrane- based spoligotyping and at affordable costs for developed countries.
European Society of Mycobacteriolog y | 30 th Annual Congress | July 2009 | Por to - Por tugal
71
Abstracts
of POSTER
PRESENTATIONS (PP)
European Society of Mycobacteriolog y | 30 th Annual Congress | July 2009 | Por to - Por tugal
73
PP-1
EVALUATION OF THE HIGH-THROUGHPUT REPETITIVE-SEQUENCE-BASED PCR
DIVERSILAB SYSTEM IN M. tuberculosis MOLECULAR EPIDEMIOLOGY STUDIES
Miotto Paolo, Baldan Rossella, Cirillo Daniela M.
Emerging Bacterial Pathogens Unit, San Raffaele Scientific Institute, Milan – ITALY
PCR-based methods have been developed to simplify and reduce the time required for genotyping M. tuberculosis by standard approaches based on IS6110-Restriction Fragment Length Polymorphism (RFLP). Of these, MIRU-VNTR complemented with spoligotyping has been proposed as an alternative. Repetitive-sequence-based PCR (rep-PCR) is useful for
generating DNA fingerprints of diverse bacterial species. Rep-PCR amplicon fingerprints represent genomic segments
lying between non-coding repetitive sequences. A commercial system (Diversilab, Biomerieux) that electrophoretically
separates rep-PCR amplicons on microfluidic chips, and provides computer-generated readouts of results has been
adapted for use with Mycobacterium species.The ability of this system to type M. tuberculosis was evaluated in comparison
with spoligotyping and MIRU-VNTR in two different panels.
First, we evaluated a 35 strains panel by MIRU-15 complemented with spoligotyping and Diversilab rep-PCR. Results
were analyzed with MIRU-VNTRplus database for spoligo-MIRU, and with Diversilab Software for rep-PCR using two
different algorithms (Pearson Correlation [PC] and Kullback-Leibler [KL]). Threshold for clusters was fixed at 98% of
similarity for rep-PCR. MIRU-15 showed a clustering rate of 11.4% whereas the rep-PCR reported a clustering rate of
28.6% (PC) and 17.1% (KL). The discriminatory power (Hunter-Gaston discriminatory index [HGDI]) for spoligo-MIRU15 resulted 0.983 whereas for rep-PCR was 0.978 (PC) and 0.983 (KL).
We also compared the two techniques on a panel composed by 8 closely related strains from a probable outbreak. In
this case the rep-PCR showed a discriminatory power of 0.571 (PC) and 0.679 (KL), compared to a HGDI of 0.643 for
spoligo-MIRU-15. Nevertheless, clustering rate for MIRU-15 was 50.0% whereas rep-PCR algorithms showed a clustering rate of 100.0% (PC) and 62.5% (KL), respectively. To understand the meaning of the discrepancies still found between
spoligo-MIRU-15 and rep-PCR, analysis of epidemiological data for the clustered patients will be taken in consideration.
Preliminary data obtained by Diversilab suggest that the KL algorithm is more appropriate for M. tuberculosis typing analysis. Nevertheless, even if rep-PCR results analyzed by KL algorithm showed a discriminatory power similar to MIRU-15,
clustering rate remains higher. This new technique could be useful for a routine use in clinical laboratories for real-time
genotyping and laboratory contaminations control.
European Society of Mycobacteriolog y | 30 th Annual Congress | July 2009 | Por to - Por tugal
75
PP-2
68 SPACERS Mycobacterium tuberculosis COMPLEX SPOLIGOTYPING :
A STUDY USING A MICROBEAD-BASED HIGH THROUGHPUT FORMAT.
Zhang J1, Abadia E1, Refrégier G1 , Ruimy R2, Boschiroli ML3, Guillard B4 and C. Sola1.
1 - Institut de Génétique et Microbiologie, UMR8621, CNRS-Université Paris-Sud (Universud), Equipe IGEPE, bât. 400, Centre scientifique d’Orsay, rue Gregor Mendel, 91405 Orsay-Cedex2 - APHP, Hôpital Bichat-Claude Bernard, Paris
3 - Agence française de sécurité sanitaire des aliments AFSSA, Maisons-Alfort
4 - Institut Pasteur du Cambodge
New captures probes for the microbead-based spoligotyping assay (Luminex) were designed to test for the presence/
absence of 25 spacers that are not used in routine spoligotyping. These spacers are expected to provide an improved
discriminatory power for Major Genetic Group I, including the « ancestral » TbD1+ MTC (Mycobacterium tuberculosis
complex) clinical isolates.
The new 68 spacers spoligotyping format developed on a Luminex platform was shown to give excellent results. Around
400 strains of MTC from 3 different centers (Bichat Hospital, AFSSA, Institut Pasteur of Cambodia) were studied. We
show that the 68 spacers format is more discriminant to study the strains of the East African Indian family (EAI) : among
86 strains of the EAI family, it could distinguish 44 clusters compared to 27 clusters by routine 43 spacers spoligotyping.
Whereas for a total of 210 clinical isolates of Mycobacterium bovis, we reported 31 types instead of 25, and for a total
of 30 “Beijing” clinical isolates, 4 clusters instead of 3 clusters were found. For Mycobacterium africanum, a total of 17
strains were assayed and 11 instead of 9 clusters were found. High-thr(East-African Indian - Indo-Oceanic clade) and
other Major Genetic Group I still-undefined spoligotyping signatures. This observation often concerns the first spacer
of a string of several missing spacers (border domains). These observations are currently under further investigation by
sequencing. Extended High-throughput spoligotyping is also a new mean to detect mutational events that could be diagnostics of clade-specific MTC evolution.
76
ESM 2009
PP-3
ASSOCIATION BETWEEN BEIJING GENOTYPE AND DRUG
RESISTANCE AMONG Mycobacterium tuberculosis
ISOLATES CIRCULATING IN THE REPUBLIC OF GEORGIA
Stefan Niemann1, G. Khechinashvili2, M. Gegia2, N. Mdivani2, and Y. W. Tang3
Molecular Mycobacteriology, National Reference Center for Mycobacteria, Research Center Borstel, Borstel, Germany
Georgian Foundation against Tuberculosis and Lung Diseases, Tbilisi, Georgia
Vanderbilt University Medical Center, Nashville, TN, USA
Rising tuberculosis (TB) rates and high levels of multidrug-resistant TB (MDR-TB) have become a major public health
problem in several parts of the former Soviet Union. High rates and transmission of MDR-TB have been noticed to be associated with the presence of Mycobacterium tuberculosis Beijing genotype strains pointing to the importance of pathogen
genetic factors for the modulation of infection outcome and epidemiology. Here we present the first data on the population structure of M. tuberculosis strains from the Republic of Georgia, a high incidence setting at the Black Sea Coast.
All strains were analysed by spoligotyping and 24-loci MIRU-VNTR genotyping. Identification of major M. tuberculosis
genotypes was carried out by using the MIRU-VNTRPlus database (www.miru-vntrplus.org) and a similarity analysis
performed to identify strains with identical genotyping profiles (clusters), which are indicative for the rate of recent
transmission. Anti-tuberculosis drug resistance was determined by in vitro antimicrobial susceptibility testing.
Genotyping profiles were successfully generated for 187 M. tuberculosis isolates which were further investigated.The most
prominent genotype found, was Beijing (29%), followed by LAM (18%), Ural (12%), and Haarlem (n=10) strains. Approx
50% of the isolates were grouped in clusters. When the distribution of drug resistance is considered, it was noticed that
MDR-TB was nearly completely restricted to Beijing strains. Further detailed analyses are currently in progress.
Our data underline the importance of Beijing genotype strains for the TB epidemiology in former Soviet Union countries.
However, Ural, Haarlem genotype and a large variety of strains of so far undefined lineages represent nearly two third of
the strains found in Georgia. Although Beijing strains are not as dominant as in others Eastern European countries such
as Kazakhstan, we confirm a clear association between MDR-TB and the Beijing genotype in Republic of Georgia.
European Society of Mycobacteriolog y | 30 th Annual Congress | July 2009 | Por to - Por tugal
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PP-4
Mycobacterium tuberculosis EPIDEMIOLOGY AND GENETIC
DIVERSITY IN THE TWIN ISLAND REPUBLIC OF TRINIDAD AND TOBAGO
Shirematee Baboolal, 1, 2 Julie Millet,3 Patrick Eberechi Akpaka, 1 Dottin Ramoutar, 4 Nalin Rastogi 3
1 - Department of Para-Clinical Sciences, Faculty of Medical Sciences, The University of the West Indies, St. Augustine, Trinidad & Tobago
2 - Caribbean Epidemiology Centre, Jamaica Boulevard, Port of Spain, Trinidad & Tobago
3 - Unité de la Tuberculose et des Mycobactéries, Institut Pasteur de Guadeloupe, Abymes, Guadeloupe
4 - Caura Chest Hospital, Caura, Trinidad & Tobago
This work describes the first application of molecular tools for studying tuberculosis (TB) epidemiology, genetic diversity,
and transmission in the twin island Republic of Trinidad and Tobago (T&T). The study population (n=132) represented
one year recruitment of all culture positive TB cases from T&T, and was characterized by a high male to female sex-ratio
of 4 (mean age 42.8 years, range 17 to 78 years), and a HIV/TB coinfection rate of nearly 30%. It mainly occurred among
African descendants who represented 37.5% of total population but 69.7% of all TB cases (p<0.001). Spoligotyping
resulted in 25 different patterns and 12 clusters (2 to 74 strains per cluster). In total, 81.3% of the isolates in our
study was defined as modern tubercle bacilli belonging to the Principal Genetic Groups 2/3. Five major lineages were
observed: East-African Indian (EAI), Latin-American and Mediterranean (LAM), X, Beijing, and Haarlem. A comparison
with international SITVIT2 database showed that the overall lineage distribution in T&T was completely different from
other Caribbean neighbors (n=2653 isolates). A high clustering rate of 90% was observed essentially due to a single large
cluster of 74 strains designated as Spoligotype International Type - SIT566 (T&T clone). Patients harboring this genotype
were overrepresented in St George Central, the capital city of Port-of-Spain, younger (mean 39.1 years vs. 47.7 years
for other genotypes, p<0.0005), and more frequently prison inmates and drug users, while those harboring “other genotypes” were older and showed diabetes as an associated factor. A study of the evolutionary relationships suggested probable relatedness of SIT566 with X1 prototype SIT119. A database search localized most of the SIT566 related patterns
in USA. Second-line typing using Mycobacterial Interspersed Repetitive Units (MIRUs) suggested the highly conserved
nature of SIT566, its phylogeographically specificity to T&T.
Acknowledgements
We thank the Caribbean Epidemiology Center, Trinidad & Tobago for support, and the staff of the Chest Clinics at the
Eric Williams Medical Sciences Complex, the San Fernando General Hospital, and Trinidad Public Health Laboratory for
assistance with data collection. SB is grateful to the University of West Indies for financial support, and JM to the Regional
Council of Guadeloupe and European Social Funds for a Ph.D. fellowship. NR and JM thank J. Driscoll for providing information regarding the SIT566 clone in USA.
78
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PP-6
SPOLIGOTYPE PATTERNS AND DRUG RESISTANT
PROFILE OF Mycobacterium tuberculosis IN SUDAN
Ghada Suliman Sharaf-Eldin 1, Imad F.Elmoula 1, Mohammed S. Ali 1, Nageeb S. Saaed 2,
Ahammed B Ali 1, Kim Mallard 3, Ruth McNerney 3, Saad Algamdi 3
1 - Al Neelain University-Sudan
2 - National Health Laboratory-Sudan
3 - London School of Hygiene & Tropical Medicine.
Sudan has a high burden of tuberculosis with an estimated 93,000 new cases each year. The purpose of this
study was to investigate the genotypic patterns of M. tuberculosis strains circulating in Sudan and to assess
their susceptibly to anti-tuberculosis drugs. Isolates from 237 smear positive tuberculosis patients were collected from different geographic regions of the country. Spoligotyping was performed by the Kamerbeek
method and results were compared with the international SpolDB4 database (Institut Pasteur, Guadeloupe).
Results revealed 28 clusters ranging in size from 12 to 57 isolates. Seventy unique (unclustered) strains were observed,
representing 30% of the strains examined.The most frequently observed spoligotype patterns belonged to the CAS family which represented 115 (48.5%) of isolates studied. T1, H3, U and Beijing strains were found in 12 (5.1%), 11 (4.6%),
7 (3%) and 6 (2.5%) patients respectively. Strains belonging to the Beijing family were found mainly in Western Sudan.
Resistance to isoniazid, rifampicin, ethambutol and streptomycin was observed in 18.1, 22.4, 22.2 and 32% of strains respectively. Twenty patients (8.4%) had MDR-TB of which 10 were new cases. Seventeen patients with rifampicin resistant
tuberculosis were infected with CAS1-DELHI strains matching SIT 25 of the SpolDB4 database and 3 were of the SIT
1 Beijing family. 15 loci MIRU-VNTR typing subdivided the 17 CAS strains into one cluster of 5, two clusters of 2 and
8 individual MIRU types. Similarly the 3 Beijing spoligotypes were differentiated into a cluster of 2 and a single strain. The use of molecular strain typing provides a proactive approach that may be used to initiate, and not just augment,
traditional surveillance outbreak investigation in Sudan. However, caution must be used when interpreting clustered
spoligotype patterns in this region.
European Society of Mycobacteriolog y | 30 th Annual Congress | July 2009 | Por to - Por tugal
79
PP-7
CONTROVERSIAL DISSEMINATION PATTERN OF THE BULGARIA
-SPECIFIC M. tuberculosis SPOLIGOTYPE ST125_BGR
Violeta Valcheva 1, 2, Igor Mokrousov 1, 3, Stefan Panaiotov 4, Elizabeta Bachiiska 4, Thierry Zozio 1, Christophe Sola 1, Nadya
Markova 2, Nalin Rastogi 1
1 - Institut Pasteur de Guadeloupe, France
2 - Institute of Microbiology, Sofia, Bulgaria
3 - St. Petersburg Pasteur Institute, St. Petersburg, Russia
4 - National Center of Infectious and Parasitic Diseases, Sofia, Bulgaria
Objective
To investigate phylogenetic position and geographic genetic diversity of spoligotype ST125 in Bulgaria.
Material and Methods
Study sample included all available 47 DNA samples belonging to spoligotype ST125 that were taken from two previously
published M. tuberculosis collections from Bulgaria (Valcheva et al., 2005, 2007, 2008abc; Panaiotov et al., 2005, 2006).
These DNA were additionally typed using new 24-loci MIRU format, IS6110-RFLP typing and LAM-PCR. Phylogenetic
analysis was done using PAUP and PHYLIP packages.
Results
Comparison with SITVIT2 database (Institut Pasteur de Guadeloupe) revealed a high gradient of ST125 in Bulgaria
(14.3%) compared to its negligible presence in the world. Typing of Bulgarian ST125 strains revealed that they: (i) did
not harbor a LAM-specific IS6110 insertion (ii) formed a monophyletic cluster in 24-MIRU tree of Bulgarian strains (iii)
grouped closely with ST34 that is a prototype of the S family. A similarity of the IS6110-RFLP profiles confirmed a true
relatedness of these ST125 strains whereas a diversity of the MIRU loci suggested a long-term evolution of this spoligotype in Bulgaria. Minimum spanning tree of the 24-MIRU-based subtypes of ST125, and comparison with their geographic
distribution revealed an enigmatic and complex dissemination pattern of this spoligotype across Bulgaria.
Conclusion
T125 likely belongs to S family and may have originated from spoligotype ST4 by a deletion of a single spacer #40 in the
DR locus. ST125 is phylogeographically specific for Bulgaria; we propose its renaming as ST125_BGR. A high diversity of
the MIRU loci suggests a long-term evolution of this spoligotype in Bulgaria.
Acknowledgments.
This work was supported by NATO grant SFP-982319 “Detect drug-resistant TB”.
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PP-8
Mycobacterium tuberculosis BEIJING GENOTYPE
AND ORIGINS OF THE BULGARIANS
Panaiotov, Stefan; Bachiyska, Elizabeta; Brankova, Nadia; Levterova,Victoria
National Center of Infectious and Parasitic Diseases, Sofia 1504, Bulgaria
Recent studies demonstrated that exist genotypes of M. tuberculosis locally distributed to specific geographic region.
Other genotypes are distributed globally or on vast geographic areas. These facts led to other recent fundamental studies and conclusions that exists certain genetic predisposition of the ethnic groups to specific M. tuberculosis genotypes,
hence genetic predisposition to tuberculosis specific genotypes is suspected. In the past and nowadays the waves of human migration coincide with expansion of tuberculosis genotypes. In our study we associated the global distribution of
MTB Beijing genotype, its’ distribution in Bulgaria, the geographic regions of historical origin of the Bulgarian tribes, and
the ethnic affiliation of the Bulgarians. For our analysis we used data published in the fourth international spoligotyping database (SpolDB4), publications, personal communications and official historical theories regarding origins of the Bulgarians.
From the literature and in SpolDB4 exists about 330 spoligotypes of Bulgarian M. tuberculosis clinical strains collected
from all over the country. Beijing spoligotype was not identified among them. We concluded that this MTB genotype is
not (or very rare) distributed in Bulgaria. In Romania Beijing genotype was not identified too (personal communication).
Other countries associated with the origins and migration of the Bulgarians where Beijing genotype is not identified is
Iran. Significant part of the Bulgarian spoligotypes are phylogenetically related to MANU family, widely distributed in India.
These facts correlate with the widely accepted theory that the origins of the Bulgarian tribes are of Indo-Iranian lineage.
In contrary, this fact does not support the theory for the Turkik lineage or more precisely the Turano-Hunnic origins of
the Bulgarian ethnos.
Beiging genotype is widely distributed in Central Asian countries, Kazahstan, Turkmenistan, Russia, Turkey, China etc.
Bulgaria has very active tourist, cultural, political, trade and immigration links with all these countries. Based on these
empiric facts we indirectly conclude that there is genetic predisposed resistance of the Bulgarian and Iranian ethnos to
MTB Beijing genotype.
European Society of Mycobacteriolog y | 30 th Annual Congress | July 2009 | Por to - Por tugal
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THE EXTENT OF THE LATIN AMERICAN-MEDITERRANEAN
Mycobacterium tuberculosis SPOLIGOTYPE FAMILY IN PORTUGAL
Suzana David 1, João Nuno Ribeiro 1,2, José-Nuno Maio 1,2, Inês João 1, António Amorim 3, Edna Pereira 3
1 - Unidade de Referência e Vigilância Epidemiológica, Laboratório de Infecções Respiratórias – Micobactérias, Instituto Nacional de Saúde Dr. Ricardo Jorge, Portugal
2 - Grupo de Microbiologia e Imunologia da Infecção, Instituto de Biologia Molecular e Celular,
Porto, Portugal
3 - Laboratório de Saúde Pública, Micobacteriologia / Tuberculose, Administração Regional de Lisboa e Vale do Tejo, Lisboa, Portugal
Portugal is classified at the intermediate level with respect to the incidence for tuberculosis. Geographical spread is
heterogeneous with the majority of cases concentrated in the large urban areas mainly in the Lisbon and Porto districts.
Previous efforts in the characterization of the population structure of Mycobacterium tuberculosis isolates stressed the
importance of this approach to tuberculosis control. Spoligotyping data restricted to the metropolitan area of Lisbon,
revealed a 51% prevalence of the Latin American-Mediterranean (LAM) spoligotype family, and a high proportion of SIT20
(LAM1) and SIT42 (LAM9) sub-families. In the present study this characterization has been extended to encompass both
the Lisbon and Porto areas. With the use of complementary data from SpotClust and Ag85C103 RFLP, the proportion of
the LAM genotype was shown to be as high as 62% of the total number of isolates. The LAM genotypes were further
characterized for the RDRio deletion detected in up to 60% of the LAM isolates. This study suggests that Portugal may
have one of the highest global proportions of the LAM family. Monitoring and further characterizing of this relevant spoligotype family is considered of major importance for the understanding of the dynamics of tuberculosis in Portugal.
82
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PP-11
FITNESS STUDY OF THE RDRIO LINEAGE AND LAM FAMILY OF
Mycobacterium tuberculosis IN A STUDY POPULATION
IN RIO GRANDE, BRAZIL
Von Groll, Andrea 1;
Martin, Anandi 1; Felix, Carolina 2; Prata, Pedro 2; Honscha, Günther 3; Portaels, Françoise 1; Almeida da Silva, Pedro 2;
Palomino, Juan Carlos 1
1 - Institute of Tropical Medicine, Antwerp, Belgium
2 - Universidade Federal do Rio Grande, Rio Grande, Brazil
3 - Laboratório Municipal de Tisiologia, Rio Grande, Brazil
RDRio is a novel Mycobacterium tuberculosis lineage member of the Latin American-Mediterranean (LAM) family. LAM has
been found worldwide but it is more predominant in South America. The aim of this study was to assess the presence
of the RDRio lineage and LAM family in the city of Rio Grande, Brazil, and to investigate the fitness of these strains based
on the determination of their rate of growth. Fifty clinical isolates of M. tuberculosis were genotyped and 43 different patterns were found by spoligotyping and MIRU-VNTR.The predominant genotypes belonged to the LAM family (54% of the
strains) followed by clade T (22%) and Haarlem (16%). The RDRio lineage represented 38% of the total strains and 70.4%
of the LAM strains found in this study. Strains belonging to the LAM family showed a fitness advantage comparing their
rate of growth with that of non-LAM. RDRio strains were predominant within the LAM family, but a significant difference
in fitness between RDRio and the non-RDRio strains was not confirmed.
European Society of Mycobacteriolog y | 30 th Annual Congress | July 2009 | Por to - Por tugal
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Genomic characterization of Lisboa
family strains by deletion analysis
João Perdigão, Carla Silva and Isabel Portugal
Centro de Patogénese Molecular, URIA, Faculdade de Farmácia da Universidade de Lisboa
Multidrug and extensive drug resistant tuberculosis poses a very serious threat for public health. Lisbon Health Region
has one of the world’s most serious situations regarding this problem. Such, is the result of a continued circulation of an
endemic and predominant strains of a particular genetic family – Lisboa family. Little is known regarding the phylogeny,
relative virulence and genetic background of these strains. The loss or deletion of specific genomic regions constitutes
the most important way by which Mycobacterium tuberculosis diverges and adapts. Several deletions, named Regions
of Difference (RD), have already been described and associated with phylogeographic lineages. The characterization of
Lisboa family in this manner may elucidate its origin and perhaps be helpful in explaining its high prevalence.
Three representative clinical isolates of different genetic clusters of strains circulating in Lisbon Health Region were
screened for the presence of 16 distinct RDs. Deletion detection was performed by PCR carried out using primers flanking each RD. Confirmation was performed by sequencing analysis.
All three isolates were found to possess four of the tested deletions: TbD1, pks15/1, RD174 and RDRIO. It was not possible to discriminate between the strains using this deletion typing approach. However, it was possible to infer on the
phylogeography of these strains. The presence of TbD1 and pks15/1 deletion positions the strains in the modern and
Euro-American lineage, respectively. On the other hand, RD174 suggests that the analyzed strains are related to the
West-African sub-lineage.
The present study point toward the fact that Lisboa strains and others circulating in Lisbon belong to Mycobacterium
tuberculosis modern lineages of the Euro-American lineage,West-African sub-lineage, therefore revealing more on Lisboa family’s origin.
84
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PP-13
IMPORTANCE OF MOLECULAR TYPING IN SUSPECTED
INTRA-FAMILIAL TRANSMISSION OF TUBERCULOSIS
Obrovac, Mihaela 1, Katalinic-Jankovic,Vera 1, Grce, Magdalena 2, Zmak, Ljiljana 1
1 - Croatian National Institute of Public Health
2 - Rudjer Boskovic Institute
Tuberculosis (TB) is most commonly transmitted from a person suffering from infectious pulmonary TB to another person
by infected droplet nuclei. The chance that a person will be infected with TB depends on the intensity, frequency and duration of the exposure to tubercle bacilli. Also, numerous studies emphasize the importance of host resistance and hereditary
susceptibility, indicating that the development of TB is a result of a complex interaction between the host and the pathogen
influenced by environmental factors. Assuming that there is prolonged duration and frequency of contact between family
members and among household contacts, it is estimated that the risk of TB transmission will be high. There are several
studies confirming intra-familial transmission using genotyping of isolated M. tuberculosis strains. However, the possibility of
unsuspected transmission should not be disregarded.We report here of two cases of suspected TB transmission in families
caused by M. tuberculosis strains that were found not to be identical according to genotyping profiles obtained by determining variabile number of tandem repeats (VNTR) of mycobacterial repetitive interspersed units (MIRU). Genotyping was performed using complete set of 24 VNTR loci. Epidemiological data were collected by contact tracing and interviewing patients. Our results show that TB cases in family do not necessarily have to be caused by the same M. tuberculosis strain. Epidemiologic
investigations need to be combined with genotyping data for better understanding of transmission dynamics.Transmission of
TB cannot be confirmed by contact investigation only, even when intra-familial transmission is suspected.
European Society of Mycobacteriolog y | 30 th Annual Congress | July 2009 | Por to - Por tugal
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DETECTION OF CLONAL COMPLEXITY IN CLINICAL M. tuberculosis
ISOLATES BY MIRU-VNTR IN CUKUROVA REGION, TURKEY
Ülkü ORAL ZEYTINLI, M. Begüm KAYAR, Ayse KARACALI, Arzu SAHAN KIPALEVErkan YULA, Fatih KÖKSAL
University of Cukurova
Purpose of the study: Tuberculosis remains one of the most prevalent infectious disease in the world . The application of molecular typing methods to the analysis of clinical Mycobacterium tuberculosis (MTB) complex isolates has
greatly facilitated the understanding of epidemiology of tuberculosis (TB) and revealed that the infection by this pathogen can be clonally complex and reinfection, coinfection. Genotyping using RFLP-IS6110 (Restriction Fragment Length
Polymorphism) is based on transposable element IS6110 and mycobacterial interspersed repetitive unit-variable number
of tandem repeat typing (MIRU-VNTR) has become a major method for epidemiological tracking of Mycobacterium
tuberculosis complex clones. Our aim was to establish the range of applicability of 12 loci MIRU–VNTR genotyping in
epidemiology of TB and evaluate the discriminatory power obtained with RFLP-IS6110 and MIRU-VNTR used alone or
in combination. Methods
In this study, we analyzed 94 clinical MTB complex isolates from sputum in patients with pulmonary TB between February
2008- February 2009 in Cukurova region, Turkey.
Results
MIRU–VNTR typing detected 45 different patterns, 61 strains were grouped into 12 clusters and 33 strains had uniqe
patterns. The largest cluster comprised 9 strains. In addition, 2 clusters contained 5 strains, 6 clusters contained 3 strains
and 1 cluster contained 2 strains. It is also determined that the loci including MIRU 04, MIRU 10, MIRU 26 and MIRU 40
have the highest allelic diversities and discriminatory power. On the other hand with IS6110-RFLP typing of same clinical MTB strains, 33 genotypes were founded. 76 strains of which were gruoped into15 clusters. 18 of the 94 strains had
unigue RFLP patterns.
Conclusion
MIRU-VNTR is more discriminative methods for phylogenetic studies than IS6110-RFLP and could define from
reinfections to reactivations important to treatment of MTB complex organisms.
86
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PP-15
MOLECULAR EPIDEMIOLOGY STUDY OF TUBERCULOSIS PATIENTS
IN A SMALL CITY OF SÃO PAULO – BRAZIL, FROM 2002 TO 2006
Leite, Clarice; Santos, Adolfo; Pandolfi, José Rodrigo; Malaspina, Ana C; Pavan, Fernando; Mendes, Natália
Universidade Estadual Paulista, Faculdade de Ciências Farmacêuticas
The molecular epidemiology study using different techniques revolutionized the understanding of the epidemiology of
tuberculosis allowing comparison between strains of Mycobacterium tuberculosis and tracking the movements of individual
lines. This project aimed to use the techniques of molecular epidemiology (MIRU) trying to understand more about the
phenomenon of transmission of tuberculosis among patients with pulmonary tuberculosis in a small city of São Paulo
- Brazil, attended the Special Health Service of Araraquara (SESA - clinic of reference in the diagnosis and treatment of
tuberculosis) in the city of Araraquara. From total 163 positive cultures received, the MIRU technique was performed in
74,2% (121/163) of the isolates from patients attended by SESA in the period from 2002 to 2006. 5 isolates were also
identified as environmental mycobacteria and 4 unidentified mycobacteria. From the 121 isolates submitted to genotyping, six did not present all alleles among the 12 loci of MIRU. From the 115 isolates submitted to the dendrogram, 29
(25,2%) are grouped into 13 clonal groups with similarity of 100%. 10 groups with two isolates each and 3 groups containing 3 isolates each.The others 86 isolates (74,8%) had a single genetic profile. About the group of 29 patients, only 10
were female and 19 males. Except for one patient, the other 28 were treated with the schedule I having cure in 82,8% of
the cases. Whereas the similarity of 83% or greater, highlighted 3 major clonal groups called A, B and C, involving 86 of
all isolates analyzed. In these large groups were included all 13 clonal groups with 100% similarity. These data suggest the
possibility the tuberculosis in Araraquara is because of the presence of persistent endemic strains responsable for 74,8%
of cases, besides the existence of recent transmission in this work was 25,2%.
Keywords: Epidemiology, Tuberculosis, MIRU
Financial support: FUNDUNESP.
European Society of Mycobacteriolog y | 30 th Annual Congress | July 2009 | Por to - Por tugal
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GENOTYPING OF Mycobacterium tuberculosis
IN NORTHWEST OF PARANÁ STATE OF BRAZIL
Leite, Clarice Queico Fujimura 1, Nogutia, Erika N 2, Malaspina, Ana Carolina 1, Santos,
Adolfo Carlos Barreto 1, Hirata, Rosáro DC 3, Hirata, Mário H 3, Cardoso, Rosilene Fressatti 2
1 - São Paulo State University
2 - Maringá State University
3 - University of São Paulo
Introduction
Worldwide, tuberculosis (TB) remains the most frequent and important infectious disease causing morbidity and death.
The molecular epidemiology study using different techniques revolutionized the understanding of the epidemiology of
tuberculosis allowing comparison between strains of Mycobacterium tuberculosis and tracking the movements of individual lines.We reported here the first insight about the genetic diversity of M. tuberculosis in northwest of Paraná State,
south of Brazil. This knowledge encourages additional prospective epidemiological study for evaluation of the Regional
Tuberculosis Control Plan in this setting.
Objectives
Provide information about the genetic diversity and prevalent genotype of Mycobacterium tuberculosis and compare the
usefully of two methodologies in epidemiological study of tuberculosis in low endemic area in south of Brazil. Material
and Methods: We used spoligotyping and MIRU-VNTR typing to genotype M. tuberculosis isolates.
Results
The 93 isolates analyzed by spoligotyping were divided into 36 different patterns and 25 were described in the SpolDB4.0
database. Latin American and Mediterranean, Haarlem and T family were responsible for 26.9%, 17.2% and 11.8%, of
tuberculosis cases respectively. From the 84 isolates analyzed by MIRU-VNTR typing, 58 showed unique pattern and 26
belonged to 9 clusters. The MIRU loci 40, 23, 10 and 16 were the most discriminatory. MIRU-VNTR and spoligotyping
combined showed 85.7% of discriminatory power (HGI=0.995).
Conclusions
Spoligotyping and MIRU-VNTR typing combined are useful tool for epidemiological study in this low endemic setting in
south of Brazil and tuberculosis predominantly develops through reactivation of latent infection.
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PP-17
SPOLIGOTYPING OF Mycobacterium tuberculosis ISOLATED FROM
PATIENTS OF CLEMENTE FERREIRA AMBULATORY IN SÃO PAULO, SP – BRAZIL
Mello, Fernado Augusto Fiuza 1, Albarral, Maria Idemar Pedrosa 1, Mendes, Natália Helena 2, Pandolfi, José Rodrigo
Cláudio 2, Santos, Adolfo Carlos Barreto 2, Almeida, Elisabete Aparecida 1, Cardoso, Rosilene Fressatti 3, Leite, Clarice
Queico Fujimura 2
1 - Clemente Ferreira Institute
2 - São Paulo State University
3 - Maringá State University
The molecular epidemiology study using different techniques revolutionized the understanding of the epidemiology
of tuberculosis allowing comparison between strains of Mycobacterium tuberculosis and tracking the movement of
individual strains. This project aims to use the technique of molecular epidemiology, Spoligotyping, trying to understand
more about the phenomenon of transmission in patients with pulmonary tuberculosis treated at Clemente Ferreira
Ambulatory (ambulatory of reference for the treatment of tuberculosis) in São Paulo city, from August 2006 to July 2008.
The clinical isolates were re-identified by molecular technique (PCR and PRA), and the strains identified as M. tuberculosis conducted by the genotyping technique of Spoligotyping. From 102 isolates, the technique of IS 6110-PCR confirmed
the identification of M. tuberculosis in 96 clinical isolates and the PRA in 99, the remaining 3, 2 isolates identified as M.
avium subtype 2 and 1 unidentified mycobacteria. The results showed that 3 clinical isolates of M. tuberculosis had not
the IS6110 insertion sequence specific of M. tuberculosis, as well as 3 isolates identified in the clinic as M. tuberculosis
by molecular techniques were atypical mycobacteria. Of 96 isolates confirmed as M. tuberculosis, were analyzed by the
technique of Spoligotyping, a total of 89 isolates, which revealed the presence of 21 strains (23.6%) with spoligotipes not
yet described in the data base world (spolDB4) and 68 (76.4%) of isolates involved in 7 different families, containing 2 to
30 isolates. The most frequent was T family with 30 isolates), followed by LAM (with 20 isolates) and Haarlem (with 10
isolates), which together accounted about 67.4% of all isolates. Were also identified 4 genotypes of the Beijing family, all
simultaneously resistant to isoniazid and rifampicin and / or more drugs.
European Society of Mycobacteriolog y | 30 th Annual Congress | July 2009 | Por to - Por tugal
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COMPARISON OF Mycobacterium BEIJING
GENOTYPE WITH VNTR, SPOLIGOTYPING AND RFLP-IS6110
Elahe Tajeddin , Parissa Farnia, Mohammad Kargar,Jamileh Noroozi, Mojtaba ahmadi,
Mehdi kazempour, Maryam Hadadi,Mohammadreza Masjedi, Aliakbar Velayati
Mycobacteriology Research Center (MRC) National Research Institute Of Tuberculosis and
Lung Disease (NRITLD), Shahid Beheshti University Medical Campus.Tehran,Iran.
Background
Beijing strains constitute more than 1/4 of Mycobacterium tuberculosis (MTB) genotypes. Beijing genotype is considered
an important genotype because of its reasonable characteristics such as: association with multi-drugs resistance TB.
Accordingly these strains are reluctant to conventional TB drugs.Therefore, it is necessary to investigate the transmission
rate among Beijing strains within the studied communities. In this study, three molecular methods (Spoligotyping,VNTR,
and RFLP-IS6110) were used to identify transmission among patients infected with Beijing strains.
Materials and Methods
The susceptibility tests were performed on 238 M. tuberculosis culture positive specimens. Thereafter, the isolated Beijing
genotype was subjected to VNTR and RFLP. The results of Spoligotyping were analysed by using SPOLDB4 database.
VNTR typing was used to identify alleles diversity in 9 locus (MPTR-A, ETR-A, ETR-B, ETR-C, ETR-D, ETR-E, ETR-F,
QUB11B, QUB3232) of isolated Beijing strains.The allelic diversity of VNTR was measured by using Hunter Gaston
Index (HGI).
Results
The spoligotyping of M. tuberculosis isolates revealed the following 8 patterns: Haarlem (27.7%), CAS1 (25.2%), EAI3
(21.8%), CAS2 (6.7%), T1 (6.3%), Beijing(5.5%) U(5%), T(0.4), EAI2 (1.2%).
The following VNTR loci (QUB3232), (QUB11b, ETR-E and ETR-F) and (other loci) were identified as most (HGI≥ 0.6),
median (HGI≥0.4-0.6) and weakest (HGI=0) distinctive loci for Beijing families respectively. Whereas the Beijing strains
demonstrated diverse patterns in RFLP,13/13(100%) and VNTR 10/13(77%).
Conclusions
Beijing is one of the dominated circulating strains in Iran and interestingly majority of infected cases were due to reactivation rather than recent transmission.The VNTR and spoligotiping methods were more efficient to detect Beijing strains
than by use VNTR and RFLP allow.
Keywords
Spoligotyping / VNTR / RFLP / Mycobacterium tuberculosis Beijing genotype.
90
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PP-19
MULTIPLY RECURRENT TUBERCULOSIS IN A PACIENT
LIVING WITH HIV: REINFECTION OR REACTIVATION?
Ritacco,Viviana 1, Reniero, Ana 2, Beltrán, Marcelo 2, López, Beatriz 1, Kantor, Isabel 3, Barrera, Lucía 1
1 - ANLIS, CONICET, Argentina
2 - Hospital Municipal de San Isidro
3 - PAHO/WHO consultant
Purpose
To determine the cause of recurrent clinical episodes of tuberculosis in a patient living with HIV. Patient: Male, 24 year-old
and illegal drug intravenous user for 10 years at the time of first consultation, assisted between 1995 and 2009 in our
outpatient clinic, San Isidro Municipal Hospital, Argentina.
Methods
Clinical-epidemiological follow up. Mycobacterial culture, identification and drug susceptibility testing. Mycobacterium
tuberculosis IS6110 RFLP and spoligo genotyping within the frame of a population-based study performed in San Isidro,
an outskirt of Buenos Aires City.
Findings
Our patient’s first diagnosis of tuberculosis was confirmed by culture in 1995, almost simultaneously with HIV serological
conversion. At that time, the isolate was found only resistant to isoniazid and its genotype matched that of a isoniazidresistant outbreak strain previously identified in two of our patient’s prison inmates, who were also members of his
intravenous-drug-user gang. One year after cure, our patient suffered from a relapse of his tuberculosis due to the same
strain, which now had added resistance to rifampicin and lost a band in the IS6110 fingerprint. In 2002, the patient suffered from a third episode of tuberculosis, this time due to a fully drug susceptible strain, identified previously in other
of our patient’s intravenous drug user friends. At that time, given his poor clinical condition, our patient underwent a
prolonged hospitalization in the Muñiz Hospital, the epicentre of a large tuberculosis outbreak caused by the notorious
multidrug-resistant strain “M”. After successful treatment and cure, our patient, who was never compliant with antiretroviral treatment, was discharged from the Muñiz Hospital. A few months later, he sought again assistance at our clinic with
active tuberculosis due to the multidrug resistant strain “M”. He is currently struggling with a relapse of disease caused
by this outbreak strain.
Conclusion
Whether recurrent tuberculosis is due to a newly acquired infection or to reactivation of a previous one is a centurylong controversial question. In our case, both conditions alternated throughout the 14 years of the patient living with
HIV. Work partially funded by FP7 grants 201690 and 223373 of the EC.
European Society of Mycobacteriolog y | 30 th Annual Congress | July 2009 | Por to - Por tugal
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PP-20
MOLECULAR EPIDEMIOLOGY OF TUBERCULosis
IN VILA NOVA DE GAIA, PORTUGAL
Tavares Magalhães, Ana1,Alves, Adriana1, Braga, Rosário2,Valente, Isabel2, Duarte, Raquel3 and Miranda, Anabela1
1 - Tuberculosis Reference Laboratory, Department of Infectious Diseases, National Institute of Health, Porto, Portugal
2 - Central Hospital,Vila Nova de Gaia, Portugal
3 - Chest Clinic,Vila Nova de Gaia, Portugal
DNA fingerprinting of Mycobacterium tuberculosis �������������������������������������������������������������������
has provided a better understanding of the epidemiology of tuberculosis. Restriction fragment length polymorphism based on IS6110 (RFLP-IS6110) has been the gold standard for typing
M. tuberculosis since 1993. In recent years, mycobacterial interspersed repetitive units variable number tandem repeat
(MIRU-VNTR) has been proposed as a first-line typing method for M. tuberculosis. This technique generates easily analyzed and portable data, has a good discrimination power and has been proven useful for studying the epidemiology of
tuberculosis and the phylogeography of tuberculosis bacilli.
In the present study we evaluated the genetic diversity of M. tuberculosis clinical strains, isolated from 115 patients from
Vila Nova de Gaia, Portugal, during the period of 2004 to 2005, using the standardized MIRU-VNTR typing method based
on 15 loci, proposed by Supply and collaborators in 2006. Strain lineage designation, allelic diversity and clustering rate
were determined using the MIRU-VNTRplus identification database. The discriminative power of the method was analysed using the Hunter and Gaston diversity index.
Based on MIRU-VNTR typing, the 115 strains were divided into 62 types as follows: 69 strains were distributed into 16
clusters containing two to eight isolates, and 46 strains had unique profiles. MIRU-VNTR revealed a clustering rate of
46% in the sample under study. The Hunter-Gaston index was 0.976. The most discriminatory loci were Mtub04, MIRU
40, MIRU10, QUB11b, Mtub30, Mtub39 and QUB26 showing an allelic diversity higher than 0,6.
The phylogenetic analysis revealed the presence of two major lineages, LAM and Haarlem, representing 60% and 27% of
the total isolates, respectively. This fact is in agreement with the M. tuberculosis phylogeography for the South of Europe.
Fourteen strains showed drug resistance but no association was established between drug resistance profile and phylogenetic family.
MIRU-VNTR showed a good discriminatory power for typing of M. tuberculosis strains in this setting and the MIRUVNTRplus database �������������������������������������������������������������������������������������������������������
is an important tool for ������������������������������������������������������������������������������
lineage identification. This typing approach offers, simultaneously, epidemiologic and phylogenetic information, as demonstrated. Overall, this study reveals that recent transmission of tuberculosis
is high in Vila Nova de Gaia, and that import of strains in this region is not a problem.
92
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PP-21
MOLECULAR STUDY OF RECURRENT TUBERCULOSIS CASES
Alves, Adriana; Miranda, Anabela; Tuberculosis Reference Laboratory Group
Tuberculosis Reference Laboratory, Department of Infectious Diseases, National Institute of Health, Porto, Portugal
Tuberculosis recurrence is frequently attributed to reactivation of the isolate responsible for the first episode of the
disease. Nevertheless, this can also be due to an infection with another isolate, or to mixed infections. Clarification of
the cause of recurrence is very important and can be achieved by molecular typing of serial isolates of M. tuberculosis.The
most appropriate methods to do so are IS6110 RFLP and MIRU-VNTR.
In this work, 39 clinical isolates of M. tuberculosis belonging to nine individuals with recurrent disease were studied
throughout time. Seven of these patients were resistant to isoniazid and rifampicin as well as to most other 1st and 2nd
line drugs. Another patient was resistant to isoniazid and streptomycin, and the last one was resistant to rifampicin only.
For some of these individuals, resistance to drugs worsened during the cause of the disease, which in some cases has
lasted for more than a decade. All 39 strains were analyzed by IS6110 RFLP. MIRU-VNTR was used to type: (i) the first
strain of each patient when serial isolates showed no change in the IS6110 RFLP profile; or (ii) each strain with a pattern
change in relation to the previous one.
RFLP results showed that only one out of nine patients displayed a change in the pattern of serial isolates with gain of
one IS6110 element. Analysis of the results using Bionumerics grouped these patients in nine clusters, being that: (i) strains
from two patients belong to the same cluster; and (ii) strains of one patient are divided in two clusters. Results of 15
MIRU-VNTR loci typing corroborate the IS6110 RFLP findings. In other words, the serial isolate that gained one IS6110
element also shows a change in MIRU-VNTR results. In this case, we observed a reduction of one repeat in loci Mtub21
and QUB11b. Finally, 40% of these strains belong to the LAM family, 10% to the Haarlem family, and the remaining 50%
did not find a match in the database.
This work shows that the cause of recurrent tuberculosis of the nine patients included in this study is due to persistence
of initial M. tuberculosis strain or to reactivation when there is more than one episode of disease. Antibiotic resistance is
the most important cause of chronic tuberculosis in these patients, since all analyzed strains are resistant to the majority
of the 1st and 2nd line drugs.
European Society of Mycobacteriolog y | 30 th Annual Congress | July 2009 | Por to - Por tugal
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GENOTYPING OF DRUG RESISTANCE IN Mycobacterium
tuberculosis USING DIAGNOSTIC MICROARRAYS
Ehricht, Ralf 1, Slickers, Peter 1, Monecke, Stefan 2
1 - CLONDIAG, Jena, Germany
2 - University Hospital Dresden
Tuberculosis is a disease of worldwide concern. Antimicrobial resistance in Mycobacterium tuberculosis is an increasing
challenge and, in contrast to other bacteria, not caused by the acquisition of certain genes, but by acquisition of single
point mutations in genes which are present in all strains. Unfortunately, culturing and subsequent growth inhibition assays
are still time demanding preventing fast detection and treatment. Genotyping methods as PCR followed by sequencing
are an alternative. Here the bottlenecks are processing time, overall costs and lack of parallelisation. Hybridisation of
such PCR products against highly discriminatory oligonucleotide probes is an alternative approach which could solve
these problems. We developed an assay using a diagnostic oligonucleotide microarray and covering probes for relevant
mutations in genes rpoB, katG, embA, and embB, for the embC/embA-intergenic region, and the mabA/inhA promoter.
PCR is employed to amplify these genes and to incorporate biotin 16-dUTP during elongation. These labeled amplicons
are hybridised to the array which are inserted into ArrayStrips, and hybridisation is visualised using dye precipitation triggered by streptavidin-peroxidase complexes bound to the biotin labels and the ArrayMate reading device.The procedure
is currently tested using DNA from previously characterized strains for which conventional susceptibility test results
and relevant sequences are available.
94
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PP-23
GENOTYPING OF MONO AND MULTI-DRUG RESISTANCE TB IN SAUDI ARABIA
Sahal Al-Hajoj1, Bright Varghese1, Mais Herbawi1 , Ruba Al-Omari1 and Caroline Allix-Béguec2
1 - King Faisal Specialist Hospital and Research Centre, Comparative Medicine Tuberculosis Research Unit Saudia Arabia
2 - Genoscreen
A total of 150 isolates collected from different regions in Saudia Arabia were the subject of finger printing using GenoScreen
MIRU-VNTR kit (do you know the timeframe?). Upon genotype the data were compared to the international MIRUVNTRplus database (www. Miru-vntrplus.org). The data showed that Saudia Arabia harbors the following major clades EAI
13.07%, Haarlem 12.42%, TUR 13.07%, Beijing 12.42%, unknown12.42%, Dehli/CAS 7.84%, LAM 7.19%, Cameroon 6.54%,
UgandaI 5.23%, S 3.92%, multiple matches1.96%, NEW-1 1.31%, URAL 0.65%, Ghana 0.65%, X, 0.65%, UgandaII, 0.65%. Indicate
the clustering rate for this panel of 150 isolates. Ongoing transmission may be an indication to the need of improving TB
control program. The unknown clades represent a considerable % (12.42%). These could represent unique endogenous
clades. However, further analysis is needed to gain insight into the nature of the unknown clades.
European Society of Mycobacteriolog y | 30 th Annual Congress | July 2009 | Por to - Por tugal
95
PP-24
early detection of MDRtb by molecular tools in the control
of drug resistant tuberculosis in portugal: a case of success
Diana Machado1, Miguel Viveiros1,2, Liliana Rodrigues1,3, Isabel Couto1,4, Leonard Amaral1,2,3
1 - Unit of Mycobacteriology, Instituto de Higiene e Medicina Tropical, Universidade Nova de Lisboa (IHMT/UNL), Lisbon, Portugal
2 - COST ACTION BM0701 (ATENS)
3 - UPMM, IHMT/UNL, Lisbon, Portugal
4 - Centro de Recursos Microbiológicos (CREM), Faculdade de Ciências e Tecnologia, UNL, Caparica, Portugal
Multidrug resistant tuberculosis (MDRTB) represents a threat to public health and a challenge to tuberculosis (TB) control programs. From 1994 to 1997, Portugal had an incidence of 48.3 new cases of TB per 100 000 inhabitants and an
average of 22.7% of these cases were MDRTB, the highest in Western Europe. In an attempt to assist the National Health
Authorities in the control of TB, we implemented in 2002 with the support of Fundação Calouste Gulbenkian, the “TB
Fast-Track” program as part of the TB Task Force of Greater Lisbon, involving 12 Lisbon hospitals and based on the use of
the BACTECTM MGIT 960 system, coupled with the direct identification of M. tuberculosis complex (MTBC) and the detection of mutations in the rpoB gene, using the INNOLiPA Rif.TB Assay (LiPA) (Innogenetics). Because mutations in the
rpoB result in resistance to rifampicin (RIF), and resistance to RIF is almost always accompanied by resistance to isoniazid
(INH), this approach allowed us to identify the MDRTB patient within 24-48 hours. A full report confirming identification
and antibiotic susceptibility (AST) of MTBC by conventional methods (BACTEC culture plus AST and Accuprobe ID)
was issued within additional 12 days.
From September 2002 to January 2006, the LiPA assay was directly applied to 630 acid fast positive respiratory specimens. The comparison between data from this assay and conventional methods revealed 84 discrepancies. The 11 false
positive results corresponded to patients with therapy already established by the time of specimen sampling, whereas
the 73 false negative resulted from inhibition of amplification. A total of 487 of the 600 MTBC positive isolates were
susceptible to all 5 first-line anti-TB drugs. The frequency of MDRTB (resistant to at least INH plus RIF) was 10%. From
the 63 MTBC resistant to RIF, 62 were detected by the LiPA as carrying mutations S531L (60 isolates), H526Y and D516V
(1 isolate each). No mutation was detected by LiPA for one sample, repeatedly identified as resistant by AST. Detection
of rpoB mutations proved to be a good surrogate marker for MDRTB, since only 2 out of 600 MTBC isolates were
monoresistant to RIF.
The early detection of active TB, particularly the detection of MDRTB, is essential for the success of any TB control
program. The application of molecular techniques for the early identification of MDRTB assisted the National Health
Authorities in the reduction of MDRTB rates in Lisbon to less than 8% (average 2003 to 2007).
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PP-25
Drug-resistance of Mycobacterium tuberculosis
at penitentiary institutions of St. Petersburg,
Russian Federation.
Vladimirov, Kirill; Zaitseva, Elena; Ivanov, Aleksandr
Institution: State Medical Academy named after I.I. Mechnikov, St. Petersburg, Russia.
Background
Morbidity with tuberculosis (TB) in Russian Federation and the whole world remains high. This index is up to 40 times
above the average level among prison population, with high prevalence of multi-drug resistant (MDR) TB.
Setting. Central hospital for prisoners in St. Petersburg.
Study design. We retrospectively reviewed data of the patients, who were admitted to the hospital for active culturepositive TB between 2005 and 2008. Between 2005 and 2007, new and re-treatment cases were admitted. In 2008, only
new TB cases were admitted. We studied the results of drug-susceptibility of Mycobacterium to Isoniazid (H), Rifampicin
(R), Ethambutol (E), Streptomycin (S), Kanamycin (K), Ofloxacin (O) in solid Lowenstein-Jensen medium. Cases of pansensitivity, drug resistance (DR), including MDR and extra drug resistance (XDR) were defined.
Results
As much as 163 cultures were studied. From 52 cases in 2005, 36.5% were pan-sensitive, 25.0% were DR and 38.5%
were MDR. In 2006, 36.0% of 50 cultures were pan-sensitive, while casualty of MDR increased to 48.0%. In 2007, number
of pan-sensitive cultures decreased to 21.4%, while 35.7% were MDR and 7.2% were XDR. In 2008, among 47 cultures
nearly half were MDR (48.9%) and 12.8% were XDR, only 27.7% cultures were pan-sensitive.
Conclusions
Among prisoners in St. Petersburg, the value of the cases of primary MDR and XDR TB increased dramatically. Introduction
of the fast methods of drug resistance detection is required.
European Society of Mycobacteriolog y | 30 th Annual Congress | July 2009 | Por to - Por tugal
97
PP-26
AN INCREASE OF DRUG RESISTANCE SINCE 2001
IN MULTIDRUGRESISTANT M. tuberculosis ISOLATES FROM BELGIUM
Karolien Stoffels, Maryse Fauville-Dufaux
Reference Laboratory of Tuberculosis and Mycobacteria, Scientific Institute of Public Health, 642 Rue Engeland, 1180
Brussels, Belgium. Tel. +32-23733210 | Fax. +32-23733281.
[email protected], [email protected]
Between January 1994 and December 2008, MDR clinical isolates of 174 patients were analyzed in our National Reference
Laboratory. They represent 90% of all the MDR-TB patients identified in Belgium during this 15-years period. Since 2000,
the number of MDR patients identified in our country is stable (in average 16 per year, i.e. an average of 1,3 % of the
patients tested for susceptibility to drugs) but the isolates are resistant to more and more second line drugs.We observe
a dramatically increase in resistance to ethambutol, rifabutin, amikacin and ofloxacin, as well as to pyrazinamid.
We divided the studied period in 2 ranges, 1994 to 2000 and 2001 to 2008. Only the first MDR clinical isolate of each patient was taken into account. So these results do not consider the evolution of the isolates during treatment in Belgium,
but only the initial MDR resistance profile.
In the second period (2001 to 2008) 75,6% of the MDR clinical isolates showed resistance to ethambutol versus
45,5% in the first period (increase of resistance of 30,1%); 75,4% were resistant to rifabutin versus 70,4% in the first
period (increase of 5%). Resistance to pyrazinamid increased from 39,6% to 55,6% (difference of 16%). Resistance
level to amikacin showed an increase of 12,2% (3,6% to 15,8%) and resistance level to ofloxacin showed an increase
of 8,6% (3,6% to 12,2%).
No primary XDR isolate was observed during the first period, but 5 were detected since 2001.Three MDR isolates developed
into XDR what the total amount of XDR strains brought to 6 and only 2 for respectively the second and first period.
Concerning the genetic families identified, 14,7% more Beijing strains were registered in the second period compared to
the first period. A decrease of the members of the LAM and Haarlem family was noted (16,4% and 17,3%).
In conclusion, an important increase of resistance to ethambutol, pyrazinamid, amikacin and ofloxacin is observed in MDR
clinical isolates detected in Belgium. This confirms the urgent need for new anti-tuberculosis drugs.
98
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PP-27
Mutational analysis of genes associated with
resistance to injectable second-line drugs in Mycobacterium
tuberculosis clinical isolates from Lisbon, Portugal
João Perdigão1, Ana Ferreira1, Ana Malaquias1, Rita Macedo2, Laura Brum2 and Isabel Portugal.1,2
1 - Centro de Patogénese Molecular, URIA, Faculdade de Farmácia da Universidade de Lisboa
2 - Laboratório de Micobactérias, Centro de Bacteriologia, Instituto Nacional de Saúde Dr. Ricardo Jorge
Objectives
Multidrug resistance (MDR) constitutes a serious problem to tuberculosis (TB) control program in Portugal. An even
more serious threat is the one posed by the high rate of extensive drug-resistant TB (XDR-TB). Our laboratory has
already shown that high rates of this form of TB exist in Lisbon. Given the fact that MDR-TB and XDR-TB are currently
associated with a limited number of genetic clusters, mainly Lisboa family clusters, the diversity of genetic polymorphisms
conferring resistance to second-line drugs is also probably limited. In this study we intended to characterize the genetic
polymorphisms associated with resistanace to second-line injectable drugs and to assess the clinical isolates clonality.
Methods
We have analyzed 19
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MDR-TB strains resistant to one or more second-line injectable drugs, collected from several hospital units across Lisbon Health Region during the year of 2005. All isolates were typed by Mycobacterial Interspersed
Repetitive Units (MIRU-VNTR) and, screened for mutations in tlyA and rrs genes.
Results
Three different mutations were identified on tlyA gene and another three at rrs gene. Overall, 9 isolates had mutations in
tlyA gene and 8 isolates had mutations in rrs gene; two isolates didn’t have any mutation in either gene.The most frequent
mutations found were A1401G in rrs gene (6/19) and 755InsGT in tlyA gene (6/19). We also verified that there was no
overlapping of mutations from different genes. The genotyping analysis revealed that the isolates could be distributed
through two different MIRU-VNTR genetic clusters: Lisboa3 and Q1. Cluster Q1 contained all clinical isolates bearing the
A1401G mutation in rrs gene, while Lisboa 3 cluster contained all isolates that had the 755InsGT mutation in tlyA gene.
Conclusion
We have identified several mutations that might be associated with resistance to different but related second-line drugs:
kanamycin, amikacin and capreomycin. The two most prevalent mutations were associated with different genetic clusters, which suggests recent transmission and, ultimately, that XDR-TB transmission is taking place. The most prevalent
mutations associated with injectable second-line drugs have therefore been defined, which opens the way for molecular
detection of resistance to second-line drugs in the region.
European Society of Mycobacteriolog y | 30 th Annual Congress | July 2009 | Por to - Por tugal
99
PP-28
MULTIDRUG-RESISTANT TUBERCULOSIS
Nuak, Joao; Ferreira, Danina; Carvalho, Teresa; Gomes, Maria Helena; Sarmento, Antonio
Hospital S. Joao, Porto - Portugal
Introduction
Multidrug-Resistant Tuberculosis (MDRTB) is caused by M.tuberculosis (MT) resistant to at least Isoniazid (INZ) and
Rifampin (RIF), and can be due to unsuitable or irregular treatment.
Purpose
To know the epidemiological and clinical characteristics of the disease in patients (pts) hospitalized with MDRTB in an
Infectious Diseases Service.
Patients And Methods
Review of clinical records of pts with MDRTB. Diagnosis was based on drug susceptibility testing.
Results
Between 1996 and 2007, 16 pts had MDRTB. Eleven were male. Ages ranged between 27-40y(X=32.2±4.33). Fifteen
(93.8%) had HIV infection (14 drug addicts; 1 sexual risk). One pt had professional contact with MDRTB.The disease was
only pulmonary in 7(44%) pt, disseminated in 5(31%), pulmonary and extra-pulmonary in 3(19%)-meningeal, lymph nodes
(LN) and urine in one each pt; another pt had only meningeal disease. Eleven (69%) pts had previous irregular antituberculosis treatment. MT was isolated in sputum in 12/16 pt (75%); CSF in 5/6 pt (83%), bronchoalveolar lavage in 2/6(33%),
blood in 3/6(50%), urine in 2/6(33%), LN in 2/6(33%), gastric aspirate and feces in one each. Most pts had MT isolated in
more than one sample. All MT were resistant to INZ and RIF. Thirteen out of 16 pt (81%) were also resistant to streptomycine, 6/12(50%) to pyrazinamide, 6/16(37%) to ethambutol, 5/12(42%) to rifabutine, 10/15(67%) to ethionamide,
4/14(28%) to kanamycin, 4/13(31%) to ofloxacilline, 3/7(43%) to PAS, 2/5(40%) to kapriomycine e 1/5(20%) to cyclocerin.
In 4 pts MT was simultaneously resistant to at least three 2nd line drugs (XDRTB). Fifteen pts had medical treatment according to the drug susceptibilities testing. In two pts pneumectomy was performed. Eight pts died: One before diagnosis
and 7 between 30-720 days of diagnosis. One pt survived for 6y. maintaining positive cultures of sputum despite medical
and surgical therapy.Two pts were treated for 18 months with clinical and radiological improvement, without evidence of
disease 1 and 5y. later. Five pts were lost to follow-up. One is on treatment with clinical and radiological improvement.
Conclusion
MDRTB is a serious public health problem. HIV, drug addiction and irregular treatment were important factors for its
development. Prognosis depends on early detection of drug resistance and institution of appropriate therapy.We emphasize the very high mortality.
100
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PP-29
CHARACTERISATION OF STREPTOMYCIN MUTATIONS IN Mycobacterium
tuberculosis CLINICAL ISOLATES IN THE AREA OF BARCELONA
Griselda Tudó1, Emma Rey1, Fernando Alcaide2, Pere Coll3, Gemma Codina4, Núria Martín-Casabona4, Michel Montemayor3,
Raquel Moure2, Margarita Salvadó5, Julià González-Martín 1
1 - Servei de Microbiologia, CDB. Hospital Clínic de Barcelona-IDIBAPS, Universitat de Barcelona
2 - Servei de Microbiologia, Hospital Universitari de Bellvitge-IDIBELL, Universitat de Barcelona
3 - Servei de Microbiologia, Hospital de la Santa Creu i Sant Pau de Barcelona, Universitat Autònoma de Barcelona
4 - Servei de Microbiologia, Hospital Universitari Vall d’Hebron. Universitat Autònoma de Barcelona
5 - Laboratori de Referència de Catalunya, Barcelona. All the authors are members of Spanish Network for the Research in Infectious Diseases (REIPI, RD06/0008).
Objective
To determine the proportion and type of mutations in Mycobacterium tuberculosis (Mtb) isolates resistant to streptomycin
(SM) and their relationship with the level of resistance and their genotype.
Methods
SM resistant isolates from an Mtb strain collection (1995-2007) were studied. Minimum inhibitory concentrations (MIC)
of SM for each isolate were determined using the proportion method with Middlebrook 7H10 medium. The entire rpsL
gene and 2 specific fragments (loop 530 and region 912) of the rrs gene were sequenced. IS6110-RFLP and spoligotyping
techniques were used to type Mtb isolates.
Results
Of 69 SM resistant isolates, 36 (52.17%) presented a mutation in either the rpsL gene and/or the rrs530 gene, with no mutation in the rrs912 region. No mutations were found in 33/69 (47.8%) SM resistant isolates (all of them with MIC≤16µg/
ml). Seventeen (24.63%) isolates showed rpsL mutations: 9 (13.04%) at position 88 (7: AAG→AGG, 1: AAG→ACG and 1:
AAG→CAG) and 8 (11.59%) in codon 43 (AAG→AGG). Isolates with mutations in the rpsL gene (94.1%) had a MIC≥512
µg/ml. Among isolates with alterations in the rrs gene (27.53%):10 (14.49%) had a 491 C→T change and low MIC level; 7
(10.1%) had a mutation at position 513 A→T or A→C and 2 (2.89%) had a 516 C→T substitution.These mutation points
were related to intermediate and high MIC levels. One isolate with a codon 88 mutation had a second mutation in the
rrs530 gene at position 491. IS6110-RFLP typing identified 4 clusters (11/69, 13%). Clusters I and II were monoresistant
to SM, with a low MIC level and a mutation at position 491 in the rrs gene. Cluster III was multidrug-resistant with a
high MIC level and a mutation in codon 88 in the rpsL gene. Cluster IV and V was monoresistant to SM with a low MIC
level and no mutation. Interestingly, all the isolates with a mutation at position 491 in the rrs530 gene were identified as
LAM3 lineage. All the Beijing family presented mutations in the rpsL gene (2 and 1 at codons 88 and 43, respectively).The
spoligotyping lineageT5-MAD2 was detected in non-mutated isolates.
Conclusions
Mutations in the rpsL and rrs genes were detected in at least 50% of SM resistant isolates. Mutations in the rpsL gene
were associated with high-level resistance while mutations in the rrs530 gene were associated with different MIC levels.
The isolates with no mutations had low-level resistance. Mutations in the rrs530 gene at position 491 were associated
with LAM3 lineage
European Society of Mycobacteriolog y | 30 th Annual Congress | July 2009 | Por to - Por tugal
101
PP-30
SURVEILLANCE OF DRUG-RESISTANT TUBERCULOSIS IN ITALY
Fattorini Lanfranco 1, Pardini Manuela 1, Cirillo Daniela 2, Borroni Emanuele 2, Miotto Paolo 2, Filippini Perla 3, Cassone
Antonio 3, TB-CCM Study Group 4
1 - Istituto Superiore di Sanità, Rome, Italy
2 - Istituto Scientifico San Raffaele, Milan, Italy
3 - Istituto Superiore di Sanità, Rome, Italy
4 - Italian network of mycobacteriology laboratories
Introduction
Drug-resistant TB is an increasing problem worldwide. In order to update the national data on drug resistance, we started a surveillance program including the most representative diagnostic centres in Italy. Purpose of the study. The study
was designed to determine: 1) Accuracy of drug susceptibility testing (DST) for streptomycin (S), isoniazid (I), rifampicin
(R), ethambutol (E). 2) Drug resistance in new cases (NC), previously treated cases (PTC), patients born in Italy (PBI),
patients born abroad (PBA). 3) Molecular typing.
Methods
1) Thirty laboratories were enrolled in 2006-7 in 18/20 regions for proficiency testing (PT), based on the amount of
DST performed every year and geographic location. The laboratories received 20 strains each, and sent DST results to
the WHO Supranational Reference Laboratory (SRL) of Istituto Superiore di Sanità (ISS) which compared them with
the judicial results of the Global Network of SRLs. 2) A questionnaire was returned to ISS with DST results of TB cases
diagnosed in 2007; strains resistant to >1 drugs and 10% of the susceptibles were collected. 3) molecular typing was
performed at the SRL of San Raffaele Hospital (Milan) by spoligotyping and MIRU-VNTR. Results. 1) Twenty-nine laboratories completed the PT. The average efficiency (correct results/total results) was high (95.5±2.8% for S, 97.6±2.9% for I,
95.7±2.9% for R, 96.2±4.4% for E). 2) In 2007, a total of 1,698 antibiograms for SIRE were examined. As to NC and PTC,
total monoresistances were 10.6 and 16.5%, respectively; MDR cases were 2.5 and 26.6%, respectively. As to PBI and
PBA, total monoresistances were 10.9 and 11.2%, respectively; MDR cases were 2.6 and 5%, respectively. 3) In 257 strains
collected in 2006-07 and examined for molecular typing, 146 spoligotypes and 29 clusters were found. Beijing genotype
was detected in 5% of cases. In 44 MDR strains typed by the MIRU-12 technique the clustering rate was 0.023 showing
low transmission rate.
Conclusions
1) PT indicated that the DST in this network of laboratories was accurate. 2) MDR rate in Italy consistently increased
in NC from 1998-2001 (1.1%) to 2007 (2.5%), a phenomenon likely related to immigration. 3) No MDR-TB outbreak
was detected. Noteworthy, unlike reported from other European countries, MDR strains were not associated with the
Beijing lineage (This work was supported by the Italian Ministry of Health, CCM Project Surveillance of resistance to
anti-TB drugs, Grant N92)
102
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PP-31
tuberculosis RESISTANCE in a general
hospital in portugal – 9 years surveillance
Sancho L.; Portugal C.; Tancredo L.; Silva M.; Dias A.; Silva F.; Sousa Germano
Laboratory of Microbiology, Department of Clinical Pathology
Hospital Fernando Fonseca – Amadora, Portugal
[email protected]
Tuberculosis remains a serious public health problem in Portugal. In 2008, the Portuguese Health Authorities reported
a TB incidence of 25,3/100.000 inhbitants (13,6% immigrants). TB Multi Drug Resistant (MDR) were 2,5%, 34% of which
were Extensively Drug Resistant (XDR).
Resistance to any of the primary drugs makes the disease more difficult and expensive to treat.
Our Hospital is located in Lisbon’s surroundings and covers a population of 750.000 inhabitants most of them with poor
socioeconomic level and immigrants from Africa and East Countries. In the Great Lisbon are located 66% of TB cases of Portugal.
Purpose
The aim of this study was to investigate the frequency of drug resistance of Mycobacterium tuberculosis Complex in a
general Hospital in Amadora, Portugal, during a 9-year period (2000-2008).
Methods
A total of 19.417 clinical specimens (15.159 pulmonary and 4.261 extra pulmonary), collected from 9.525 patients, were
cultured for mycobateria.
Molecular genetic identification of M.tuberculosis Complex and its resistance to Isoniazid and/or Rifampicin was made
with the technology Genotype MTBDR plus (HAIN-Lifecience-Germany).
Antimycobacterial susceptibility test to the primary drugs, Streptomycin (STR), Isoniazid (INH), Rifampicin (RIF),
Ethambutol (ETB) was performed in BACTEC MGIT 960 System
Results
In 19.417 cultured clinical specimens for mycobateria, 1094 (14,2%) were positive by cultural methods.
1029 were identified as M.tuberculosis Complex; 783 (76,1%) were strains without resistance, 150 (14,6%) with one resistance, 73 (7.1%) were MDR, being more than 25% XDR.
The proportion of M.tuberculosis strains resistance rate to antituberculosis drugs during the 9-year period was: Isoniazid
11,1% (114), Streptomycin 20.2% (208)), Rifampicin 7.2% (74), and Ethambutol 4.7% (48); but in 2008 was: Isoniazid 4,8%,
Streptomicin 17%, Rifampicin 4,1% and Etambutol 2%.
On our tuberculosis population (661), in the last 6-years (2003-2008), we have compared the resistance rate related to
3 parameters: sex, age and HIV.
The tuberculosis (661) and MDR (38) populations have the same incidence rate: in male (67%) and in females (33%).
The age distribution in the MDR population (38) was 0% [0-15], 5% [16-25], 29% [26-35], 39% [36-45], 13% [46-55], 11%
[56-65], 0% [66-75], 3% [76-100]; and in patients without resistance (623) was 3% [0-15], 13% [16-25], 30% [26-35], 20%
[36-45], 14% [46-55], 8% [56-65], 7% [66-75], 5% [76-100].
The HIV parameter results were analysed on a 554 tuberculosis population. The MDR incidence rate for the HIV group
(213) was 7%, and for the no HIV group (341) was 4%.
Conclusion
The level of resistance in our population (MDR 7%) is significantly higher than Portugal’s average (2,5%)
The Multi Drug Resistance tends to be lower in the last years. The same can be observed in each of the tested drugs.
HIV infection, age and sex patient are factors that contributed to the variation of tuberculosis/MDR incidence rate.
Comparing the resistance rate by sex parameter, we didn’t found differences for tuberculosis or MDR populations; they
both have a bigger incidence in the male sex (67%).
The major incidence of tuberculosis is among active population, between 26 and 45 years old, but, it is between 36 and 45
that we found most of the MDR strains (39% of all), mostly because of drug abuse and HIV infection in this age group.
The incidence of MDR tuberculosis is clearly bigger in HIV positive (7%) than in HIV negative (4%).
Discussion
In spite of our population have a level of resistance above the Portuguese average, we noted that the number of tuberculosis cases, including MDR, decreased 7,8% during this 9-year period analysed, which is comparable to 2008 national
data (-7.2% in the last decade).
European Society of Mycobacteriolog y | 30 th Annual Congress | July 2009 | Por to - Por tugal
103
PP-32
DOES A MUTATION IN THE RPOB MEAN THAT THE
M.tuberculosis IS RESISTANT TO RIFAMPICIN?
Yates, Malcolm; Brown, Tim; Drobniewski, Francis
HPA National Mycobacterium Reference Unit
Testing for mutations in the “hot spot” region of the rpoB gene is gaining momentum for the diagnosis of rifampicin
resistant strains of M.tuberculosis and as a surrogate marker for MDRTB.
The use of PCR combined with hybridisation to commercial strips (Hain Lifesciences, Innolipa)has decreased time and
increased ease of use so that these investigations are being performed by more and more laboratories.
The strips consist of a series of overlapping probes that cover the whole region (S bands) and are all present in Wild Type
isolates. There are also a series of probes (R bands) covering the commonest mutations which are linked with rifampicin
resistance and with the deletion of the corresponding S band.
Occasionally an S band is deleted with no R band appearing. We report these as “unidentified mutations”. The question
is whether these isolates are resistant or sensitive to rifampicin.
Recently we identified three patients from Sao Tome, an island off the West Coast of Africa (population 55000),
with a strains of M.tuberculosis that had the same S band deleted. Sequencing data showed that the strains had the
same synonymous mutation, VNTR/MIRU profiles were identical, and all strains were fully sensitive to first line drugs.
On analysis of our database, 466 strains were found to have rpoB mutations, 103 (22%) of these were unidentified mutations of which 13% were sensitive to rifampicin. A further 15 isolates gave a WT result but were rifampicin resistant.
All strains were sequenced to identify the mutation. Before rpoB mutations can be used as a surrogate for MDRTB the rate of mono-resistance to rifampicin in the area must
be determined: e.g. in the London area 30% of rifampicin resistance is mono.
Care must, therefore, be taken when reporting these rpoB mutation results to Clinicians as
1) unidentified mutations do not always correspond to rifampicin resistance and 2) rpoB mutations may not always correspond to MDRTB
104
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PP-33
GENOTYPIC DETECTION OF ISONIAZID AND RIFAMPIN
RESISTANCE IN Mycobacterium tuberculosis CLINICAL ISOLATES
Maitane Aranzamendi Zaldumbide, Carlos Fernandez Mazarrasa , Luis Martinez-Martinez, Jesus Agüero Balbin.
Hospital Universitario Marques de Valdecilla, Servicio de Microbiologia, Santander, Spain.
Background
The emergence of Mycobacterium tuberculosis resistant to first-line drugs underlines the urgent need for new resistanceprofiling methods that would allow for timely determination of proper treatment. The aim of this study was to evaluate
the development of targeted and fast molecular diagnostic method suitable for specific genome regions responsible for
isoniazid (INH) and rifampin (RAMP) resistance in M. tuberculosis clinical isolates.
Methods
79 strains known to be resistant to INH (n=71) or INH+RAMP (n=8) by the automated system BacT ALERT (bioMérieux)
were selected from a stock collection of clinical isolates (January 2000- March 2009). The genome regions associated
with INH-R (including the codon 315 of the katG gene and the fabG1(mabA)-inhA regulatory region) and RAMP-R (81-bp
hot spot region of the rpoB gene called RRDR) were amplified by PCR and the DNA sequences were studied.
Results
Of the 79 isolates, 22 (27.84%) had the mutation S315T in the katG gene, 5 (6.32%) showed changes at -15 nucleotide of
the fabG-inhA regulatory region and 2 (2.53%) presented both mutations.A significant proportion of strains, 50 (63.29%), had
no detectable alterations at the studied loci. INH + RAMP-R strains were associated with mutations in the RRDR of the rpoB
gene in all cases. From these, majority (5 of 8, 62.5%) presented the mutation S531L, whereas the others involved changes
at the codon 516: mutations D516V and D516F, which were identified in 1 (12.5%) and in 2 (25%) strains respectively.
Conclusions
Our results demonstrated a low sensitivity of this method to detect INH-R strains, and points to the need of finding
out other mutant regions. On the other side, confirm the usefulness of this strategy for fast assessment of resistance to
RAMP, which in turn is a marker for multiresistance. This analysis can also be done with assays based on reverse line blot
hybridization detecting the same mutations, except D516F, which is not targeted.
European Society of Mycobacteriolog y | 30 th Annual Congress | July 2009 | Por to - Por tugal
105
PP-34
PHYSIOLOGICAL FITNESS AND TRANSMISSION POTENTIAL OF MULTI-DRUG
RESISTANT Mycobacterium tuberculosis CLINICAL ISOLATES IN HONG KONG
CHAN Chiu Yeung Raphael 1, CHAN Wai Chi Edward 1, AU Tai Kong Mike 1, LAI Wai Man Raymond 2, YEW Wing Wai 3,
YIP Chi Wai 4, KAM Kai Man 4.
1 - Department of Microbiology, the Chinese University of Hong Kong;
2 - Department of Microbiology, Prince of Wales Hospital
3 - Tuberculosis & Chest Unit, Grantham Hospital,
4 - Tuberculosis Reference Laboratory, Department of Health, HKSAR, Hong Kong.
This study evaluated the infectivity and transmission potential of multi-drug resistant Mycobacterium tuberculosis (MDRMTB) strains by determining (i) whether resistance developed at a physiological cost which rendered them less capable
of surviving environmental stress and infecting human host, and (ii) the degree of genetic relatedness shared by resistant
organisms, which indicated the extent by which they spread among humans.The relative growth rates of selected isolates
were measured using the MGIT system and compared to that of drug-sensitive strains. Our data showed that their average initial growth rate, measured within 7 days of inoculation, was inversely proportional to the number of mutations
they harbored in key resistance genes, with strains carrying 5 mutations growing at a rate 46% slower than that of the
wild type. These findings suggested that resistance gene mutations in MTB imposed a range of physiological cost characterized by reduced growth fitness. However, results of epidemiological typing showed that 34% of the 402 MDR-MTB
isolates analyzed exhibited genetic relationship to other strains, indicating that such fitness cost did not significantly affect
the ability of MDR-MTB to transmit between human hosts and cause infection. Importantly, the identification of five major clusters of 50 strains strongly suggests that infection due to dissemination of ‘parental’ MDR-MTB clones is common.
On the other hand, the majority of test strains displayed a unique genetic profile, indicating that MDR-MTB might also
emerge independently through drug selection within individual patient. The DNA fingerprints and growth fitness data of
local resistant isolates may be used for matching the genetic identities, predicting transmission potential, and tracing the
routes of dissemination of future MDR-MTB isolates in the community.
This work was supported by the Research Fund for the Control of Infectious Diseases (Project code 6902191 and 6902200)
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Mycobacterium tuberculosis: 1999-2008 ANTITUBERCULOSIS DRUGS
SURVEILLANCE IN CLINICAL ISOLATES FROM PATIENTS IN THE
LARGEST HOSPITAL IN THE NORTH OF PORTUGAL
Sousa, Ana Sofia; Pinheiro, Maria Dolores; Carvalho, Teresa; Gonçalves, Helena
Laboratório de Microbiologia do Serviço de Patologia Clínica. Hospital de S. João, Porto, Portugal
Introduction
Tuberculosis, whose causal agent is Mycobacterium tuberculosis (MT), is still an important cause of morbidity and a leading
cause of death by infection worldwide. Multidrug resistant (MDR) and extreme resistant drug (XRD) strains of MT are frequently isolated.They have become an emerging global public health problem and an obstacle for tuberculosis (TB) control.
Insights on prevention of disease dissemination and its empirical treatment may be obtained from resistance surveillance and monitorization of its trends.Therefore, in our study we present the susceptibility data of the strains isolated in
Hospital de S. João over the past ten years.
Material and Methods
A prospective ten year study was done using susceptibility data of MT strains isolated in the hospital. Susceptibility testing
to first line drugs (streptomycin, isoniazid, rifampin and ethambutol) was made on the first isolate of each patient, using
the proportion method (in Lowenstein-Jensen medium until 2000, from 2001 to 2003 in Bactec 460TB and in Bactec
MGIT thereafter). When MDR strains were present, second line drugs (ethionamide, cycloserine, capreomycin, kanamycin, amikacin, ciprofloxacin and rifabutin) were tested.
Results
In these 10 years, 1493 MT strains isolated for the first time in the same number of patients, including 1103 males, were
tested for in vitro susceptibility. Streptomycin was the drug with high index of resistance (8,8%); isoniazid (6,0%) was
the second one; rifampin (2,4%) the third and ethambutol (1,3%) was the less resistant. We found no resistance in 1328
(88,9%) of our patients, but in 29 (1,9%) we isolated MDR strains and 13 (0,8%) patients had XDR ones. From 1999 to
2005, the number of MDR strains isolated was relatively stable (an average of 3 patients/year); in 2006, 2007 and 2008 the
number of patients with MDR was 8, 3 and 1 respectively. In what XDR strains are concerned, we had no isolates from
1999 to 2002; in the following years, 2003-2008, XDR strains were isolated in 1,0,2,7,2 and 1 patients.
Conclusion
The results obtained in our patients are similar to previous reports in Portuguese and international literature, supporting the view that tuberculosis is currently a serious public health problem. The knowledge of susceptibility results will
provide evidence in support of preventive health policies. It also emphasizes the role of Laboratory as a cornerstone in
diagnosis, management of individual patients and effective TB control.
European Society of Mycobacteriolog y | 30 th Annual Congress | July 2009 | Por to - Por tugal
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FITNESS COST OF Mycobacterium tuberculosis CLINICAL
ISOLATES RESISTANT TO FLUOROQUINOLONES
VON GROLL, Andrea 1; MARTIN, Anandi 1; JUREEN, Pontus 2; HOFFNER, Sven 2; PORTAELS, Françoise 1; PALOMINO,
Juan Carlos 1; ALMEIDA DA SILVA, Pedro 3
1 - Institute of Tropical Medicine, Antwerp, Belgium
2 - Swedish Institute for Infectious Disease Control, Solna, Sweden
3 - Universidade Federal do Rio Grande, Rio Grande, Brazil
Fluoroquinolones (FQs) have been used as effective second-line drugs in the treatment of the tuberculosis. However, the
emergence of M. tuberculosis resistant to FQs has contributed for the occurrence of XDR-TB.This study investigated the
fitness cost related to the mechanism of resistance to FQs in M. tuberculosis clinical isolates. A total of 37 isolates had
the ofloxacin, moxifloxacin and gatifloxacin susceptibility determined by the proportion method and were sequenced
to look for mutations in gyrA and gyrB. The role of efflux pumps was evaluated by determining the minimal inhibitory
concentration of the FQs in the presence and absence of verapamil by resazurin microtiter assay. Growth curves of the
isolates were obtained using the MGIT960 automated system and the lag phase time and rate of growth were established
to compare the fitness. On the 25 FQ resistant isolates (FQR), the most frequent mutation was at Ala-90→Val, followed
by mutations at Asp-94 to Gly, Tyr, Ala and Asn. Some unusual mutations were identified at Asp-89→Asn, Asn-533→Thr
(gyrB) and deletion of the codon 678 and 679 in gyrB. The isolate with mutation at Asn-533→Thr was the only case of
no whole cross resistance among the three FQs tested. One FQR isolate was wild type for the region investigated. The
efflux mechanism was indentified in 36% of the FQR isolates, being more frequently found in moxifloxacin and gatifloxacin.
In regard to the fitness parameters, the mutation at Asp-94 showed a longer lag phase while the mutation at Asn-90 had
not any significant difference related to the wild type FQS. The establishment of FQR isolates without fitness cost warns
for the possibility of a continue emergence of XDR-TB and highlights for a more rational use of FQs, not only for the
treatment of TB, but also, for other bacteria.
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IN VITRO ACTIVITY OF OFLOXACIN, MOXIFLOXACIN AND GATIFLOXACIN
AGAINST Mycobacterium tuberculosis BY THE RESAZURIN COLORIMETRIC METHOD
VON GROLL, Andrea 1; MARTIN, Anandi 1; JUREEN, Pontus 2; HOFFNER, Sven 2; PORTAELS, Françoise 1; ALMEIDA DA
SILVA, Pedro 3; PALOMINO, Juan Carlos 1
1 - Institute of Tropical Medicine, Antwerp, Belgium
2 - Swedish Institute for Infectious Disease Control, Solna, Sweden
3 - Universidade Federal do Rio Grande, Rio Grande, Brazil
The in vitro activity of ofloxacin, moxifloxacin and gatifloxacin was tested against 41 strains of Mycobacterium tuberculosis by the resazurin microtiter assay (REMA) plate and the proportion method on 7H11 agar. A critical concentration
of 2.0 µg/ml for ofloxacin and 0.5 µg/ml for moxifloxacin and gatifloxacin was obtained by the proportion method on
7H11 agar. For REMA we propose a critical concentration of 2.0 µg/ml for ofloxacin and 0.25 µg/ml for moxifloxacin and
gatifloxacin. Full cross-resistance among the three fluoroquinolones could not be confirmed since one strain resistant
to moxifloxacin and gatifloxacin was still susceptible to ofloxacin. This finding could have important implications for the
treatment of tuberculosis patients.
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RAPID DETECTION OF EXTENSIVELY DRUG-RESISTANT Mycobacterium
tuberculosis BY THE RESAZURIN MICROTITER ASSAY PLATE
Paasch, Fabienne; Martin, Anandi; Docx, Sven; Fissette, Kristina; Portaels, Françoise; Palomino, Juan Carlos
Institute of Tropical Medicine, Antwerp, Belgium
Introduction
A major concern for tuberculosis control programs is the emergence of multidrug-resistant (MDR) tuberculosis and
especially extensively drug-resistant (XDR) tuberculosis. Conventional drug susceptibility testing (DST) requires 3 to 6
weeks to yield results. Therefore, there is an urgent need of new timely and accurate detection of first and second line
anti-tuberculosis drug resistance.
Purpose of the study
The aim of this study was to evaluate the first and second line drugs rifampicin (RIF), isoniazid (INH), ofloxacin (OFX), kanamycin (KAN), amikacin (AMK) and capreomycin (CAP) with clinical isolates of M. tuberculosis by
the colorimetric resazurin microtiter assay (REMA) plate in comparison to the indirect proportion method (PM).
Method
A total of 150 clinical isolates were studied. DST by PM was performed on Löwenstein Jensen for RIF and INH and on
7H11 agar for the other drugs. The minimal inhibitory concentration obtained by REMA was compared with the PM.
Results REMA results were easily determined visually after 8 days compared to 21 to 42 days by the PM. Out of 150 isolates 92
were MDR and 20 were XDR. After defining the critical concentration for each drug by the colorimetric assay, excellent
results were obtained for first and second line drugs with levels of specificity and sensitivity between 93% and 100%.
Conclusion
In this study drug resistance detection by REMA has shown high level of agreement with the conventional PM.Therefore,
REMA could be a reliable alternative method for rapid detection of MDR and XDR M. tuberculosis.
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DETECTION OF EMBB GENE CODON 306 MUTATIONS IN ETHAMBUTOL
SUSCEPTIBLE AND RESISTANT Mycobacterium tuberculosis STRAINS.
Montoro, Ernesto 1;Yzquierdo, Sergio 1; Lemus, Dihadenys 1; Echemendia, Miguel 1; Takiff, Howard 2
1 - Institute of Tropical Medicine Pedro Kourí (IPK), Havana, Cuba
2 - Venezuelan Institute for Scientific Research (IVIC), Caracas,Venezuela
Ethambutol (EMB) is one of the first line drugs in the treatment of tuberculosis. The major mechanism of acquisition of
resistance to EMB in Mycobacterium tuberculosis seems to be associated with points mutations in the embCAB operon encoding different arabinosyl transferases. In particular, amino acid replacements at embB gene codon 306 occur frequently
in EMB-resistant M. tuberculosis strains. However, this alteration has been also reported in multidrug-resistant (MDR)
strains susceptible to EMB. The aim of this work was to detect the most frequently mutation at codon 306 in EMBresistant strains as well as MDR strains. For this purpose, the indirect Proportional Method (PM) on Löwenstein-Jensen
was carried out as susceptibility test to isoniazid (0,2 µg/mL), streptomycin (4 µg/mL), EMB (2 µg/mL) and rifampicin (40
µg/mL) on 86 M. tuberculosis strains from the collection at the National Reference Tuberculosis Laboratory. All strains
that showed resistance to EMB by PM, all MDR strains and a selection of EMB-susceptible strains were extracted the
DNA to obtain a fragment of 803 bp by PCR, corresponding to embB gene codon 306. All this fragments were sequenced
by automatic form using appropriate primers and they data were assembled, edited electronically, and compared with
wildtype gene sequences. From 34 MDR strains, only 18 showed mutations (53%) being 8 resistant and 10 susceptible
strains to EMB. The 61,5% of EMB-resistant strains showed mutations at codon 306 whereas EMB-susceptible strains no
had alterations in this fragment. In conclusion, the results confirm that embB gene codon 306 is responsible of majority
EMB-resistant in M. tuberculosis. All mutations were founded in MDR strains and because of this, the simple detection of
changes in this fragment could be considered as multidrug-resistance marker.
European Society of Mycobacteriolog y | 30 th Annual Congress | July 2009 | Por to - Por tugal
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TOLERANCE OF MOXIFLOXACIN IN ROUTINE
CLINICAL TREATMENT OF TUBERCULOSIS
Yew, Wing Wai 1,YAN, See-Wan 1, FUNG, Siu-Leung 1, CHAU ,Chi-Hung 1, CHAN, Chiu-Yeung 2
1 - Tuberculosis and Chest Unit,Grantham Hospital, Hong Kong, CHINA
2 - Department of Microbiology. The Chinese University of Hong Koong, Hong Kong, CHINA
From Sep 07 through Feb 09, in a tertiary centre of pulmonary diseases, 65 patients [all male, age 70.0;16.6 yrs (mean;SD)]
with tuberculosis (TB) were commenced on moxifloxacin treatment as part of their routine drug regimens, due to intolerance /contraindication to standard first-line anti-TB agents or drug-resistant disease. 96.9% of patients received
400mg moxifloxacin once daily. The duration of moxifloxacin treatment for all patients was 51.2;37.3 days, except one
who received the drug for 330 days. 28 patients (43.1%) had completed their designated duration of moxifloxacin treatment. 9 patients had nausea, thrush and headache not necessitating drug withdrawal. 15 patients (23.1%) developed
probably moxifloxacin-related adverse events requiring drug cessation: QTc prolongation (5), skin rash (3), arthralgia (2),
neutropenia /thrombocytopenia (2), vomiting (1), angioedema (1) and ECG T-wave inversion (1). Time to occurrence of
these events was 43.7;42.0 days. A 93-yr old patient died of stroke and myocardial infarct after 3 days of treatment. 2
other patients died of pneumonia during moxifloxacin therapy. A 82-yr old patient who developed ECG T-wave inversion
had sudden death 9 days after drug cessation. Another 6 patients died of serious comorbidities, such as lung carcinoma,
1–80 days after moxifloxacin cessation. Otherwise all patients responded favourably to their anti-TB therapy, with clinical, radiographic and bacteriologic improvement. Univariate but not multivariate analysis reveals that older age (>65 yrs)
might increase the risk of moxifloxacin associated adverse effects (P=0.07). Similarly, mortality is associated with the
number of comorbidities (P=0.025). Thus, patient tolerance to “long-term” moxifloxacin use in treatment of TB appears
reasonable. However, in older individuals with significant comorbidities, vigilance should be exercised regarding putative
drug-associated events of potential severity, especially those of cardiovascular nature.
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Characterization of gidB gene in Mycobacterium
tuberculosis isolates in Lisbon Health Region:
role in streptomycin resistance and epidemiological markers
João Perdigão 1, Ana Sabino 1, Catarina Milho 1, Rita Macedo 2, Laura Brum 2, Isabel Portugal 1,2
1 - Centro de Patogénese Molecular, URIA, Faculdade de Farmácia da Universidade de Lisboa, Portugal
2 - Departamento de Doenças Infecciosas, Instituto Nacional de Saúde Dr. Ricardo Jorge, Lisboa, Portugal
Objectives
Streptomycin (STP) was the first antibacillary drug introduced in the treatment of tuberculosis in 1944. With the development of further antibacillary drugs, streptomycin has become less used. Development of STP-resistance is usually
explained by the acquisition of mutations in rpsL gene or in the rrs gene. Our laboratory regularly isolates STP-resistant
strains without any mutation in the referred genes. Recently, mutations occurring in a rRNA methyltransferase (encoded
by gidB gene) were shown to be involved in the acquisition and resistance to STP. In this study, we examined the gidB
gene of STP-resistant isolates in search of mutations that may explain the acquisition and STP low-level resistance on
these strains.
Methods
We have analyzed by sequencing and/or endonuclease analysis the gidB gene of 57 STP-resistant clinical isolates and 30
STP-susceptible clinical isolates, recovered in 2005 and 2006 from different hospital units. The entire rpsL ORF of all
isolates was amplified and screened for mutations by endonuclease and sequencing analysis. All clinical isolates were also
genotyped by MIRU-VNTR.
Results
The gidB gene of 19 STP-resistant isolates was sequenced and two missense mutations, A80P and F12L, were found in 5
and 1 out of 19 isolates, respectively. We have found that these gidB mutations were only present in isolates without rpsL
mutations.The remaining isolates were screened by endonuclease analysis for mutations A80P and K43R in gidB and rpsL
genes, respectively. Overall, mutation A80P in gidB gene was found in 10/57 STP-resistant isolates; 11/14 STP-susceptible
multidrug resistant isolates; and, none of 16 pansusceptible isolates. GidB mutation A80P was also associated with MIRUVNTR genetic cluster Q1, although an independent occurrence has been identified.
Conclusion
We conclude that gidB mutations may in fact explain the high number of STP-resistant strains with no mutation in rpsL
or rrs, isolated in our laboratory. These mutations probably confer STP low-level resistance that may pass undetected in
regular drug susceptibity testing. The independent occurrrence suggests however, that the acquisition of such mutations
present an adaptative advantage.
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FLUORQUINOLONE RESISTANT Mycobacterium tuberculosis
ISOLATES AND THEIR MOLECULAR CHARACTERISTICS
I Gaile 2, G Skenders 1,V Leimane 1, I Jansone 2, M Bauskenieks 2, I Pole 1, V Baumanis 2
1 - State Agency of Tuberculosis and Lung Diseases, Stopini , Riga District, Latvia, LV 2118
2 - Latvian Biomedical Research and Study Centre, University of Latvia , Ratsupites 1, Riga LV 1067, Latvia
Tuberculosis incidence decreased in Latvia from 74 per 100.000 populations in 1998 till 40.3 in 2008. However, multi drug
resistance (MDR) is still high and ~10% of it is extensive drug resistance (XDR). The goal of present study is analysis of
fluoro quinolone (Q) resistance and genetic characteristics of an appropriate isolates.
60 MDR cultivated and resistant to Q isolates from 2001-2007 were analysed for mutations in the gyrA gene, 25 of
them using primary clinical material also. Mutations were evaluated by sequencing or using in house developed modified
(Giannoni et.al.2005) reverse hybridisation method, but kanamycine (K) resistance by sequencing of rrs gene fragment.
48 isolates were confirmed as typical XDR then. Genotyping by PvuII restriction and spoligotyping was performed as
well retrospective case control studies.
35 isolates (58%) contained mutations in the codone 94 (D94 to G, A or H), 14 (23%) in the 90 and 3 (5%) in the codone
91. In 8 cases (13%) mutations were not found. 8 samples (14%) contained whether two mutations or wild type sequences also. 15 K resistant isolates (of 48) contained mutation in the codone A1400G, the rest were wild type. Genotyping
revealed 10 clusters with 2-6 isolates in each. 62% of isolates were of Beijing genotype, but 33% to other typical in Latvia
MDR genotype – C (related to LAM). The clustering rate (47%) indicates on transmission, however, small cluster size
shows, that it is more among hospitalised persons or imprisoned ones. 23 patients suffered from primary XDR TB.
Treatment outcome of XDR patients is 28% cured, failures 55%, the rest defaulted or died.
More profound biochemical and genetic properties of these MDR and XDR isolates ought to be studied in order to
improve the cure rate. Typical genotypes mutations in the gyr gene in Q resistant isolates indicate on the necessity to
search among Q line new drugs affecting other metabolic processes also.
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DESIGN OF A RAPID METHOD OF IDENTIFICATION OF A HIGHLY
TRANSMITTED STRAIN BASED ON THE LOCALIZATION OF IS6110
Sofia Samper 1, Isabel Millan 2, Ana I. Lopez-Calleja 3, Patricia Gavin 1, M. Antonia. Lezcano 4
1 - Hospital Univesitario Miguel servet / I+CS / CIBER enfermedades respiratorias
2 - Universidad de Zaragoza / Hospital Universitario Miguel Servet / I+CS / CIBER enfermedades respiratorias
3 - Hospital Universitario Miguel Servet / I+CS
4 - Hospital Universitario Miguel Servet / CIBER enfermedades respiratorias
Efficient molecular methods allowed the detection of a large and unsuspected tuberculosis outbreak, involving 85 patients in Zaragoza (Spain), caused by a strain named Mycobacterium tuberculosis Zaragoza “MTZ”, representing nearly 20%
of the isolates in 2001 and being still present among the isolates from our tuberculosis population.
We mapped and localized 8 of the insertion sites of IS6110 in its genome observed by RFLP. The insertion sequence
6110 besides being a very useful tool in molecular epidemiology, induces loss of gene activity either by mediating deletion
events or disrupting coding sequences and regulatory domains. It also could modulate expression of neighboring genes
by acting as a promoter sequence, driving or enhancing their expression.
In the present work, we designed a rapid method for identifying this particular M. tuberculosis MTZ. For this purpose,
different pair of primers which targeted the flanking sites of IS6110 in MTZ were designed. One hundred isolates among
clinical isolates already molecular typed were chosen randomly. and were tested in these isolates. Separate PCR reactions
were performed for these isolates. Among the 8 sites localized, 4 of them were previously described as preferential locus
for IS6110 transposition.
After testing with different locations finally one intragenic insertion in Rv2823c was selected for rapid diagnosis. Only
4 of the 100 hundred samples tested were positives, being the four positives isolates MTZ. None more of the isolates
were positive, the other 96 showed the same size of the amplified product than the positive control used H37Rv, indicating that IS6110 was not present.
Conclusion
the mapping of the IS6110 insertion sites in the genome of “MTZ” strain resulted in both, offers clues for better
understanding of the adaptability and virulence of M. tuberculosis, and the design of a rapid method for identifying this
particular M. tuberculosis MTZ.
Presentations Type: Poster
European Society of Mycobacteriolog y | 30 th Annual Congress | July 2009 | Por to - Por tugal
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DRIVING FORCES ON THE EVOLUTION OF THE
PROGENITOR OF M. tuberculosis
M. C. Gutierrez 1,2, R. Brosch2, M. Marceau1, J.Tap2, E. Bourdon2, S. Brisse2, S. Mangenot3, G. Salvignol3,V. Barbe3, C. Médigue3, and P. Supply1
1 - Institut Pasteur de Lille –INSERM
2 - Institut Pasteur, Paris
3 - CEA/DSV/IG/Génoscope, Evry, FRANCE
Genomic and functional plasticity of agents of tuberculosis (TB) suggest that they are the legacy of extremely long
evolutionary fight for survival which started with their environmental ancestors that evolved to the exclusively human
intracellular parasite of our days. Almost certainly, TB has impacted on humankind through pre-history. Mycobacterium
tuberculosis and its earlier relatives have probably been co-evolving with Homo sapiens and its earlier relatives for hundred
of thousand of years. Despite extensive research, the cause of M. tuberculosis speciation and the factors that have led to
its predominance as a human pathogen are still unknown.
Previous studies of rare human TB clinical isolates from East-Africa (Van Soolingen et al., Int J Syst Bacteriol 1997; Fabre
et al., J Clin Microbiol, 2004; Gutierrez et al., PLoS Pathogens 2005) showed that they share many properties of other
agents of TB, but radically differ in terms of a more diversified population structure and obvious traces of intra-species
horizontal gene transfer (HGT).These features suggest that these smooth TB bacilli are extant representatives of a much
broader and older progenitor species, named “M. prototuberculosis”.
We performed c���������������������������������������������������������������������������������������������������
omparative genomics on a comprehensive collection of 56 strains of “M. prototuberculosis” to understand the roles played by various evolutionary processes in shaping the structure of M. tuberculosis genome and of its
population. Multi-locus sequence typing of 16 house-keeping genes and three intein-encoding sequences confirmed the
large genetic diversity of the TB bacilli and suggests that the M. tuberculosis complex (MTBC) is just a particularly succesful clonal lineage that has emerged from the M. prototuberculosis progenitor pool, probably involving multiple HGT
episodes (clonal epidemic structure). Preliminary analysis of whole-genome sequences from the four most genetically
distant strains of smooth TB bacilli indicate extensive chromosomal rearrangements and the existence of multiple genomic islands compared to MTBC genomes. Genome downsizing, mutations, HGT of genomic islands, and intra-species
recombination appear as major driving forces on the evolution of the ancestor of extant M. tuberculosis.
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Resistance, MDR and XDR of M. tuberculosis in Spain in the last years.
P. Ruiz, M. Causse, F.J. Zerolo, J. Gutierrez, M. Casal
Mycobacteria Reference Center. Department of Microbiology. Faculty of Medicine. HospitL “Reina Sofía” . Córdoba. Spain.
Tuberculosis is among the leading causes of death worldwide. The World Health Organization (WHO) estimates that
32% of the world population is infected with Mycobacterium tuberculosis . The resistance to antituberculous drugs is a big
problem to the control of the illness . The emergence of multidrug-resistant strains (MDR), extensively drug-resistant
(XDR) and extreme drug-resistant (XXDR) strains, is a global problem that has made a considerable alarm. There is an
increasing demand to determinate in vitro susceptibilities of clinical isolates to antimicrobial agents other than those
considered primary drugs.
The purpose of this study, was determinate the resistance, MDR and XDR strains in our Reference Center in the four last years.
Material and method
We are studied 624 samples from patients suspects of tuberculosis. 355 strains of M. tuberculosis were isolates , in
BACTEC MGIT 960 and Lowenstein -Jensen medium and identified using Accuprobe and GENOTYPE MYCOBACTERIA.
The susceptibility testing was made for primary drugs and:Amikacin (AK) 1.0 µg/ml; Kanamycin (K) 1 µg/ml; Capreomycin
(CM) 2.5 µg/ml; Ethionamyde (ETH) 5.0 µg/ml; Ofloxacin (OF) 2.0 µg/ml; Ciprofloxacin (CI) 2 µg/ml ; Moxifloxacin (MX)
2 µg/ml; Levofloxacin (LE) 4µg/ml; Rifabutin (Rb) 0.5 µg/ml ;Rifapentine 5 µg/ml and Linezolid (Lz) 1.0 µg/ml. The Bactec
MGIT technique with standart protocol was strictly followed as recommended,
Results
From 624 samples, in 355 were isolated M. tuberculosis and 55 (15,49 %) of theme were resistant to some of the antimicrobial agents studied. The resistance to streptomycin was 3´38 %, to rifampin 7,88%. ethambutol 1,12 %, isoniazid 11.26
% and pyrazinamide 1,97%. A 7,6 % of strains were resistant to some of the second line drugs.The MDR was 5,6 % and
the XDR 1,4 %. No XXDR were isolated.
Conclusion
The resistance to secondary drugs made necessary the in vitro studies.This method, BACTEC MGIT 960 is a reliable and
rapid method to determinate the secondary drugs.
European Society of Mycobacteriolog y | 30 th Annual Congress | July 2009 | Por to - Por tugal
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DETECTION OF NON TUBERCULOUS MYCOBACTERIA IN
SURFACE WATERS: COMPARISON OF CULTURE METHODS
Radomski, Nicolas 1, Lucas, Francoise 1, Cambau, Emmanuelle 2, Moulin, Laurent 3, Haenn, Sophie 3, Régis, Moilleron 4
1 - Leesu, Universite Paris-Est AgroParisTech
2 - CNRMYC, CHU Saint-Louis de Paris
3 - Crecep, Etude biologie
4 - Leesu, Universite Paris-Est AgroParisTech
Since there is no evidence for person-to-person transmission, environment is considered a likely source of non tuberculous mycobacteria (NTM) infections. Particularly, environmental water, from river, lake, pond or hot spring seems being a
major source of NTM. NTM has been also isolated from wastewater, from sources of drinking water, from drinking water
distribution system, from tap water, and even from bottled mineral water. Among human infections caused by NTM from
water origin, pulmonary infections and cutaneous infections are often described. These NTM human infections linked
to water could stem from changes in water use, from human vulnerability or from increase of virulence level among
environmental strains. Also, it seems necessary to determine the origin of these NTM in the environment, in order to
evaluate the importance of non-clinical habitats and to detect the emergence of virulence. Lack of knowledge about life
cycle of harmful bacteria from surface water, particularly NTM, require more analytical tools which are not currently
standardized or adapted to environmental samples. The aim of this study is to propose an improved culture method
that could be used for counting and isolating NTM from surface water and wastewater. Based on literature, we selected
different bacteriological methods from medical applications that had previously been applied to water samples. Samples
from the river Seine (Paris, France) were used to select a culture method that will prevent the growth of interfering
microbiota, with minimal inhibition of mycobacteria. The effect of antibiotics (polymyxin B, amphotericin B, nalidixic acid,
triméthoprime, azlocillin, vancomycin) and chemical decontamination process (methods using acids, bases, detergent or
cetylpyridininium chloride) on have been compared. Results will be discussed and a method adapted to water with high
densities of interfering bacteria will be proposed.
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TYPING OF Mycobacterium avium subsp. avium FROM
DIFFERENT SOURCES USING PVUII–PSTI–IS901 RESTRICTION
FRAGMENT LENGTH POLYMORPHISM (RFLP) IN CROATIA
Spicic, Silvio 1, Cvetnic, Zeljko 1, Pate, Mateja 2, Duvnjak, Sanja 1, Zdelar-Tuk, Maja 1, Racic, Ivana 1
1 - Croatian Veterinary Institut, Zagreb
2 - Veterinary Faculty Ljubljana, Ljubljana, Slovenia
A total of 9 M. avium subsp. avium strains isolated from bovines (N=1), pigs (N=2), wild boars (N=2) and poultry (N=4)
were genotyped by PvuII–PstI–IS901 restriction fragment length polymorphism (RFLP) analysis. The isolates were collected in the period from 2001 to 2006. Revealed profiles were designated according to the nomenclature established
and used in the OIE reference laboratory for avian tuberculosis in Brno, Czech Republic. Digestion with restriction endonuclease PvuII resulted in 3 PvuII RFLP profiles (F, Q and M), comprising 8-9 bands. Digestion with restriction endonuclease PstI was successfully accomplished in 8 isolates demonstrating 4 different profiles of 11-13 bands. Among them, 3
were found to be new in the database (A31, A32 and A33). Combination of PvuII–PstI digestion revealed 4 RFLP profiles
(A29/F, A31/F, A32/F, A33/M). No epizootiological connection was found between the isolates expressing the predominant
profile (A29/F), found in pigs, wild boars and poultry. This is the first research in the field of genotyping M. avium subsp.
avium strains isolated from different animal species in Croatia.
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TUBERCULOSIS IN PETS AND WILD ANIMALS LIVING IN URBAN ENVIRONMENT
Spicic, Silvio; Duvnjak, Sanja; Obrovac, Mihaela; Zdelar-Tuk, Maja; Katalinic-Jankovic,Vera; Racic, Ivana; Cvetnic, Zeljko
Croatian Veterinary Institut Zagreb
Apart from pets and domestic animals humans are often in direct or indirect contact with wild animals, especially
those living in Zoos. Because of their origin these animals are a possible source of infection with mycobacteria atipical for certain regions. In that manner, in 2004. M. africanum type I was isolated from organs of a hyrax (Procavia
capensis) that died in zoological garden in Zagreb. The hyrax had been imported from United Arab Emirates (UAE).
Also, in the same year, Mycobacterium tuberculosis infection was diagnosed in a dog. This was a pet who lived it’s
intire life in a city (Zagreb, Croatia) and whose owner was negative on tuberculosis. Both isolates were MIRU typed
on 12 locuses (2, 4, 10, 16, 20, 23, 24, 26, 27, 31, 39 i 40). Sinse no extensive researsch was done in the past with
regards to molecular typisation of M. africanum with MIRU typing, we compared our results to the ones available
online (data base www. miru-vntrplus. org )(ALLIX-BÉGUEC et. al., 2008). M. africanum type I isolated from the hyrax had a unique code (235424253422). MIRU type of M. tuberculosis isolated from the dog was identical to 8 human isolates originating from all over Croatia in the period from 2004.-2006.
Out^ of these 8 ^cases, 3 originated from
^
c
Zagreb and County of Zagreb; 3 from neighbouring counties (Sisacko – moslava ka,
Karlovacka and Varaždinska) and
2 from more distant counties (Medimurska and Primorsko – Goranska). Considering that the highest tuberculosis incidence of the same MIRU genotype was localised in a 50 km radius in as many as 6 human cases we deducted that
it’s very likely that these people were source of infection for this dog. According to these examples, pets and zoo animals are important links in the chain of import and spread of pre-existing mycobacteria species in urban environment.
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IS1245-RFLP Based Genetic Relatedness Of
The Mycobacterium avium subsp. hominissuis STRAINS
ISOLATED FROM HUMANS, ANIMALS AND ENVIRONMENT IN CROATIA
Spicic, Silvio 1, Cvetnic, Zeljko 1, Pate, Mateja 2, Katalinic-Jankovic,
Vera 1, Duvnjak, Sanja 1, Ocepek, Matjaz 2, Zdelar-Tuk, Maja 1, Krt, Brane 2
1 - Croatian Veterinary Institut, Zagreb, Croatia
3 - Veterinary Faculty Ljubljana, Ljubljana, Slovenia
Significance of infections with Mycobacterium avium, in particular subspecies M. a. hominissuis, in animals and humans
is constantly increasing. Susceptibility of humans, cattle and swine to various mycobacteria and the prevalence of these
bacteria in the environment, although very important from the aspect of epizootiology and epidemiology, have not been
sufficiently investigated in Croatia.
This study is based on massive tuberculin skin tests of cattle and swine from large farms, bacteriology and molecular
identification of M. a.hominissuis isolated from domestic and wild animals, humans and environment. Genotyping of the
23 isolates was conducted by IS1245 RFLP method. The selected cut-off value for cluster designation was similarity level
of 75%. A total of 5 clusters containing 86.9 % of all genotyped strains were identified. High similarity level of the profiles
within a cluster was found for the isolates from man, swine from intensive breeding, deer, wild boar and sawdust from
various counties. As no epizootiological links between animals and humans were identified, humans and animals could be
considered as dead-end hosts. According to our results, the source of infection for humans and animals is most probably
the environment.
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DEVELOPMENT OF REAL-TIME PCR ASSAY FOR
QUANTIFICATION OF MYCOBACTERIA IN SURFACE WATERS
Lucas, Francoise 1, Radomski, Nicolas 1, Cambau, Emmanuelle 2, Moulin, Laurent 3, Haenn, Sophie 3, Moilleron, Regis 1
1 - Leesu, University Paris-Est
2 - CNRMYC, CHU Saint-Louis de Paris
3 - Etude Biologie, Crecep
Most non-tuberculous mycobacteria (NTM) are saprophytes living in natural environments. However, some species
are opportunistic pathogens involved in various human diseases. An increase in incidence of mycobacteriosis has been
recognized worldwide, probably linked to changes in water use, population vulnerability. One important question arising from the increasing occurrence of NTM diseases is the origin of these pathogens. Several cases showed that water
play a significant role in the transmission of NTM. Indeed NTM can be found in various aquatic ecosystems, and also in
water distribution systems. However their number and diversity in environmental water bodies and in wastewaters are
often underscored by the actual detection methods and no standard protocol exist. Cultural studies are time consuming
and poorly specific for NTM growth. Few quantitative PCR have been developed for NTM species. However, these PCR
methods have only been applied to clinical samples and may not be adapted for environmental samples. The aim of this
study is to develop a real-time PCR assay to quantify NTM in surface water samples. First the specificity and sensitivity towards NTM of several published target genes, such as 16S rRNA, rpoB, hsp65, ITS and gyrA and gyrB have been
evaluated using the algorithm BLAST. This first screening resulted in the selection of 8 primer pairs, which specificity and
sensitivity were empirically evaluated by DNA amplification of 50 microbial isolates from the river Seine (France), which
belong to Firmicutes, Proteobacteria, Actinobacteria and Bacteroidetes and Mycetes. This strain library was completed
with 25 NTM and other 8 Actinobacteria strains from national collections. This second step of screening resulted in the
selection of two highly specific primer pairs targeting gyrB and rrs genes, showing respectively 94,83% et 93,10 % of
specificity. The efficiency of the real time PCR was evaluated for both primer pairs using the strain libraries.
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Nontuberculous Mycobacteria, isolated from patients with
lung disease, from Lisboa e Vale do Tejo region, during 2008
António Amorim1*, Rita Macedo, Edna Pereira
Laboratório de Saúde Pública - Micobacteriologia/Tuberculose, Administração Regional de Saúde de Lisboa e Vale do Tejo,
I.P., Lisboa, Portugal
*
Presenting author. Phone: 00351213602520. E-mail address: [email protected]
Despite the clinical relevance of most nontuberculous mycobacteria (NMT) in pulmonary infection in immunocompetent
patients is still unclear, this mycobacteria play an increasing significant pathogenic role in HIV-positive, and other immunocompromised patients. Nevertheless, no data about NTM species isolated from patients with lung disease, from Lisboa e
Vale do Tejo region, are published or available until now.
We carried out a study during the entire year of 2008 with the purpose of identify, determine the incidence of each
specie, and correlate this data with some epidemiological data in patients with lung disease from Lisboa e Vale do Tejo region.
A total of 46 patients with lung disease were detected with NTM infection. ������������������������������������������
Among this patients the isolated nontuberculous mycobacteria were M. intracellulare (n=7, 15,2%), M. fortuitum (n=6, 13%), M. kansassi (n=6, 13%), M. chelonae (n=4,
8,7%), M. gordonae (n=4, 8,7%), M. avium (n=3, 6,6%), M. peregrinum (n=3, 6,6%), M. spp (n=3, 6,6%), M. abscessus (n=2,
4,3%), M. mucogenicum (n=2, 4,3%), M. szulgai (n=2, 4,3%), M. triplex (n=2, 4,3%), M. lentiflavum (n=1, 2,2%) and M. simiae
(n=1, 2,2%).The patients with lung disease that we identified as infected with NMT have a median age of 51,6 years, 45,7%
(21/46) were male and 54,3% (25/46)were female. Despite 43,5% (20/46) of our patients were HIV-state unknown, in the
group of known HIV-state, 88,5% (23/26) were HIV-negative, and only 11,5% (3/26) were HIV-positive.
Our results suggest that the pattern of NTM pulmonary infection, in Lisboa e Vale do Tejo, has no significant differences
from those reported in the rest of Europe and USA, and also, no significant correlation with HIV status. We carried out
the study during only one year, so, further studies are needed to better clarify the NMT infection in patients with lung
disease, in Portugal, in both immunocompromised and immunocompetent patients.
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NONTUBERCULOUS MYCOBACTERIA INFECTIONS IN
THE STATE OF PARÁ, AMAZON REGION, BRAZIL
Lima, Karla Valéria Batista 1, Lopes, Maria Luíza 1, Furlaneto, Ismari Perini 2, Lima, Elys Juliane
Cardoso 2, Conceição, Emilyn Costa 2, Sousa, Maísa Silva de 2, Costa, Ana Roberta Fusco 1
1 - Instituto Evandro Chagas, Belém, Pará, Brazil
2 - Universidade Federal do Pará, Belém, Pará, Brazil
Introduction
The genus Mycobacterium currently has more than 130 species. This genus includes M. tuberculosis complex and M. leprae and other organisms referred to as nontuberculous mycobacteria (NTM). In recent years, there has been a marked
increase in the number of cases of human disease due NTM, and the NTM diseases seems to be related to the geographic distribution of these species in the environment. Purpose of the study
The aim of the present study was to describe the diversity of NTM from clinical isolates received at the Instituto Evandro
Chagas, Pará, Amazon Region of Brazil, between 2004 and 2008.
Methods
NTM included in this study were isolated from clinical specimens of 95 patients, whom 84 had pulmonary infection, and 11 infections cases related to other sites (lymphonod, biopsy and abscess fluid). Löwenstein-Jensen
medium was used for the recovery of mycobacteria from clinical specimens. Genetic characterization to species level was determined by PCR-RFLP analysis of hsp65 gene (PRA), and 16S rDNA and hsp65 sequencing.
Results
Ninety five patients presented NTM infections, of whom 88.4% (84/95) manifested pulmonary symptoms, 1.1% (1/95)
presented lymphadenopathy and 10.5% (10/95) represented cases of healthcare-associated infections. A total of 13 species were identified and included: M. abscessus; M. bolletii; M. massiliense; M. fortuitum; M. avium; M. intracellulare; M.
scrofulaceum; M. colombiense; M. kansasii; M. simiae; M. interjectum;M. smegmatis and M. szulgai. M. chelonae-M. abscessus
(26), M. avium-M. intracelullare-M. scrofulaceum (27) and M. simiae (23) complexes were the most frequent in pulmonary
infections. Lymphadenopathy case was caused by M. fortuitum infection.We encountered M. chelonae-M. abscessus complex (6), M. smegmatis (2) and M. fortuitum (2) in cases of healthcare-associated infections.
Conclusion
We showed the diversity of species associates the NTM infections isolated at the Instituto Evandro Chagas and we encountered high variety of species in isolated from pulmonary samples. A finding was the presence of M. simiae complex
species in human infections, which is not common.
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EVALUATION OF HSP65, TB ,SP REGIONS IN
IDENTIFYING MYCOBACTERIUM OTHER
THANTUBERCULOSIS( MOTT);USING PCR-RFLP
Noorolhoda Saadaee Jahromi ,Shima Seif, Parissa Farnia, Mehdi Kazempour,Mohammad Kargar, JamilehNowroozi, Mehdi
Kazempour, Mohammadreza Masjedi,Aliakbar Velayati Mycobacteriology Research Center (MRC),National Research
Institute Of Tuberculosis and Lung Disease(NRITLD),Shahid Beheshti University Medical Campus.Tehran,Iran.
Background
Mycobacteria Other Than Tuberculosis (MOTT) are frequent causes of pulmonary infections resembling TB, but differ
from MTB complex by being opportunistic pathogens and are acquired mainly from the environment. Recent investigators reported an increasing cause of pulmonary infection by MOTT species, in Iran. Identification of MOTT by conventional biochemical methods is cumbersome and time-consuming. Therefore in the present study,the capabilities of 3
different regions (Tb,Sp,Hsp65) were examined using three primers. We demonstrated that Hsp 65 genotyping would be
very helpful to identify mycobacteria at the species level.
Material & Method
DNA were extracted from 121 culture positive specimens during the year 2007-2009.The amplification carried out
using following primers ( Tb ; Tb 115’-ACCAACGATGGTGTGTCCAT-3’ ,Tb 12 5’-CTTGTCGAACCGCATACCCT) ,(Sp
; Sp 15’-ACCTCCTTTCTAAGGAGCACC-3’ ,Sp 2 5’- GATGCTCGCAACCACTATCCA-3’), ( Hsp ; HSP F3 5’-ATCGCCAAGGAGATCGAGCT-3’ , HSP R4 5’-AAGGTGCCGCGGATCTTGTT-3’)
The PCR products (Tb: 439bp ,Sp: 250-330 bp ,HSP: 644 bp) were digested using restriction enzymes with BsteII and
HaeIII for Tb ,HaeIII for Sp and AvaII ,HphI,HpaII for HSP regions.The digested patterns were analyzed on 2% agarose gel.
Result
The results demonstrated different sensitivity ratio for various Mycobacterium species by Tb ,Sp and Hsp regions. For
RGM , Rapid Growing Mycobacteria , group(e.g., M. furtitium and M. chelonei ) the sensitivity of Tb primer was the highest
among the other two regions(92%). Although, for slow growing mycobacterium(nonphotochromgen & phtochromgen)
the combination of two primers(i.e., Tb+Sp) was required . Thereafter ,the sensitivity for identification of such species
reached upto 94%. Incontrast , scotochromoghen(e.g M. gordonae and M. scrofalceum) were differentiated more precisely
using Sp primer(74.3% ).
Conclusion
The recent increase in MOTT species within the country , underline the need to rapidly distinguish such Mycobacteria
from tuberculosis complex .We demonstrate the use of combined primers for accurate identification of mycobacterium
up to species level.
Key Word: Identification,Atypic Mycobacteria,PCR-RFLP,RGM
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Mycobacterium avium alveolitis after cleansing hotel spa whirlpools
Svensson; Erik 1; Ridell; Malin 1; Åkerström; Magnus 2; Andersson; Eva 2
1 - Institute for Biomedicine, University of Gothenburg
2 - Department of Occupational and Environmental Medicine, University of Gothenburg
Hotel staff cleaning spa whirlpools and filters became ill in a disease suspected to be the so-called hot tub lung, which is an
allergic alveolitis-like granulomatous lung disease. In total seven employees at three hotels were involved. Mycobacterium
sp. was suspected to be the cause. Cultures from patients and from water and outlet filters were done. A quantitative
culture method was developed and water from different parts of the equipment was analysed.
One patient had definite allergic alveolitis and M. avium was isolated. Two other employees from the same hotel had
suspected alveolitis, but no cultivation for mycobacteria was done. In this hotel the cleansing of the spa filters was done
with high-pressure washers.
Two employees at another hotel had fever, chills and dyspnea related to cleansing the equipment. Their disease was not
regarded as allergic alveolitis, but both of them were colonized with M. avium
In the third hotel, two employees were colonized with M. avium, but no one hade symptoms related to work. One patient,
though, had flu like symptoms shortly after bathing in the pool.
In respiratory samples from five of the seven patients M. avium was isolated. The pool water and the surface films of the
water filters contained M. avium, often mixed with other, rapidly growing mycobacteria, however a pure culture was never
obtained. In the quantitative water culture 0 - 16000 CFU/mL of M. avium was isolated.
These are the first reported cases of hot tub lung alveolitis (hypersensitivity pneumonitis) in Sweden. Cultures from
patients, filters and water have contained M. avium. The symptoms of the patients have presently ceased. The cleaning
practices have been changed and the pool filter equipment has been rebuilt.
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IDENTIFICATION OF NONTUBERCULOUS MYCOBACTERIA
IN CLINICAL SAMPLES USING MOLECULAR METHODS: A THREE-YEAR STUDY
Isabel Couto1,2*, Diana Machado1, Miguel Viveiros1,3, Liliana Rodrigues1,4 and Leonard Amaral1,3,4
1 - Unit of Mycobacteriology, Instituto de Higiene e Medicina Tropical, Universidade Nova de Lisboa (IHMT/UNL), Lisbon, Portugal
2 - Centro de Recursos Microbiológicos (CREM), Faculdade de Ciências e Tecnologia, UNL, Caparica, Portugal
3 - COST ACTION BM0701 (ATENS)
4 - UPMM, IHMT/UNL, Lisbon, Portugal
Although Mycobacterium tuberculosis, the etiologic agent of human tuberculosis is the main cause of mycobacteriosis in
Man, other species of mycobacteria may also cause infection in humans. The increasing importance of nontuberculous
mycobacteria (NTM) is now consensually recognized and demands for faster methods for their identification and selection of appropriate therapy. In this work we report our experience on the identification of NTM received from 12
hospitals of the Lisbon Health Region (Portugal) over a three-year period using the GenoType Mycobacterium (CM/AS)
assays (HAIN Lifescience). From 1 January 2005 to 31 December 2007, our laboratory received a total of 1192 acid-fast
bacilli (AFB) positive isolates from 1174 patients presenting with presumptive active mycobacteriosis. All isolates were
processed for Ziehl-Neelsen staining and inoculated into MGIT tubes of the BACTEC MGIT 960 system. M. tuberculosis
isolated from culture were identified by the Accuprobe system (Gen-Probe). Full-grown AFB cultures, negative for M.
tuberculosis, were identified by GenoType Mycobacterium (CM/AS) kits. Out of the 1192 specimen received, 1181 were
identified as members of the Mycobacterium genus. From these, 1032 cultures (87.4%) were positive for M. tuberculosis
complex. The remaining 149 cultures were NTM, corresponding to 12.6% of the total number of cultures from which
mycobacteria were isolated. During the study period, NTM prevalence increased steadily, starting with 8.7% in 2005 and
rising to 15.2% in 2007. The joint use of the CM and AS kits identified 96.6% of all NTM isolates tested. Among the 18
NTM species identified, M. avium complex was the most frequent, although it accounted for only 34% of all NTM. In
countries with high incidence of tuberculosis and, particularly, multidrug resistant tuberculosis (MDRTB) such as Portugal,
therapeutic failure with isoniazid and rifampicin is anticipated to be due to an MDRTB strain. Since many NTM species
are resistant to these drugs, the identification of the mycobacteria causing therapeutic failure (MDRTB versus NTM) is of
major importance. The introduction of molecular methods for the identification of NTM in our laboratory has resulted
in an increased awareness of the importance of being able to rapidly identify NTM as potential pathogens and the key
role played by the laboratory in assisting the selection of therapeutic modality.
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MYCOBACTERIA IN ANIMALS IN SLOVENIA –
AN OVERVIEW OF THE LAST DECADE
^
C 2, Matjaž OCEPEK1
Mateja PATE1, Darja FERME1, Manca ŽOLNIR
DOVC
^
c
1 - Veterinary Faculty Ljubljana, Gerbiceva
60, SI-1115 Ljubljana, Slovenia;
e-mail: [email protected] ; fax: +386 1 4779 352
2 - University Clinic of Respiratory and Allergic Diseases Golnik, Golnik 36, SI-4204 Golnik, Slovenia
Successful national control program carried out between 1962 and 1973 contributed to eradication of bovine tuberculosis (BTB) in Slovenia. Since then, infections with the causative agents of BTB were seldom detected. The main role of
veterinary mycobacteriologists has therefore become the detection and identification of the causative agents of avian
tuberculosis and opportunistic mycobacterial pathogens. The aim of this study was to summarize the work done in the
past ten years (1999-2008) in the field of veterinary mycobacteriology in Slovenia.
Identification of the isolates was based on the following features and tests: colony morphology and growth characteristics, biochemistry, PCR and commercial identification kits AccuProbe (Gen-Probe) and GenoType (Hain Lifescience).
From 1999 to 2008, a total of 292 mycobacteria were isolated from domestic, pet and wild animals in captivity. The vast
majority of isolates were represented by M. avium (80.1%), identified to subspecies level in 92.7% (M. a. subsp. avium –
36.3%, M. a. subsp. hominissuis – 56.4%), while 7.3% isolates remained identified as M. avium. These mycobacteria were
found predominantly in pigs, followed by cattle, poultry and exotic birds. M. caprae was found in 1.4% cases and was
related to an outbreak in a zoo, affecting bisons and camels, and to a single culture-positive case of BTB in cattle in the
last 15 years. M. tuberculosis was found in one cow (0.3%) as a consequence of human-to-animal transmission proven by
means of molecular epidemiology. Other species detected included M. terrae (0.7%, pigs), M. fortuitum (0.7%, pig & cattle),
M. scrofulaceum (0.3%, bison) and M. celatum (0.3%, pig). A relatively large proportion of isolates (16.1%) originating from
pigs, cattle, sheep, goat, mouflon, bison and monitor lizard were identified to the genus level only. This could be partly
attributed to the lack of reliable identification methods in the past.
Development of diagnostic kits based on molecular tests led to improved and easier diagnostics of mycobacteria, especially of the species commonly found in the environment, therefore reducing the burden of unidentified mycobacteria in
a routine laboratory.
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ISOLATION AND IDENTIFICATION OF Rhodococcus AND Nocardia
GENDERS IN SPUTUM SAMPLES WITH TUBERCULOSIS SUSPECT
Leite, Sérgio Roberto A; Silva, Paulo; Sato, Daisy Nakamura; Santos, Adolfo; Carlos Barreto; Miyata, Marcelo; Leite, Clarice
Queico Fujimura
São Paulo State University
Species from Rhodococcus and Nocardia genders are partially alcohol acid resistant and could be isolated from clinical samples (sputum, bronchial-alveolar rinsing and lung biopsy). These genders grow successfully in Löwenstein-Jensen
medium and can cause pulmonary infections similar to tuberculosis cases. Our purpose was to evaluate the presence of
these bacteria from 1636 sputum samples of patients with clinic suspect of pulmonary tuberculosis at Ribeirão Preto city,
São Paulo state, Brazil, between the years 2000 and 2002.The samples were analyzed in the laboratory of Instituto Adolfo
Lutz, Ribeirão Preto unit. From 426 acid-fast bacilli positive sputum samples, applying phenotypic methods and chemotaxonomy, we isolated 296 mycobacteria (identified as 224 M. tuberculosis and 72 NTM), 29 Nocardia sp., 51 Rhodococcus
equi and 09 non-identified partially acid-fast bacilli (PAFB). The success of the treatment depends on early diagnosis, thus
the differential diagnostic between Mycobacterium, Nocardia and Rhodococcus genders deserves special importance.
Due to the high incidence of tuberculosis and the similarity in symptoms, probably the nocardiosis and rodococosis are
under-notified in Brazil.Therefore human health professionals need to observe carefully the importance of Rhodococcus
equi and/or Nocardia sp., that were found on 12% and 6.8% respectively of patients suspected with tuberculosis, attended
at Ribeirão Preto city between 2000 to 2002.
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PCR-RFLP OF hsp65 FOR IDENTIFICATION OF
Mycobacterium leprae DIRECTLY FROM A CLINICAL SAMPLE
Neonakis Ioannis K1, Kontos Fanourios2, Gitti Zoe1, Baritaki Stavroula1, Bazigos Stavros1, Mihailelis Efstratios1, Zerva
Loukia2, Spandidos Demetrios A1
1 - Microbiology Labolatory, University Hospital of Heraklion, Heraklion, Greece.
2 - Clinical Microbiology Laboratory, Medical School of Athens, “Attikon” University Hospital, Athens, Greece.
Introduction
Mycobacterium leprae cannot be cultured in vitro. The application of PCR-RFLP analysis of hsp65 for identification of M.
leprae had been previously proposed (Rastogi et al., J Clin Microbiol, 1999, 37, 2016-19) and, to our knowledge, the
method has been used only once.
Objective
The identification of M. leprae directly from a clinical sample by application of PCR- Restriction Fragment Length
Polymorphism analysis (PCR-RFLP) of hsp65 gene.
Materials and Methods
A sample, taken with a swab from open lesions with exudates from a 51-y old patient suspected of suffering from leprosy, was suspended in sterile water. Mycobacteria were heat-inactivated at 80o C for 1 h and the DNA was extracted
using the guanidinium thiocyanate lysis buffer (Casas et al., J Med Virol, 1995, 47, 378-385). PCR amplification of a 439-bp
fragment of hsp65 was performed using the protocol and primers Tb11 and Tb12 as previously described (Telenti et al.,
J Clin Microbiol ,1993, 31, 175-178). The PCR product was further analyzed by sequencing and RFLP. The amplified PCR
product was digested using the Hae III and BsteII (New England Biolabs) restriction enzymes and the mixtures were
electrophoresed on a 3% Metaphor agarose.
Results
The BstEII digestion produced two fragments of 315 and 135 bp and the HaeIII digestion produced two fragments of 265
and 130 bp. This profile matched the one previously reported for M. leprae. Sequencing of the PCR product (GenBank
accession number: FJ497239) verified the identity of M. leprae.
Conclusions
PCR-RFLP could be a useful molecular tool as an adjunct to careful clinical and pathological assessment of patients suspected of suffering from leprosy.
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A CASE-REPORT OF Mycobacterium thermoresistibile FROM GREECE.
Neonakis Ioannis K1, Kontos Fanourios2, Gitti Zoe1, Baritaki Stavroula1, Kosmadakis Georgios1, Baritaki Maria1, Zerva
Loukia2, Spandidos Demetrios A1
1 - Microbiology Laboratory, University Hospital of Heraklion, Heraklion, Greece.
2 - Clinical Microbiology Laboratory, Medical School of Athens, “Attikon” University Hospital, Athens, Greece.
Mycobacterium thermoresistibile is a non-tuberculous mycobacterium that was first recovered in Japan by Tsukamura in
1966. Although it is strongly associated with pulmonary and dermal diseases, there have only been six reports of its isolation from clinical samples. Here we report on the first case from Greece.
The mycobacterium was isolated from the solid culture (Lowenstein-Jensen slant at 37oC) of a sputum sample after 14
days of incubation. The sample was taken from a 67-year-old male, who was a heavy smoker (1pack/day for 30 years)
and had a history of COPD, type II respiratory distress syndrome, diverticulosis and diabetes, and was presented to our
hospital with fever (38o C), productive cough, dyspnea, weakness, and acute purpura. The chest radiograph revealed an
elevated cardiothoracic ratio, as well as chronic obstructive lung disease, peribronchial infiltrations, consolidations in the
right middle and lower lung zones and a small blunt at the left pneumodiaphragmatic angle.
The Accuprobe Mycobacterium tuberculosis complex assay (Gen-Probe, San Diego, CA) was negative. Accuprobe also
yielded negative results for the Mycobacterium avium complex and Mycobacterium gordonae. Biochemical profile could
not distinguish it from other mycobacteria. The banding patterns obtained with GenoType CM and GenoType AS (Hain,
Lifescience, Nehren, Germany) were not species-specific [GenoType CM: 1,2,3 and 10, and GenoType AS: 1,2,3 and 12].
The identification of M. thermoresistibile was achieved with the amplification and sequencing of the 16S rRNA gene and
the 16S-23S internal transcribed region (GenBank accession: FJ236481).
Moreover, a 439-bp fragment of the 65-kDa heat shock protein (hsp65) gene (GenBank accession: FJ236482) was further
used for restriction fragment length polymorphism analysis with Hae III (New England Biolabs) and BsteII (New England
Biolabs). The BstEII digestion produced two fragments of 235 and 210 bp and the HaeIII digestion produced four fragments of 180, 135, 70 and 50 bp.
Although M. thermoresistibile is considered pathogenic, it is rarely isolated from clinical samples. Molecular techniques are
essential for its identification.
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ISOLATION AND FREQUENCY OF Mycobacterium sp IN A
GENERAL HOSPITAL DURING A 9-YEAR PERIOD
Portugal C.; Sancho L.; Dias A.; Tancredo L.; Silva M.; Sardinha T.; Sousa Germano
Laboratory of Microbiology, Department of Clinical Pathology
Hospital Fernando Fonseca – Amadora, Portugal
[email protected]
Tuberculosis remains a major public heath problem in Portugal with an incidence rate of 25,3/100.000 inhabitants in 2008,
being the majority of the cases in the surroundings of the two major cities (Lisbon and Oporto).
Our Hospital is located in the Lisbon’s surroundings and covers a population of 750.000 inhabitants most of them with
poor socioeconomic level and immigrants from Africa and East Countries.
Purpose
The aim of this study was to investigate the isolation frequency of Mycobacterium sp. in a general Hospital in Amadora,
Portugal, during a 9-year period (2000-2008).
Methods
A total of 19.417 clinical specimens (15.159 pulmonary and 4.261 extra pulmonary), collected from 9.525 patients, were
cultured for mycobateria. All specimens were processed by the NaCl-NaOH method as recommended by CDC, stained
by Ziehl-Neelsen, cultured on Lowenstein-Jensen and liquid medium MGIT. Identification was made with the technology
Genotype MTBDR plus (HAIN-Lifecience- Germany).
Results
Of the 19.417 cultured specimens for mycobateria, 2751 (14,2%) were positive by cultural methods.
The positive rates by clinical specimen were: 17% (279/1684) in respiratory specimens and 6% (26/476) in extra pulmonary tuberculosis; 21% (5/23) in pus, 11% (2/14) in biological liquids, 8% (5/66) in biopsy, 6% (7/112) in blood, 5% (1/22)
in ascitics fluid, 4% (1/22) in mieloculture, and 3% in urine (4/133) and cerebrospinal fluid (2/84).
In 9525 suspected TB patients, 1094 were effective tuberculosis cases (122 TB cases/year average, 97 cases in 2008). The
incidence rate by patient was 11,5%.
In comparison with the positive results of the culture, Ziehl-Neelsen stain was positive in 39% of the samples (47% of
the patients).
96% (1029) of mycobateria isolated were Mycobacterium tuberculosis Complex, 2% (20) M.avium, 2% (20) other species
(M.chelonae, M.intracellulare, M.alsiensis / malmoense / szulgai, M.scrofulaceum, M.lentiflavum).
Conclusion
In spite of the increase of TB suspected patients (+11,3%), the number of effective tuberculosis cases have decreased
(-7,8%) for a 9-year period, which is comparable to 2008 national data (-7,2%) in the last decade.
Tuberculosis is a major problem in this area with an recovery rate of 14,2% in the clinical specimens that we receive and
in 11,5% of the patients, most of them with Mycobacterium tuberculosis Complex (96%).
We have one positive patient every 3 days.
In attempt to minimize the impact of this disease, that depend on a large number of factors, we know that the laboratory
plays an important role in making a definitive diagnosis in a short time period (ASAP). We also know that is important a
close relation between the microbiology laboratory and the hospital doctors and also with the centre that follows the
patients in the community (CDP). This is our policy.
132
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LABORATORY MICROBIOLOGY CONTRIBUTION TO Mycobacterium spp.
DIAGNOSIS IN THREE DISTRICT COUNCILS OF SETúBAL
(PORTUGAL), AN AREA WITH HIGH MYCOBACTERIAL INFECTION PREVALENCE.
José Diogo, Ana Rodrigues, Isabel Nascimento, Elisabete Sardinha, Ana Raposo, Rita Figueira, Isabel Monge, Kátia Silva,
Maria José Gil, Susana Rodrigues.
Laboratory Microbiology, Hospital Garcia de Orta (Almada)
The purpose of this work is to evaluate the incidence of Mycobacterium spp. infection in three district councils of Setubal
(Almada, Seixal and Sesimbra) with a high prevalence of this disease, superior to the average in Portugal. The number
of patients with mycobacterial infection and positive mycobacteriological study in Laboratório de Microbiologia (LM),
Serviço de Patologia Clínica, Hospital Garcia de Orta, E. P. E. (HGO) between 1993 and 2008 were evaluated.
Each biological sample was processed as follows: 1) for acid-fast stain with Kinyoun’s carbolfuchsin and microscopic observation with a 100x immersion oil objective; 2) inoculation in Lowenstein-Jensen medium with aerobic incubation at
37ºC and/or inoculation in Middlebrock 7H9 and incubation in MGIT System (Bactec®); and 3) species identification and
first line antimycobacterial susceptibility testing.
In these sixteen years, LM processed more than 25000 biological products, 3813 were positive and isolated from biological products of 1704 patients.
The age, gender, positive samples for BAAR and number of cases of tuberculosis (including variation per year) in Almada,
Seixal and Sesimbra was determinated. A relation was established between the disease localization (pleuro-pulmonar,
extra-pulmonar and disseminated) by clinical and microbiological criteria. The mean age of the patients was 40,6 years,
1199 (70.3%) were male and 505 (29,7%) were female.
Species identification (since 2004) was: 664 (84,1%) M. tuberculosis, 67 (8,5%) M. gordonae, 20 (2,5%) M. avium-intracelular
and 39 (4,9%) other mycobacteria.
The first line antimycobacterial resistance was determinated in 649 M. tuberculosis strains. The resistance level was: 116
(17,9%) to streptomycin, 76 (11,7%) to isoniazid, 28 (4,3 %) to rifampicin, 17 (2,6%) to ethambutol and 14 (2,2%) to pyrazinamide. The multiresistance (simultaneous resistance to isoniazid and rifampicin) was detected in 27 (4,1 %) strains. Six patients had extensively drug-resistance tuberculosis (XDR-TB). Susceptibility pattern variation per year was also evaluated.
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Mycobacterium lentiflavum AS A CAUSATIVE AGENT OF ADENOPATHY
Santos, Claudia, Mendes, Ana Constança, Fernandes, Sandra João, Ramos, Maria Helena
Centro Hospitalar do Porto - Hospital Santo António
We report the case of a 23 year old female patient, HIV positive, presenting with mesenteric adenopathies of unknown
origin. A biopsy was performed and sent for microbiological study. Results turned out negative for aerobic and anaerobic
bacterial culture, but with positive AFB examination. Following this information, patient initiated anti-tubercular therapy –
Isoniazid, rifabutin, pyrazinamide and ethambutol. Molecular detection of Mycobacterium tuberculosis complex, directly
from the clinical sample, was negative.
We then performed an in house universal real time PCR targeting a 370 bp region of 16S rDNA bacterial gene, witch
gave a positive signal. In order to identify the amplified product, sequencing was performed using the BigDye® Terminator
v3.1 Cycle Sequencing Kit (Applied Biosystems), in ABI PRISM 310 Genetic Analyser (Applied Biosystems). The obtained
sequences were analyzed, and identified as Micobacterium lentiflavum. This information led to treatment alteration – (ciprofloxacin, clarothromycin, rifabutin and ethambutol).
Conventional mycobacterial cultures (MGIT™ and Lowenstein-Jensen medium) turned out negative after incubation time. Three months later a new biopsy was sent for microbiological study, with positive AFB examination, but negative cultures.
Sample was insufficient for molecular study. Sequence based bacterial identification has been widely used to identify microorganisms isolated in clinical samples, particularly when applied to poorly described, rarely isolated or phenotypically aberrant species.We report sequence based
bacterial identification of Mycobacterium lentiflavum directly from a clinical sample. Lack of cultural growth did not allow
susceptibility testing, and clinical specimen was insufficient for further molecular tests, including testing for mutations
associated with antibacterial resistance. However resistance to rifampin has been reported, as has been susceptibility to
isoniazid, ethambutol, clarithromycin, amikacin and ciprofloxacin, in vitro susceptibility patterns whose clinical relevance
remains uncertain. 134
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TEACHING OLD BONES NEW TRICKS; SINGLE NUCLEOTIDE POLYMORPHISM
ANALYSIS OF EUROPEAN ARCHAEOLOGICAL M. LEPRAE DNA
Watson, Claire, Lockwood. Diana
LSHTM - London School of Hygiene and Tropical Medicine, Keppel Street, London, UK
Background
Leprosy was common in Europe eight to twelve centuries ago but molecular confirmation of this has been lacking. We
have extracted M. leprae DNA from medieval bones and SNP typed the DNA, this provides insight into the pattern
of leprosy transmission in Europe and may assist in the understanding of M. leprae evolution. Methods and findings
Skeletons have been exhumed from 4 European countries (the United Kingdom, France, Denmark and Croatia) and
are dated around the medieval period (476 to 1350 A.D.). we tested for the presence of 5 previously identified single
nucleotide polymorphisms (SNPs) in 50 aDNA extractions chosen at random from the collection. M. leprae aDNA was
extracted from 20 of the 50 bone samples. SNP analysis of these 20 extractions were compared to previously analysed
European SNP data using the same PCR assays. Testing for the presence of SNPs in M. leprae DNA extracted from ancient bone samples is a novel approach to analysing European M. leprae DNA and the findings concur with the previously
published data that European M. leprae strains fall in to one group (SNP group 3). Conclusions
These findings support the suggestion that the M. leprae genome is extremely stable and show that archaeological M.
leprae DNA can be analysed to gain detailed information about the genotypic make-up of European leprosy, which may
assist in the understanding of leprosy transmission worldwide. European Society of Mycobacteriolog y | 30 th Annual Congress | July 2009 | Por to - Por tugal
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INTERPRETATION OF POSITIVE M. tuberculosis ANTIGEN
SPECIFIC IFNΓ RELEASE ASSAYS IN TUBERCULOSIS DIAGNOSIS
Greib Carine 1, Lazaro, Estibaliz 1,Viallard, Jean-François 1, Pellegrin, Jean-Luc 1, Maugein, Jeanne 2
1 - Medecine Interne et Maladies Infectieuses CHU Haut-Leveque Bordeaux
2 - Laboratoire de bactériologie, CHU Haut-Leveque Bordeaux
QuantiFERON-TB Gold in tube test is an accurate test to detect immune responses against active Mycobacterium
tuberculosis infection (TB) and has the advantage to eliminate false positive outcomes due to BCG vaccination and non-TB mycobacteria. Howevever there are still some false positive tests which remain unexplained. In this study we aimed to focus on the prevalence and the potential explanation of false positive tests among a cohort of
patients accurately screened without TB. A total of 250 adults patients with suspicion of active tuberculosis were enrolled in this study between January 2007 and
December 2008.
Among them, 88 had positive result in accordance with manufactured interpretation (Nil ≤ 8I/mL, TB antigen ≥ 0.35 IU/
mL and ≥ 25% of Nil value). For 28 patients, tuberculosis was confirmed by culture of Mycobacterium tuberculosis in 26
cases and Mycobacterium bovis in 2 cases. For 9 patients, diagnosis of active tuberculosis was made according to a clinical
and paraclinical body of arguments. For 10 patients without active tuberculosis, we could suspect a technical mistake to
explain the positive result of the test (TB antigen near 0.35IU/mL for 4 patients and mitogen < 0.5IU/mL for the 6 others). Among the other 41 patients out of 88 with a positive test without any argument for TB, we found that
12 had previous diagnosis of active tuberculosis in the past and 13 came from tuberculosis endemic areas.
Finally, 16 positives results out of 88 (18 %) remain without explanation. Errors of diagnosis, bias in collection of medical
information (previous or latent tuberculosis infection), or technical mistakes could be possible reasons. In conclusion, quantiFERON-TB is a useful tool for diagnosis of active TB. However the results have to be carefully analysed according to the high rate of false positive diagnosed in this study. 136
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DIRECT IDENTIFICATION OF Mycobacterium tuberculosis
COMPLEX, Mycobacterium avium COMPLEX
AND Mycobacterium kansasii IN SMEAR-POSITIVE CLINICAL SPECIMENS
SUXING WANG, ZHI YU NEO, KE XIN MAK, MARIA DOLORES QUIENG, LI HWEI SNG
Central Tuberculosis Laboratory, Department of Pathology,
Singapore General Hospital
GenoType@ Mycobacteria Direct (GTMD) was used for direct identification Mycobacterium tuberculosis complex (MTBC),
M. avium, M. intracellulare, M. kansasii in 136 specimens from 122 patients. All 136 specimens were AFB smear positive with
ranges from 1+ to 4+. The GTMD correctly detected and identified 134 of 136 of the mycobacteria present. Compared
to results from culture, this indicates a sensitivity and specificity of GTMD assay of 98.5 and 100%, respectively. These
values increased to 100% when specimens with only MTBC isolation were considered. There were two discrepant results during the study. The first was sputum from which M. avium complex was isolated which had been kept at –70oC for
nearly 3 years. The test was negative for inhibitors and another sputum collected from same patient at same period was
detected as being positive for M. avium by GTMD. This may have been accounted for by degradation of the RNA or a
sampling issue. The second was a stool specimen from a HIV-positive patient who had M. avium complex isolated from
his stool. Both RNA isolation and amplification were repeated for the same specimen, and the presence of inhibitor was
confirmed. Two stool specimens from one HIV-positive patient were initially identified as containing M. avium complex
by AccuProbe. GTMD results showed bands matching M. intracellulae and M. kansasii, indicating possible co-infections in
this HIV-positive patient.This was confirmed when DNA probe was performed on growth from the LJ slant even though
there were no pigmented colonies and both specimens turned out to be positive for M. kansasii. It was most likely that
the mixed infection was not detected by routine methods as the M. intracellulare in this case, had outgrown M. kansasii as
the predominant organism in broth and solid cultures.
Direct identification by GTMD takes about 5 working hours compared to 3 to 5 weeks required for culture isolation
and species identification by conventional methods.
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RAPID DIAGNOSIS AND DRUG SUSCEPTIBILITY TESTING OF
TUBERCULOSIS INFECTION: MTD-TEST2 AND BACTEC MGIT 960 SYSTEM
Müllerova, Maria
Klinlab Ltd, U vojenske nemocnice 1200, 169 00 Prague 6, Czech Republic, [email protected]
The Czech Republic is a country situated in the heart of the Europe. It has a low incidence of tuberculosis in the last 10
years. However, the increasing number of migrants from countries with high incidence of TB is changing the situation.
Samples were collected from patients in the Central Bohemian Region and in a part of Prague (2 millions inhabitants).
All samples were tested by MTD-Test2 and conventional methods, partly also by BACTEC MGIT 960, for the detection
of M.TB complex.
Drug susceptibility tests were performed by conventional methods and BACTEC MGIT 960 (S.I.R.E.+PZA).
For the differentiations of separate species of M.TB complex, the GenoType Mycobacteria MTBC test was used.
MTD-Test2 for detecting the M.TB complex takes only 3.5 hrs and its performance is highly sensitive and specific.
From December 16, 1999 to December 31, 2008 a total of 39,592 different samples were collected for detecting the
M.TB complex, comprising of 23,582 sputa, 10,910 bronchoalveolar lavages, 1,492 laryngeal swabs and 3,662 non-pulmonary samples.
From 35,930 pulmonary samples were MTD-T2 pos., cult. neg. 416 (1.16%), MTD-T2 neg., cult. pos. 116 (0.32%), MTD-T2
pos., cult. pos. 1,108 (3.08%), and MTD-T2 neg., cult. neg. 34,290 (95.44%).
Of the 1,296 isolated strains of M.TB tested for susceptibility to basic AT (S.I.R.E.+PZA), 1,109 (85.57%) strains were
susceptible and 187 (14.43%) were resistant for varying combinations of AT; however, further 78 strains (6.02%) were
resistant to INH+RIF, i.e. MDR TB.
The number of multiresistant patients is rising, especially in the last 3 years: in 2006 10.17% patients were resistant, and
from them 4.81% MDR TB, in 2007 8.78% res., 5.36% MDR TB and in 2008 35.51% res., 13.76% MDR TB. These patients
are mostly young males, 25-35 years old, from foreign countries.
The increase in the number of MDR TB is becoming a problem in our country in view of the need of using alternate AT.
138
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FIRST EXPERIENCE WITH GENOTYPE MTBDR
ASSAI FOR RAPID EVALUATION OF MDR CASES
Klavdia Levina, Anna Dementieva, Maret Saluotsa
North Estonia Medical Centre, Tallin
Tuberculosis (TB) incidence in Estonia decreased from 56.6 per 100.000 population in 1998 till 30.7 in 2008. However,
multidrug resistance (MDR) is still very high ~ 12%. Therefore rapid determination of Rifampin (RMP) and Isoniazid
(INH) resistance in M.tuberculosis (MTB) isolates remains actual for the initiation of effective chemotherapy to break
the transmission of the MDR strains.
Drug susceptibility testing (DST) by conventional methods is time-consuming. More rapid results could be achieved by
using molecular methods.
The goal of our study was to investigate the possibility of application of the Hain Lifescience GenoType MBTDRplus assay
as a rapid diagnostic tool for detection of RMP and INH resistance in MTB isolates by comparing the results with those
obtained by conventional phenotypic resistance studies.
61 smear positive clinical material and 97 MTB strains recovered from smear negative patients’ specimens by culture
have been studied by MBTDRplus assay.
Additionally DR was analyzed by standardized DST method on BACTEC MGIT 960 system when culture was later available.
Among tested MTB strains,108 were phenotypically sensitive to INH and RMP,39 were resistant to both drugs,9 and 2 have got
mono resistance to INH and to RMB correspondently.Wild type patterns were found using the MTBDRplus assay in 107 samples.
Specific mutations associated with resistant patterns MTBDRplus assay were displayed in 48 samples.
MTBDRplus assay and the DST prepared from strains isolated by cultural methods from smear negative specimens
showed 100% agreement between the two methods.
Concordant results between MTBDRplus test directly from smear positive samples and conventional DST from later
obtained culture strains was found in 95.7 % of cases tested, agreement of the results regarding RMPwas100%.
In two (3.4%) smear positive samples resistance-linked mutations were found, but no phenotypically resistance was detected. One strain recovered from AFB positive specimens was INH resistant, but had not displayed mutations conferring
INH resistance by MTBDRplus assay directly from the same smear positive specimens.
Our results suggest that GenoType MTBDRplus assay is a valuable alternative for rapid detection of resistance and in
agreement with the classical methods gives opportunity to break the transmission of the MDR strains.
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DRUG RESISTANT TUBERCULOSIS IN SLOVENIA AND EVALUATION
OF GENOTYPE MTBDRplus TEST IN CLINICAL LABORATORY
Natasa Fajfar, Manca Zolnir - Dovc
University Clinic of Pulmonary and Allergic Diseases Golnik, Golnik 36, SI-4204 GOLNIK, SLOVENIA
Slovenia is one of the countries with relative low rate of drug resistant tuberculosis (TB). In the period 1995-2008 the
rate was 4.2% (1.6-6.5) and only 0.70 % (0.0-1.8) of Slovenian TB patients had MDR TB in the same period.The aim of our
retrospective study was to evaluate the performance of MTBDRplus assay (Hain Lifescinece GmbH, Nehren, Germany)
for the detection of rifampicin (RMP) and isoniazid (INH) resistance in comparison to phenotypic drug susceptibility
testing method.
A total of 48 drug-resistant Mycobacterium (M.) tuberculosis isolates were included in the study. M. tuberculosis drug-resistant strains were obtained from patients living in Slovenia between 1995 and 2008. In total, 20 RMPr/INHr, 27 RMPs/INHr,
1 RMPr/INHs strains were analysed with MTBDRplus assay. The assay was performed according to the manufacturer’s
instructions.
In comparison to conventional drug susceptibility testing MTBDRplus was able to identify RMP resistance in 21 of the 21
strains (100%), but for INH the accordance of both methods was lower - only in 37 of 47 strains (79%). The most common mutation carried in RMP-resistant isolates was rpoB MUT3-S531L (48%) followed by mutation rpoB MUT1-D516V
(33%). In INH-resistant strains dominating mutation was in the gene katG in 60% (MUT1-S315T1 in 68%, MUT2-S315T2
in 29%) and the mutations in the inhA promotor gene in 19%. Comparative analysis demonstrated very good agreement between molecular and phenotypic drug susceptibility results
for RMP resistance, but not so for INH. Thus MTBDRplus assay is useful method for the rapid detection of drug resistance, but the results should always be confirmed by the phenotypic methods as well.
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PP-69
EVALUATION OF A NEW REAL-TIME PCR KIT FOR
THE DIAGNOSIS OF TUBERCULOSIS INRESPIRATORY SPECIMENS
Causse, M; Gutierrez-Aroca, JB; Casal, M
Mycobacteria Reference Center. Microbiology Department. Reina Sofia University Hospital, Cordoba (Spain)
Introduction
Early diagnosis of tuberculosis is one of the major objectives of the World Health Organization. In its latest update, the
Center for Disease Control and Prevention (CDC) recommends the use of a rapid molecular diagnostic technique on
at least one sample per patient.
Objectives
To evaluate a new kit (COBAS Taqman MTB®, Roche) for the diagnosis of tuberculosis in respiratory samples, and compare results with those obtained by the COBAS Amplicor MTB kit. Culturing (Lowenstein-Jensen or BACTEC MGIT
960) was used for reference purposes.
Material and methods
A total of 170 respiratory and 19 non-respiratory samples were processed. Specimens decontaminated were stained
with auramine and cultured. A single manual extraction was performed using the AMPLICOR Respiratory Specimen
Preparation Kit; eluate aliquots were then amplified using the COBAS Amplicor MTB and the COBAS TaqMan MTB kit.
Automatic sample extraction was also performed, using the Ampliprep�������������������������������������������������
TNAI kit, ��������������������������������������
200-µl aliquots being previously incubated at 95ºC for 15 minutes.
Results
All 77 smear-positive samples were classified as positive by both kits.
Of the 170 respiratory samples tested using the TaqMan, 2 false positives (FP) and one false negative (FN) were recorded.
Of the 169 tested using the Amplicor kit, there were 2 FP (different samples than TaqMan) and 2 FN.
TaqMan amplification with automatic extraction yielded one false negative and no false positives.
Real-time PCR sensitivity and specificity were 98.7% and 97.7% respectively. Use of TaqMan with automatic extraction
yielded 98.8% sensitivity and 100% specificity. Kappa indices were 0.95 and 0.97 with respect to the comparator.
For smear-negative samples, sensitivity declined to 80% and specificity remained at 97% for the TaqMan kit.
For the 19 non-respiratory specimens, all three techniques tested recorded one false negative.
Conclusions
TaqMan MTB kit is a real-time PCR method offering the same reliability as the Amplicor MTB kit��������������������
. TaqMan
������������������
MTB kit appeared to display good sensitivity using non-respiratory specimens.
It cuts down time-to-results from 6 to 2.5 hours
Automatic extraction reduced the number of false positives and considerably shortened handling time.
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QUANTIFERON-TB GOLD ASSAY (QFT) AND TUBERCULINE SKIN TEST (TST)
CLINICAL PERFORMANCE FOR THE DIAGNOSIS OF ACTIVE TUBERCULOSIS
Karabela Simona, Papaventsis Dimitrios, Nikolaou Stavroula, Konstantinidou Efthimia, Sainti Asimina, Ioannidis Panayotis,
Kanavaki Sofia
National Reference Center for Mycobacteria, “Sotiria” Hospital, Athens, Greece
Objective
The purpose of this study was to evaluate and compare Quantiferon-TB Gold In-Tube (QFT, Cellestis, Australia) and the
tuberculine skin test (TST) in patients with active TB, with and without previous BCG vaccination.
Methods
Patients with symptoms compatible with active TB were included. The TST was performed according to the Mantoux
method and the QFT assay according to the manufacturer’s instructions.The cut-off value for a positive result was ≥0.35
IU/ml interferon-gamma (IFN-γ). Sensitivity, specificity, positive and negative predictive values were calculated and compared for QFT and TST tests. Agreement between QFT and TST was assessed by the kappa (κ) coefficient.
Results
A total of 296 patients were enrolled in the study. One hundred eighty-nine had a record regarding BCG vaccination.
Forty-four (23%) of the 189 patients had been vaccinated. In total, the sensitivity and specificity of QFT, excluding those
with indeterminate results, was 79% (52/66; 95% CI: 66-88%) and 66% (153/167; 95% CI: 58�������������������������������
���������������������������������
-������������������������������
73����������������������������
%), respectively. The sensitivity and specificity of TST was 72% (46/64; 95% CI: 58-83%) and 67% (156/232; 95% CI: 59-74%), respectively.The overall
concordance between the QFT and TST tests was 70.2%, with a kappa value of 0.46 (95% CI: 0.289-0.524). In the BCGvaccinated subgroup, agreement between the two assays was 66%, with a kappa value of 0.350 (95% CI: 0.103-0.597).The
difference with the non-vaccinated subgroup (κ=0.463; 95% CI: 0.303-0.623) was considered to be not quite statistically
significant (p>0.05). Initial TST positive screening followed by a QFT positive result was found to have greater sensitivity
and specificity in the non-vaccinated [sensitivity=79% (95% CI: 59-92%); specificity=81% (95% CI: 71-88%)] compared to
the BCC-vaccinated subgroup [sensitivity=67% (95% CI: 30-92%); specificity=75% (95% CI: 57-89%)].
Conclusion
This study confirmed previous reports that QFT assay has higher sensitivity for detecting active TB compared to TST.
An overall moderate agreement between TST and QFT was found. The difference in agreement between non-vaccinated
and BCG-vaccinated subgroups could be attributed to TST influence by vaccination. In patients with active TB and no
BCG-vaccination history, TST screening followed by subsequent QFT testing proved to present the highest sensitivity
and specificity for TB diagnosis. Larger prospective studies are needed to confirm our results.
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CLINICAL PERFORMANCE OF QUANTIFERON-TB GOLD ASSAY (QFT) FOR THE
DIAGNOSIS OF LATENT TUBERCULOSIS IN DIFFERENT PATIENT GROUPS
Karabela Simona, Papaventsis Dimitrios, Nikolaou Stavroula, Konstantinidou Efthimia, Sainti Asimina, Ioannidis Panayotis,
Kanavaki Sofia
National Reference Center for Mycobacteria, “Sotiria” Hospital, Athens, Greece
Objective
The purpose of this study was to evaluate the performance and usefulness of Quantiferon-TB Gold In-Tube (QFT,
Cellestis, Australia) in the diagnosis of latent tuberculosis and to compare it with the tuberculin skin test (TST).
Methods
A cohort of 395 high risk adults was prospectively evaluated. Study groups consisted of 139 neoplastic patients, 98 with
autoimmune diseases (SLE, rheumatoid and psoriatic arthritis), 20 immunossupressed (e.g. HIV, hemodialysis) and 26
Intensive Care Unit (ICU) patients, and 112 patients with no underlying disease.The TST was performed according to the
Mantoux method and the QFT assay according to the manufacturer’s instructions. The cut-off value for a positive result
was ≥0.35 IU/ml interferon-gamma (IFN-γ). QFT and TST were simultaneously performed in 297 patients.
Results
Overall, QFT gave a positive result in 72/395 (18.22%) patients. Among the different risk groups, ICU and immunosuppressed patients represented the highest positive rates (19% and 20%, respectively). Indeterminate results (7% on
the whole) were more often seen in ICU (34.6%), neoplastic (7.2%) and in patients with autoimmune disease (7.2%).
Indeterminate results represented <1% in patients with no underlying disease. Strength of agreement between GFT and
TST results was poor (agreement=55.44%, kappa=0.171; 95% CI: [0.064-0.278]).
Conclusion
QFT is a very usefully method in TB diagnosis because in contrast to TST, it reduces over diagnosis of latent TB in previously BCG vaccinated, distinguishing truly tuberculosis cases from BCG vaccinated individuals and/or non-tuberculous
infections. As a consequence, QFT provides valuable information for therapeutical decision-making.
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TUBERCULOSIS DIAGNOSIS BY QUANTIFERON TB GOLD
ASSAY IN AREAS WITH DIFFERENCES IN TB INCIDENCE
Nikolaou Stavroula, Karabela Simona, Papaventsis Dimitrios, Sainti Asimina, Konstantinidou Efthimia, Ioannidis Panayotis,
Kanavaki Sofia
National Reference Center for Mycobacteria, “Sotiria” Hospital, Athens, Greece
Objective
Evaluation of Quantiferon TB Gold in Tube method (QFT) for the latent TB diagnosis in patients originated from countries
with high (group A) and low (group B) incidence of TB.
Material
948 whole blood samples from 153 (16,15) individuals belonging to group A and 795 (83,9%) to group B.
Method
Performance of Quantiferon TB Gold in Tube (Cellestis, Australia) method according to the manufacturers’ instructions.
Results
QFT positive results was detected in 93/153(60.8%) individuals of group A and 273/795 (29,35) of group B (p<0,0001).
Clinical information for previous BCG vaccination was available in 351 cases: 65 of group A and 286 of group B, where
QFT confirmed latent TB in 36/65(50,8%) and 46/286(16,1%) of group B (p<0,0001). As it was expected, Tuberculin Skin
Test (TST) was positive in all 351 cases with previous BCG vaccination.
Conclusion
QFT is a highly diagnostic and useful method, especially in patients originated from areas with high incidence of TB. In
contrast to widely used TST, it reduces overdiagnosis of latent TB in previously BCG vaccinated.
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QUANTIFERON® -TB GOLD IN-TUBE TEST USED IN PRAGUE
PATIENTS LISTED IN THE NATIONAL TUBERCULOSIS REGISTER
Havelkova, Marta 1, Bartu,Vaclava 2, Kubin, Milan 3
1 - National Institute of Public Health – NRL for mycobacteria
2 - Charles University , Faculty of Medicine, Faculty Thomayer Hospital
3 - Prague Hygiene Institute
In 2006-2007, 65 (29.0%) of 224 patients registered in the A15, A16 and A18/19 categories (58.5%, 32.3% and 9.2% of all
patients, respectively) were assessed using both the QuantiFERON – TB Gold In-Tube (QFT) method and the tuberculin
skin test (TST) with 2 TU PPD.
After stimulation with tuberculosis-specific antigens, a low level of interferon-gamma (IFG) production (< 0.35 IU) was
found in 18 (27.7%) patients and moderate or high levels (> 0.35 IU) in the remaining 47 (72.3%) patients. After incubation with a non-specific mitogen, a low level of IFG production was recovered in 13 (20%) individuals, with moderate or
high levels being found in the remaining 52 (80%) patients.
The TST revealed low levels of skin infiltrate reaction (0-5 mm) in 25 patients (39.1%) and moderate or high levels of skin
reaction (range 6 - > 30 mm) in the remaining 39 individuals (60.9%).
The high proportion of low reagent levels in both the QFT and tuberculin skin tests can be explained by a high number of
older patients, comorbidity with malignant tumours, diabetes or general fatigue in pre-terminal patients, and the immune
systém insufficiency related to these factors.
^
This presentation was fully supported by project KAN 200520702 of the Grant Agency of Czech Academy of Sciences (GAAV CqR).
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PRACTICAL EXPERIENCE OF USING A DNA AMPLIFICATION
ASSAY FOR RAPID DETECTION OF Mycobacterium
tuberculosis COMPLEX IN RESPIRATORY SPECIMENS
J. Cacho1, A. García-Cañas1, A. González Torralba1, I. Cano2, A. Pérez Meixeira3, A. Ramos Martos2, and M. Sánchez-Concheiro1
1 - Servicio de Microbiología, Hospital Universitario de Getafe, Madrid
2 - Servicio de Neumología, Hospital Universitario de Getafe, Madrid
3 - Servicio de Salud Pública, Comunidad de Madrid, Madrid.
Objectives
To evaluate experience in a clinical microbiology laboratory of using a DNA amplification assay for routine detection
of Mycobacterium tuberculosis complex (MTC) performed once weekly, and to compare this method with microscopy
and culture.
Methods
A total of 507 respiratory specimens from 419 patients were screened for tuberculosis (TB). Smear examinations, culture
and polymerase chain reaction (PCR) were performed on each sample. Specimens were processed according to standard
laboratory protocols. All samples were processed exactly as described in Cobas Amplicor MTB Methods Manual (Roche
Molecular Systems, USA). This method was performed once per week.
The following two groups of samples were considered to be true positive: (1) samples which were culture positive for
MTC; and (2) all samples which were culture negative for MTC but positive to PCR, provided that one or more of the
following criteria were met: (i) the samples originated from a patient whose other samples were culture positive; (ii) the
patient’s clinical history provided evidence of TB sufficient to warrant initiating treatment for TB.
Results
Inhibition of PCR was seen in 15 (2.9%) specimens. A total of 37 (7.3%) samples were considered to be true positive:
33 samples grew MTC in culture and 4 were culture negative for MTC but positive for PCR and met the criteria as described previously. The overall sensitivity and specificity of PCR as compared to true positive samples was 83.8% (31/37)
and 99.1% (466/470), respectively; that of culture 89.2% (33/37) and 100% (470/470), respectively; and that of direct
microscopy 64.9% (24/37) and 99.8% (469/470), respectively.
The average time to reporting for true positive samples was 6 days for positive PCR and 11.4 days for positive culture. Of
the smear-positive, true positive samples, 91.7% (22/24) were PCR positive. Of the smear-negative, true positive samples,
69.2% (9/13) were PCR positive. The 9 samples classified as true positive and smear negative were from 9 different patients.Treatment was started earlier in 44.5% of these patients because positive PCR findings were obtained.The average
time to reporting was 5.4 days for positive PCR findings and 17.4 days for positive culture findings.
Conclusion
MTC infection was confirmed for PCR in 91.7% of smear-positive specimens. Although PCR was performed once weekly,
treatment in 44.5% of patients was initiated earlier because of positive PCR results from smear-negative samples.
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REAL-TIME POLYMERASE CHAIN REACTION FOR THE DIRECT
DETECTION OF MYCOBACTERIUM TUBERCULOSIS IN CLINICAL SPECIMENS
Karen, Morgan
Joint Clinical Research Centre, Kampala, Uganda
Introduction
The resurgence of tuberculosis is a leading cause of death worldwide. Since M. tuberculosis, is slow growing, methods
of diagnostic testing based on culture are delayed. This study describes the development of a real-time polymerase chain
reaction (RT-PCR) assay and subsequent clinical testing.
Methods
Patients were recruited from routine TB suspects in the Monterey County Public Health laboratory, CA, USA. Initially,
using Bactec MGIT 960 cultures as the gold standard, 231 respiratory specimens composed of 76 specimens from culture-confirmed tuberculosis cases and 155 culture negative specimens were analyzed by RT-PCR. Over the next 2 years
206 respiratory patient specimens were tested from 81 flurochrome smear AFB positive and 125 negative sputa. DNA
extraction was performed directly from patient specimens by the modified Qiagen mini-Amp kit (Valencia, CA). The RTPCR was performed on the Roche LightCycler instrument (Mannheim, Germany). The assay was designed to target the
ITS region of the 16S rRNA gene of M. tuberculosis using Taqman hybridization probes for detection of amplicons. Results
The developed RT-PCR assay yielded results in one day and achieved a sensitivity of 85.5% and specificity of 100%. In
subsequent clinical use over the two year period the RT-PCR assay achieved a sensitivity of 92.3% and specificity of 100%
in direct detection of MTB from 206 respiratory patient specimens,while in contrast fluorochrome smears achieved
only a sensitivity of 60.5%% for non-specific AFB detection in the same population. The RT-PCR assay detected MTB in
64.7% of the smear negative but culture confirmed patient specimens. The RT-PCR assay costs $14 per test, inexpensive
compared to commercial MTB assays ($60). Conclusion
RT-PCR methodolgy can be used to detect MTB directly in patient specimens within eight hours, without waiting for
culture growth. This rapidly increases and enhances specific detection and treatment of MTB.
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THE UTILITY OF MOLECULAR TESTING IN
ROUTINE MYCOBACTERIOLOGY DIAGNOSIS
Fanourios Kontos, Loukia Zerva.
Clinical Microbiology Laboratory, Medical School of Athens, “Attikon” University Hospital, Athens, Greece.
Objective
Molecular methods increasingly replace phenotypic tests in Mycobacteriology. This study describes a “molecular” strategy that was developed for routine testing of clinical samples and isolates with the goal of shortening turnaround time
(TAT) and providing accurate results.
Methods
All samples submitted for mycobacterial cultures (from 12/2006 to 3/2009) were processed and cultured by standard
methodology. The first smear (+) specimen of any patient and every first (+) culture were tested by an in-house IS6110PCR in order to differentiate between Mycobacterium tuberculosis complex (MTBC) and nontuberculous mycobacteria
(NTM) (TAT 4 hours). IS6110-PCR (+) specimens or cultures were tested by Genotype MTBDRplus (Hain-LIFESCIENCE)
for MTBC species confirmation and detection of Isoniazid and Rifampicin resistance (TAT 2 days). Subsequent culture
(+) specimens from the same patient were tested by Accuprobe MTBC (Biomerieux) (TAT 2 hours). The susceptibility
testing of MTBC isolates was performed by MGIT960 (Becton Dickinson). If a (-) result was obtained by the IS6110PCR, identification of isolates proceeded using both Genotype Μycobacterium CM and AS (Hain-LIFESCIENCE) (TAT
two days). For NTM species confirmation a PCR-RFLP analysis of the hsp65 gene was applied (TAT 3 days); additionally,
sequencing of the 16S rRNA gene (TAT 5 days) represented the reference identification method for selected isolates. Results
Out of 4.000 specimens, 160 were culture positive (4%) including 85 MTBC positives (63.5% acid fast stain [AFS] positive) and 75 NTM positives (26.7% AFS positive). Five samples contained more than one NTM species, which were identified only by molecular testing. There was complete agreement between all molecular identification methods; however
16S rRNA sequencing was more informative (examples: M. fortuitum and M. lentiflavum, M. celatum). All MTBC isolates
were Rifampicin susceptible, two were high- and one low-level Isoniazid resistant by MGIT960; only the latter was not
recognized by Genotype MTBDRplus.
Conclusions
Focusing on molecular methodology resulted in fast and accurate final reporting. The low incidence of MTBC positivity
among tested specimens (2,1%) justified the decision not to use indiscriminately a direct molecular test for tuberculosis
diagnosis. The frequent occurrence of NTM (48,5% of all culture positive samples) necessitates direct molecular testing
of all AFS (+) specimens.
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DETECTION OF Mycobacterium tuberculosis DNA IN
FORMALIN-FIXED, PARAFFIN-EMBEDDED TISSUE SPECIMENS
BY SPOLIGOTYPING: APPLICATION TO HISTOPATHOLOGICAL DIAGNOSIS.
Salas S1, Hernández J1, Ojeda P2, Awad C2, de la Hoz F3, Murcia MI1.
1 - Departamento de Microbiología, Facultad de Medicina. Universidad Nacional de Colombia. Bogotá, Colombia
2 - Hospital Santa Clara E.S.E., Bogotá, Colombia
3 - Departamento de Salud Pública, Facultad de Medicina. Universidad Nacional de Colombia. Bogotá, Colombia
In Colombia, Tuberculosis (TB) remains as a public health problem with an incidence of 25 per 100 000 population while
extra-pulmonary TB has increased. The Spoligotyping method has demonstrated to be a good tool to improve the TB
extra-pulmonary diagnosis.
Purpose of study
To identify DNA of Mycobacterium tuberculosis from Formalin-fixed paraffin embedded Tissues specimens (FFPET). Methods
We examined 160 FFPET storaged for 13 years and with a suspected diagnosis of Tuberculosis infection according to
histopatological analysis. Spoligotyping was carried out as previously described with some modifications. DNA extraction
with CHELEX was used and human DNA was negative control. The Spoligotype films were scanned and classified by using
GeneTools software followed by manual editing and confirmation. Statistical analysis was performed using SPSS V.15.0.
Results
Of the 160 samples analyzed by Spoligotyping, 78 (48.8%) cases showed absence of signal at the spot 33 to 36 compatible with M. tuberculosis. Nevertheless SPOL-DB4 was made with DNA obtained from cultures, we in tented to compare
our patterns obtained and we founded only 4 samples compatible with pattern previously described LAM (2), U (1) and
Haarlem lineage (1). Incomplete patterns were observed in 34 (21.2%) and no patterns were obtained in 48 (30.0%).
Negative controls did not yield any spoligopatterns. Exact Fisher’s statistic showed a significant difference between the
positivity of Spoligotyping and storage time (P <0.05) while for the kind of tissue found no differences (P> 0.05).
Conclusions
In 48.8% cases we confirmed the diagnosis of M. tuberculosis. The spoligotyping method is a tool that provides some
information about FFPET, the results of this can be affected by storage time. In order to obtain better results is necessary
to examine the samples immediately after her obtention.
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Pott´s Disease: an ancient disease?
Cardoso, Sara, Coelho, Rui, Paulo, Cristiana, Abreu, Candida, Silva, Susana, Gomes, Helena, Sarmento, António
Hospital S.João
Introduction
Skeletal tuberculosis is a rare disease in developed countries although it´s still a significant cause of disease in Portugal
due to the high prevalence of tuberculosis and an insidious and non-specific presentation of this form of disease. Clinical
report: We report 3 cases of Pott´s disease.
Case 1: Male, 62y. History of multiple sclerosis under imunomodulator and leg trauma with disability. He went several
times to the emergency room due to intense lombalgy without trauma and discharged with non-steroidal anti-inflammatory (NSAIs) medication, without relief. D12-L1 fracture was diagnosed and the patient was discharged under conservative treatment. Fifteen days later, he appeared with paraparesia, fever, asthenia and weight loss and was submitted
to surgery. Histology of the bone fragments revealed acid fast bacilli. The direct and cultural exam of gastric lavage (GL)
were positive for M.tuberculosis complex (MTC). Thorax X-ray (XR) was normal.
Case 2: Female, 88y. History of previous ribs and femur trauma fractures. She had complaints of lombalgy and progressive
paraparesia during the last year and was chronically medicated with NSAIs. Three months before admission anorexy and
weight loss appeared. She had no fever.The magnetic ressonance revealed D6-D7 vertebral body fracture with cavitation
and medular compression. She was submitted to surgery. The bone cultural exam was positive to MTC. The thorax XR
was suggestive of pulmonary involvement but the GL mycobacteriology exam was negative.
Case 3: A 77y woman with past history of pulmonary tuberculosis and recent history of dorsal trauma with D12 fracture, treated conservatively. Later she was admitted with paraplegia and submitted to surgery.The bone culture, bronchoalveolar lavage culture and DNA (polymerase chain reaction) were positive for MTC.The cerebrospinal fluid had 24cells/
uL, high ADA (123U/L), proteins (10,20g/l) and low glucose (0,14g/l), but DNA for MTC and culture were negative. All
three patients were treated surgically and medically with rifampin, isoniazid, ethambutol and pyrazinamide. All of them
had come with dorsal or lombar pain and progressed to paraparesia and plegia, which showed some improvement with
antibiotics and physiotherapy.
Conclusion
Skeletal tuberculosis is a silent re-emergent disease, for which our doctors are not aware, leading to delayed diagnosis
and causing severe neurological damage like Pott´s paraplegia as it was observed in our patients. 150
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OSSEOUS TUBERCULOSIS AT AGE OF 9 MONTHS
Loureiro, Carla 1, Matos, Gabriel 1, Balacó, Inês 1, Mota, Marta 2, Nogueira, Célia 2, Lemos, Sónia 1, Rocha, Graça 1
1. Pediatric Department/H. Pediatrico, CHC
2. Mycrobiology Laboratory, Coimbra Medicine Faculty
Background and Aims
Osseous tuberculosis is rare, mainly as a primary disease in a previously healthy infant. Several mycobacteria may be
involved such as M. tuberculosis, M. bovis and M. avium. When there is no association with a primary pulmonary disease
in a child vaccinated with Calmet-Guérin Bacillus, M. bovis may be the causal agent. Case Report
A previously healthy 11 month-old infant BCG vaccinated at birth was admitted with a 2 month evolution of a right elbow
tumefaction. The X-ray presented destruction of the trochea, severe periostic reaction and soft tissue tumefaction, and
the ecography a fluid-filled cavity. MRI suggested neuroblastoma or Ewing sarcoma. Sedimentation rate and specific enolase were elevated. On surgery a whitish soft mass associated with necrotic fluid compressing the cubital nerve was found.
The histological examination revealed characteristic features of caseum and allowed the diagnosis of osseous tuberculosis. Mycobacterium tuberculosis complex was identified by Real-Time PCR in osseous biopsy. Gastric fluid and urine
were negative. Our patient recovered after surgical debridement and combination drug therapy. He developed a local
cutaneous fistula (no agent identified on fluid culture) and posterior soft tissue calcification. At 22 months age he remains
with no other relevant infections.
Conclusions
Skeletal tuberculosis with extravertebral location is rare. Tuberculous osteomyelites of the limb bones requires a high
index of clinical suspicion along with radiological and histopathological investigation in order to establish the diagnosis. Tuberculosis is still an important differential diagnosis in unusual bone conditions
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DIFFERENCES IN DIRECT ANTITUMORAL CAPACITY AMONG
THE VARIOUS Mycobacterium bovis BCG SUBSTRAINS
SP Secanella, M Luquin, E Julián
Dept. Genètica i Microbiologia, Facultat de Biociències, Universitat Autònoma de Barcelona, Bellaterra (Barcelona)
The administration of Mycobacterium bovis Bacillus Calmette-Guérin (BCG) is the first treatment option for avoiding the
recurrence of bladder cancer.Various BCG substrains are used. These substrains differ genetically and display differential
antigenic determinants, which it has been suggested may influence the efficacy of BCG as vaccine in tuberculosis studies.
In tuberculosis, evolutionary early strains are more efficacious than are the more attenuated evolutionary late strains.The
impact of these differences on BCG antitumoral capacity in bladder-cancer cell lines has not been addressed.
We aimed to compare the direct antitumoral activity of different BCG substrains by inhibiting cell proliferation and by
triggering the production of cytokines in bladder-cancer cell lines.
T24, J82 and RT4 �����������������������������������������������������������������������������������������������������
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uman bladder-cancer cell lines were cultured with different doses of BCGs. We tested three evolutionary early BCG substrains, Japan, Moreau and Russia, and five evolutionary late strains, Connaught, Danish, Glaxo, Phipps,
and Tice. Inhibition of cell proliferation was assessed by using a colorimetric assay at different time points; the production
of interleukin (IL)-6 and IL-8 was measured in cell culture supernatants using enzyme-linked immunosorbent assay.
Among the different BCG substrains, in the case of T24 and J82 cell lines, Connaught and Russia induced both the highest inhibition of proliferation and cytokine production. In contrast, Glaxo and Phipps (for the T24 cell line) and Glaxo
and Tice (for the J82) were the least efficacious both in reducing cell viability and in inducing cytokine production. The
remaining BCGs behaved differently depending on the cell line.
Finally, for the RT4 cell line, all BCG strains inhibit cell proliferation at the same level, except for Danish and Glaxo,
which were seen to be less efficacious. As regards cytokine production, IL-6 production was not detected in any culture,
whereas low levels of IL-8 production were observed, the lowest being for Danish and Glaxo cultures.
The results showed that Connaught and Russia are the most efficient BCGs and that Glaxo is the least effective, both
in the inhibition of tumoral-cell proliferation and the induction of cytokine production. No correlation was observed
between BCG antitumoral efficacy and genotypic evolutionary classification.
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ROLE OF TNF-a GENE POLYMORPHISMS IN HOST GENETIC
SUSCEPTIBILITY TO PULMONARY TUBERCULOSIS
Anoosheh, Saber 1, Farnia, Parissa 1, Noruzi, Jamileh 2, Kargar, Mohammad 3, Kazempour, Mehdi 1, Seif, Shima 1, Masjedi,
Mohammad Reza 4,Velayati, Ali Akbar 4
1 - Mycobacteriology Research Center, NRITLD, Shahid Beheshti University (M.C)
2 - Microbiology Department, Iran University of Medical Science
3 - Microbiology Department, Jahrom Azad University
4 - National Research Institute of Tuberculosis and Lung Disease, Shahid Beheshti University (M.C)
Preface and goal
Tuberculosis is one of most common infectious diseases and it causes death of more than 3 million people a year, worldwide. It caused by Mycobacterium tuberculosis and approximately one - third of world population are infected with this
bacteria, but only 5 - 10 % of them develop active TB.Therefore, individual differences in susceptibility to TB are expected.
These differences might be due to host factors especially genetic diversity between populations. TNF-α as a pro-inflammatory cytokine, play a key role in host defense against tuberculosis. Presence of mutation in this gene can influence the
effectiveness, performance and capability of immune Responses against infection.The Aim of this study was to investigate
the frequency of TNF-α alleles and relationship between susceptibility to TB and TNF-α gene variations.
Materials and methods
A case-control study was conducted and 65 healthy controls and 65 TB patients were enrolled. Genotype of TNF-238,
TNF -244, TNF-308, TNF -857 and TNF-863 were distinguished using by PCR-RFLP method. The results were analyzed by SPSS
v.16, Fisher exact and Hardy-Weinberg tests.
Results
Obtained results showed that,TNF-308,TNF -857 and TNF-863 were as a high frequency mutation regions in population levels,
and also we found a significant differences at TNF-308 and TNF -857 between two group of controls and patients ( P-value
< 0.05 ).
Conclusion
presence of mutation in TNF-308 and TNF -857 regions probably increases host susceptibility to mycobacterial infection and
Genotyping of these regions can be used for screening of high risk persons. Also according to high frequency distribution
of mutations in TNF -857 and TNF-863 regions, further studies on association of these regions is suggested.
Key words
Tuberculosis, Genetic Susceptibility, Cytokines, Gene Polymorphism
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MYCOLACTONE INTERFERES WITH THE PROTECTIVE
IFN-g-DEPENDENT ACTIVATION OF MACROPHAGES
DURING INFECTION WITH Mycobacterium ulcerans
Egídio Torrado1; Alexandra G. Fraga1; Elsa Logarinho1; Teresa G. Martins1; Jenny A. Carmona1; José B. Gama1; Maria A.
Carvalho2; Fernanda Proença2; António G. Castro1; Jorge Pedrosa1
1 - Life and Health Sciences Research Institute (ICVS). School of Health Sciences, University of Minho. Braga, Portugal
2 - Chemistry Research Center. School of Sciences. University of Minho. Braga, Portugal
Mycobacterium ulcerans is the etiological agent of a necrotizing cutaneous disease, known as Buruli ulcer. The pathology
caused by this pathogen is associated with the production of the lipidic exotoxin mycolactone. Following our recent
demonstration of an intramacrophage growth phase for M. ulcerans, we investigated the biological relevance of interferon-gamma (IFN-γ), as well as the mechanisms activated by this cytokine in M. ulcerans-infected macrophages.
We used three different M. ulcerans strains selected based on their virulence for mice and the type of mycolactone
produced: the low virulent mycolactone-negative strain 5114; the intermediate virulent, mycolactone C-producing strain
94-1327; and the highly virulent, mycolactone D-producing strain 98-912.
IFN-γ-deficient mice showed an increased susceptibility to infection only with strains 5114 and 94-1327, suggesting that
this cytokine plays a protective role in infections with the low and intermediate virulent strains of M. ulcerans, but not
with the highly virulent strain. In line with this, IFN-γ-activated mouse primary bone marrow-derived macrophages controlled the proliferation of the low virulent and the intermediate virulent strains, the latter only at low multiplicities of
infection. The effector mechanisms induced by IFN-γ in infected macrophages leading to M. ulcerans growth restriction
involved both phagosome maturation and acidification, as well as increased nitric oxide production. In agreement, the
addition of mycolactone D, purified from cultures of the highly virulent strain, led to a dose-dependent inhibition of phagosome maturation and nitric oxide production in IFN-γ-activated cultured-macrophages infected with the mycolactonenegative strain, resulting in an increased bacterial burden.
Our results suggest that the protection mediated by IFN-γ during the intramacrophage phase of M. ulcerans infection
depends on the type and amount of mycolactone produced.
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VIRULENCE, IMMUNOGENICITY AND PROTECTION INDUCED BY ´Mycobacterium
habana´ STRAINS IN A MURINE MODEL OF PULMONARY TUBERCULOSIS.
Montoro, Ernesto 1;Valdés, Iliana 1; Aguilar, Diana 2; Orozco, Hector 2; Hernández-Pando, Rogelio 2
1. Institute of Tropical Medicine “Pedro Kourí”(IPK), Havana, Cuba
2. National Institute of Medical Science and Nutrition ¨Salvador Zubirán¨. Mexico DF, Mexico
Mycobacterium habana’ was first isolated in Cuba by Valdivia, in 1971. Later, was demonstrated its protection capacity
against M. tuberculosis and other mycobacteria. We studied the virulence, immunogenicity and protection of 3 strains
of ‘M. habana’ using Balb/c mice. The first experiment was done to know the virulence potential of ‘M. habana’ using a
progressive pulmonary TB model. In the 2nd assay mice were vaccinated with 3 doses of bacilli. The grade of immunogenicity was related with the induction of IFNγ by the stimulation of the main organs with antigens of M. tuberculosis. The
best doses that induced immunogenicity were used in the 3rd experiment for animal vaccination. Two months later mice
were challenged with M. tuberculosis H37Rv and Beijing genotype. All the animals infected with M. habana TMC-5135 and
IPK-337 were alive until the end of the experiment. IPK-220 strain showed about 20% of death to the seven week postinfection. All the strains had significative differences when we compared with the control group infected with H37Rv
strain. The values of the colony forming units (CFU) were in correspondence with the survival rate. The percentage of
pneumonia was higher for ‘M. habana’ IPK-220, showing final values similar to mice infected with H37Rv strain. Due to
the virulent behaviour of ’M. habana’ IPK-220 we discharged it for the coming assays. The most important results of
the 2nd experiment were the statistical differences in the IFNγ production founded in groups vaccinated of ‘M. habana’
IPK-337 and TMC-5135 strains, respectively, with the BCG group. The lung CFU for these doses showed a decreasing
tendency with a total sterilization to the final of the experiment. Animal vaccinated with ‘M. habana’ strains and challenged with M. tuberculosis H37Rv had the highest survival. These results are in accordance with the low percentage of
pneumonia with both stains. Nevertheless this differences was not statistical significative in comparison with BCG group.
With the Beijing challenge we observed differences between vaccination with TMC-5135 and BCG group. The lungs of
the animals that received BCG and challenged with Beijing showed more than 70% of pneumonia and a lower granuloma
area.The CFU reveals lower lung bacilli load in mice vaccinated with ‘M. habana’ strains.The final results demonstrate the
potential of ‘M. habana’ to protect against TB infection.
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DENDRITIC CELLS DIFFERENTIALLY EXPRESS IL12-FAMILY CYTOKINES
AFTER INFECTION WITH Mycobacterium tuberculosis OR M. bovis BCG
Margarida Saraiva, Carole Sousa, Jenny A. Carmona, Andrea Cruz, Jorge Pedrosa, A. Gil Castro
Life and Health Sciences Research Institute (ICVS), School of Health Sciences, University of Minho, Braga, Portugal
IL-12 family is a group of heterodimeric cytokines, composed of 4 related cytokine members: IL-12, IL-23, IL-27 and IL-35.
These cytokines, that share some functions and receptor components of IL-12, act on CD4+ T cells modulating the type
of T helper and T regulatory responses, and initiating the development of the acquired T cell response to intracellular
pathogens, such as Mycobacterium tuberculosis (MTb) or M. bovis BCG. Bone marrow derived dendritic cells (BMDC)
originating from wild-type mice were exposed to live MTb strain H37Rv or to BCG for different periods of time. The
kinetics of mRNA production of each monomer that form the cytokines of the IL-12 family (p19, p28, p35, p40, EbsteinBarr-Virus-induced gene 3 (Ebi-3)) was determined. We show that MTb-stimulated BMDC were strong producers of p40,
p35 and p19, whereas BMDC exposed to BCG expressed much lower levels of these cytokines. The main TLR involved
in the recognition of MTb and BCG and activation of BMDC is TLR2, since in its absence the expression of the various
monomers was nearly abrogated. A consequence of this differential activation of BMDC was reflected on the distinct
type of T helper responses developed when MTb- or BCG-infected BMDC presented OVA peptide to TCR-transgenic
CD4 T cells. MTb-infected BMDC were able to induce the development of both Th1 and Th17 responses, whereas BCGinfected BMDC induced Th17 responses. We are currently addressing the molecular mechanisms that differentiate the
response of BMDC in the context of an MTb or a BCG infection. Understanding the details of DC activation by MTb or
BCG and its consequences on the CD4+ T cell arm of the immune response will help to reveal important aspects to be
improved for a better vaccination strategy.
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ANALYSIS OF M. smegmatis MUTANTS RESISTANT TO MS6 INFECTION
Simões, Marta Filipa; Jordão, Luísa; Teles, JMM; Couto, S; Moniz-Pereira, José; Pimentel, Madalena
Centro de Patogénese Molecular-Unidade dos Retrovirus e Infecções Associadas, Faculty of Pharmacy, University of
Lisbon, Portugal
Mycobacteriophages represent excellent model systems for studying mycobacterial hosts. Ms6 is a temperate mycobacteriophage that infects Mycobacterium smegmatis. The first step of a dsDNA phage infection is adsorption to a host
receptor followed by injection of DNA into the cytoplasm. Characterization of the phage resistant mutants represents
a common genetic strategy for the identification of mycobacterial genes involved in the synthesis of parietal phage receptors. The study of these genes is of highly importance, as phage receptors are also involved in the entry of others
molecules, such as antibiotics, into the cell. In order to identify the Ms6 receptor we started to select from a mutant
library obtained after a transposition event using the pCG79 system (Guilhot et al, 1994), M. smegmatis mutants to a
Ms6 infection. DNA obtained from these mutants was submitted to an enzymatic restriction analysis, which allowed the
selection of four mutants with different profiles.
In order to understand which step of the phage infection was affected, adsorption and infection assays with DAPI stained
phages were performed.
We found different profiles among the selected mutants suggesting that different steps of the infection are affected.With
these results, our goal is to identify the mycobacterial genes disrupted by the transposition event.
Identification of the affected genes is an important achievement which may contribute to the characterization of the
phage receptor.
Guilhot, C., I. Otal, I. Van Rompaey, C. Martín, and B. Gicquel. 1994. Efficient transposition in mycobacteria: construction
of Mycobacterium smegmatis insertional mutant libraries. J. Bacteriol. 176: 535-539.
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HUMORAL RESPONSE IN TUBERCULOUS PATIENTS AGAINST
THE MYCOLIC ACIDS OF Mycobacterium tuberculosis
Julián, Esther Gómez 1; Rodríguez-Güell, E 1; del Val-Romero, B 2; Clivillé, R 2; Cañete, C 3; Navarro, A 3; de Gispert, FX 3;
Luquin, M 1; Alonso, C 2
1 - Dept. Genètica i Microbiologia, Facultat de Biociències, Universitat Autònoma de Barcelona, Bellaterra (Barcelona)
2 - Dept. Microbiologia, Consorci Sanitari Integral, L’Hospitalet de Llobregat (Barcelona)
3 - Unitat de Respiratori, Consorci Sanitari Integral, L’Hospitalet de Llobregat (Barcelona)
Although numerous serological tests have been developed for TB serodiagnosis, none of these have shown adequate
levels of accuracy; in consequence, they have not been widely implemented.
Diverse mycobacterial antigens have been evaluated in these tests, among which are cell-wall glycolipidic antigens such
as cord factor (CF). This molecule is made up of a trehalose residue sterified to two mycolic acids (MAs); it has been
established that MAs are the epitopes for the anti-CF antibodies recognition. Furthermore, a possible cross-reaction
between anti-MAs antibodies and cholesterol (COL) in TB patients has recently been published.
The purpose of this study was to detect and compare the presence of IgG, IgM and IgA antibodies against MAs with
regard to the presence of anti-CF and anti-COL antibodies in TB patients.
To address this question, MAs and CF were purified from M. tuberculosis H37Rv (ATCC 27294T), and commercially available COL was used. An ELISA was developed to detect IgG, IgM and IgA antibodies to MAs and COL, and an ELISA
previously developed for CF was performed.
The presence of IgG, IgM and IgA anti-MAs, CF and COL in the sera of 31 HIV-negative TB patients at the time of TB
diagnosis and throughout anti-TB prophylaxis, 20 PPD-positive donors, 20 PPD-negative donors and 20 patients affected
by other pneumonias were determined.
No antibodies to MAs were determined in any sera of the groups studied, whether for TB patients at the onset or during
prophylaxis, or in control groups.
Anti-CF IgG and IgA antibodies in sera from TB patients were detected, following a downward kinetic from the beginning
to the end of the prophylaxis. Test sensitivity and test specificity were 41% and 78%, respectively, for IgG detection, and
19% and 95%, respectively, for IgA detection. Additionally, anti-CF IgM antibodies were detected in all the groups.
In contrast, neither IgG nor IgA anti-COL antibodies were detected in any of the groups; however, IgM anti-COL antibodies were also detected in sera from all the groups studied.
Each serum showing anti-CF and COL antibodies was individually analysed, and their titres against each antigen were
compared. Analysis showed that response profiles were different against the two antigens. Moreover, given that the sera
reacting against COL or/and CF did not react against MAs, it is therefore possible to rule out a cross-reaction between
MAs and COL.
The overall results invalidate MAs as possible antigens for the serodiagnosis of TB.
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Mycobacterium tuberculosis
Arley Gómez López
Infectious Diseases and Tropical Medicine Unit; Universidad del Rosario
TlyA protein has a controversial function as a virulence factor on Mycobacterium tuberculosis (Mtb). Updated studies
have not demonstrated a possible hemolytic activity conferred by TlyA and contrary to recent evidence have suggested
a function enzymatically similar to RNA methyltransferase at ribosomal level which confers antibiotic susceptibility to
capreomycin and viomycin.
Our aim was to determine the In vitro hemolytic activity of Mtb TlyA overexpressed and purified from E. coli BL21-AI and
to carry out an in silico phylogenetic and structural analysis.
Based on tlyA gene sequence (Rv1694) from Mtb H37Rv specific primers were designed. The amplification product was
ligated on pEXP5-CT/TOPO vector (Invitrogen), transformed and overexpressed on E. coli BL-21-AI. After purification by
affinity chromatography, TlyA-His6 recombinant protein was analyzed by SDS-PAGE under denaturing conditions which
was detected as 28 KDa single band. Recombinant protein as recognized by anti-His monoclonal antibody.
Protein structural characterization by circular dichroism was carried out. On the other hand, hemolytic activity assays
using TlyA-His6 purified were negative as well as on bacterial lysates.
Hemolytic assays with TlyA-His6 supplemented with calcium and magnesium were negative suggesting lack of specific
requirements for this activity.
By using bioinformatics tools, a ribosomal binding called S4 located between 5 and 68 residues and FtsJ among 62 and
247 residues were identified on TlyA. These domains have been described on proteins associated with ribosomal translational machinery. The Hidrophobicity profile was in disagreement with a possible transmembrane helix although non
polar amino acid composition suggesting that TlyA might not be membrane attachment. Nevertheless, three-dimensional
model (Structural homology with 1L9K model) reveals a consensus structure with a common core, comprising a parallel
β-sheet of six strands, sandwiched between two layers of α-helices corresponding to a RNA methyltransferase structure.
Phylogenetic analyses showed that TlyA is highly conserved among mycobacteria species and it does not exhibit changes
among Mtb complex strains. tlyA gene evolution might operate under purifying selection model. Additionally differences
were observed among TlyA and bacterial pore forming proteins.
This evidence supports a link between ribosomal modifications to posttranslational level and suggests a functional annotation error of this family protein at GENBANK as well as missannotation on several genomes as Mtb genome. Studies
on resistance mechanisms of Mtb mediated by ribosomal proteins might be useful for understanding new alternative
therapeutic approaches for tuberculosis control applied to knowledge of second line antibiotics such as capreomycin.
Key words
TlyA, Mycobacterium tuberculosis, hemolysin, Structural modeling, RNA methyltransferase.
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Evaluation of the applicability
of serodiagnosis for tuberculosis in Portugal
Carina Ferreira 1, Andrea Afonso 2, Raquel Duarte 3, Konstantin Lyashchenko 4, Anabela
Silva 5, Filomena Rodrigues 5, Anabela Miranda 5, Margarida Tavares 6, Cátia Caldas 6, Fátima
Valente 2, António Valente 2, Olga Vasconcelos 7, Joana Amado 7, Margarida Correia-Neves 1
1 - Life and Health Sciences Research Institute (ICVS), University of Minho, Braga, Portugal
2 - Centro de Saúde de Bragança, Bragança, Portugal
3 - Centro de Diagnóstico Pneumológico de Vila Nova de Gaia, Portugal
4 - Chembio Diagnostic Systems, New York, USA
5 - Centro de Tuberculose e Micobactérias, Instituto Nacional de Saúde Dr Ricardo Jorge, Delegação do Porto, Portugal
6 - Serviço de Doenças Infecciosas, Hospital de São João, Porto, Portugal
7 - Hospital Joaquim Urbano, Porto, Portugal
Diagnosis of tuberculosis requires laboratory techniques to complement clinical information. Among the different techniques in use, the only accurate diagnostic method is based on the search for Mycobacterium tuberculosis growth in
cultures of human biological samples, usually sputum. However, this approach is long, lacks sensitivity for samples with
low bacterial load, and is invasive in the case of non-pulmonary tuberculosis. In order to overcome these constraints,
several novel methodological approaches are under evaluation. Among these is the determination of tuberculosis-specific
antibodies in the sera - serodiagnostic methods.Validation of serodiagnosis tests in any given country is mandatory, since
sensitivity and specificity vary depending on factors such as the composition and frequency of environmental mycobacteria, previous vaccination with BCG, prevalence of tuberculosis and other diseases, and host genetic background. We
evaluated the specificity and sensitivity of two serodiagnosis tests: TB STAT-PAK II - an immunochromatographic test for
the detection of antibodies to Mycobacterium tuberculosis; and MAPIA - Multi-Antigen Print Immunoassay (both developed at Chembio Diagnostics, NY, USA). The specificity of the tests was very high, ranging from 94 to 100% depending
on the test and control population analysed. The sensitivity was 37 and 54% for TB STAT-PAK II and MAPIA, respectively,
and it increased during the first 3 months of treatment, for TB STAT-PAK II. Interestingly, when the smear results were
combined with the ones from the TB STAT-PAK II, the sensitivity increased from 71 (just smear) to 79%.Thus, taking into
consideration the great specificity of the TB STAT-PAK II, its use for smear negative patients could increase the rate of
detection in early TB diagnostic.
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IMPLEMENTATION OF THE THIN LAYER AGAR (TLA) FOR THE DIAGNOSIS OF
SMEAR NEGATIVE PULMONARY
TUBERCULOSIS IN A HIGH HIV PREVALENCE SETTING
Martin, Anandi 1, Munga Waweru, Peter 2, Babu Okatch, Fred 2, Amondi Ouma, Naureen
2
, Bonte, Laurence 2, Palomino, Juan Carlos 1,Varaine, Francis 2, Portaels, Françoise 1
1 - Institute of Tropical Medicine, Antwerp, Belgium
2 - Médecins Sans Frontières, Paris, France
Early diagnosis of smear-negative pulmonary tuberculosis in countries with high incidence of TB/HIV co-infection is
crucial to limit the mortality and control the disease. The objective of this study was to evaluate the performance of a
low-cost method, the Thin Layer Agar (TLA), for the diagnosis of smear-negative patients compared to the gold standard
Löwenstein-Jensen method. TLA relies on microscopic detection of cording growth that is characteristic of M. tuberculosis and is able to differentiate between M. tuberculosis and non-tuberculous mycobacteria. This prospective study
was performed in Homa Bay district Hospital in Kenya. Out of 1584 smear-negative sputum samples, 212 were positive
by LJ (13.5%) and 220 positive by TLA (14%). The sensitivity of LJ and TLA was 71% and 74 % respectively. With further
improvement for decreasing the contamination rate in both methods, TLA could become an affordable method for the
diagnosis of smear-negative tuberculosis in resource-limited settings allowing the simultaneous detection and identification of M. tuberculosis, within 2 weeks.
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DIRECT DETECTION OF RIFAMPIN RESISTANCE IN
Mycobacterium tuberculosis BY THE NITRATE
REDUCTASE ASSAY APPLIED DIRECTLY IN SPUTUM SAMPLES
Regina Ferro e Silva 1, Maria de Lourdes Shikama 1, Gleize Villela 1, Daisy Nakamura Sato 1, Carmen
Maria Saraiva Giampaglia 1, Maria Conceição Martins 1, Anandi Martin 2, Juan Carlos Palomino 2
1 - Instituto Adolfo Lutz (IAL), São Paulo, Brazil
2 - Institut of Tropical Medicine, Antwerpen, Belgium
Resistance to rifampin (RIF) is an important predictor for the early diagnosis of MDRTB. Conventional methods for
DST of M. tuberculosis require several weeks to give results.Thus, an alternative in vitro method which can detect drug
susceptibility directly from sputum and presents quick results and low cost, will be very useful for tuberculosis diagnosis.
The nitrate reductase assay uses the detection of nitrite as an indication of growth when used as a drug susceptibility test.
Objective:to compare the nitrate reductase assay (NRA) with the proportion method (PM), considered as gold standard,
to detect RIF resistance in M. tuberculosis directly from sputum samples. Method: the study was carried out by 4 regional
laboratories from the state of São Paulo, Brazil. A total of 206 sputum samples tested smear positive from patients with
pulmonary tuberculosis. The sputum was decontaminated by Petroff method and DST to RIF was carried out using the
PM and the NRA. Nitrate Reductase Assay: Sputum samples, after decontamination were inoculated in one tube control
(without drug) and one tube containing 40 µg/ml of rifampicin. Findings: the results of the DST obtained directly from
sputum were: 6 samples resistant to RIF and 200 samples susceptible. The comparison between NRA and the traditional
gold standard method showed agreement of 100%. The sensitivity and specificity of the NRA for rifampicin was 100%.
Results were available in 10 days for 66 (34%) samples, 15 days for 102 (53%) samples and 20 days for 24 (13%) samples
while the results of PM took 30 days to be available. Conclusions: the NRA proved to be a promising method for the
screening of suspect MDRTB cases directly from sputum samples. The simplicity of the method, its low cost and celerity
to give the results make it a good alternative method for laboratories in resource-poor settings.
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DIRECT DETECTION OF RIFAMPIN RESISTANCE IN
Mycobacterium tuberculosis BY THE NITRATE REDUCTASE
ASSAY APPLIED DIRECTLY IN SPUTUM SAMPLES
Ferro Regina Silva 1, Shikama Maria de Lourdes 1, Villela Gleize 1, Sato Daisy Nakamura 1, Giampaglia Carmen Saraiva
Martins Maria Conceição 1, Martin Anandi 2,3, Palomino Juan Carlos 2, Telles Maria Alice da Silva 1
1 - Instituto Adolfo Lutz, São Paulo, Brazil
2 - Institute of Tropical Medicine, Antwerp, Belgium
3 - Médecins Sans Frontières, Paris, France
1,
A cost-effective and rapid drug susceptibility testing (DST) method is required to guide TB treatment. Commercially
available systems such as BACTEC MGIT 960 and MB/BacT are faster but demand costly equipment and supplies, therefore, are not feasible in most resource-poor settings. Resistance to rifampin (RIF) is an important predictor for the early
diagnosis of MDRTB. To compare the nitrate reductase assay (NRA) with the proportion method (PM) to detect RIF
resistance in M. tuberculosis directly from sputum samples. The study was carried out by 4 regional laboratories from the
state of São Paulo, Brazil. A total of 210 sputum samples tested smear positive from patients with pulmonary tuberculosis. The sputum was decontaminated by Petroff method and DST to RIF was carried out using the PM and the NRA.
The results of the DST obtained directly from sputum were: 6 samples resistant to RIF and 204 samples susceptible. No
discordance was observed between the two methods.The sensitivity and specificity of the NRA was 100%. Results were
available in 10 days for 75 (36%) samples, 15 days for 107 (51%) samples and 20 days for 28 (13%) samples.The results of
PM took 30 days to be available. The NRA proved to be a promising method for the screening of suspect MDRTB cases
directly from sputum samples. The simplicity of the method, its low cost and celerity to give the results make it a good
alternative method for laboratories in resource-poor settings.
Acknowledgements
This study was partially funded by INCO-Dev ICA4-CT-2001-10087.
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MTBDRPLUS ASSAY IS A USEFUL TOOL TO SCREEN FOR
MULTI-DRUG RESISTANT TUBERCULOSIS IN A NATIONAL SURVEY
De Haas, Petra 1, Ramona Zenhorst 2, Pike Mwamba 2, Mweemba Muvwimi 3,
Winnie Mwanza 2, Grace Mbulo 2, Nathan Kapata 2, Helen Ayles 1
1 - Zambart project, Lusaka, Zambia and LSHTM London
2 - Zambart project, Lusaka, Zambia
3 - National Reference laboratory, Lusaka, Zambia
Background
The World Health Organization requests that countries conduct tuberculosis drug resistance surveys (DRS) every five
years to monitor trends of drug resistance and to determine rates of multi-drug resistant tuberculosis (MDR-TB). Zambia
conducted its second nation-wide DRS in 2008. The objective of this study is to investigate whether the MTBDRplus
assay (HAIN), a new molecular assay performed directly on sputum, is a useful tool in conducting a DRS.
Method
Throughout Zambia approximately 900 sputum specimens were collected from consecutive smear-positive TB patients and transported to the TB Reference Laboratory in Lusaka. Specimens were decontaminated and concentrated smears were prepared. MGIT and LJ cultures were inoculated. Drug susceptibility testing (DST) was performed on positive cultures. Remaining decontaminated sputum was heat-killed, sonicated
and stored at -80C. The MTBDRplus assay was performed using a 1:5 or 1:10 dilution of decontaminated sputum. Results: Of the first 340 specimens tested using the MTBDRplus assay, 307 (90.3%) showed no evidence of resistance,
while thirty-three (9.7%) showed mutations consistent with resistance: 10 were MDR-TB, 20 were isoniazid (INH)
mono-resistant and 3 were rifampicin (RIF) mono-resistant. MGIT DST results were obtained from 271 (79.7%) of
340 specimens with an MTBDRplus result. We were unable to obtain MGIT DST results from the remaining 69 (20.3%)
specimens due to contamination or lack of growth. Thirteen (39.4%) of 33 specimens that showed mutations consistent
with resistance in the MTBDRplus assay failed to yield a DST result on MGIT. Six out of these 13 samples are according
to the MTBDRplus assay MDR, 5 are INH mono-resistant and 2 RIF mono-resistant . One sample that showed RIF monoresistance using the MTBDRplus assay, showed both isoniazid and rifampicin resistance uses the MGIT DST.
Conclusion
In our study, the MTBDRplus assay performed directly on sputum was more rapid and cost-effective than culture to
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CONTRIBUTION OF LABORATORY FACTORS TO HIGH MGIT
CULTURE CONTAMINATION RATE IN ZAMBIA
de Haas, Petra 1, Moyoyeta, Monde 2, Samutela, Mulemba 2, Mwanza, Winnie
2
, Musunsa, Alan 3, Mbulo, Grace 2, Muvwimi, Mweemba 3, Ayles, Helen 1
1. Zambart project, Lusaka, Zambia and LSHTM, London
2. Zambart project, Lusaka, Zambia
3. National reference laboratory, Lusaka, Zambia
Background:
An evaluation study of the MGIT liquid culture system in Zambia found a high contamination rate. It was suggested that factors such as delayed sample processing due to long distances, inadequate storage of samples once submitted, poor laboratory infrastructure and inexperienced staff may have contributed to the high contamination.The objective of this study was
to investigate the contribution of laboratory factors to the higher than expected rate of MGIT contamination.
Method:
As part of the National drug resistance survey, 917 sputum specimens were collected from smear-positive TB patients
and transported to the TB Reference Laboratory in Lusaka. After decontamination using NALC-NAOH (1,5% final concentration) MGIT and LJ cultures were inoculated. In addition 584 of the decontaminated samples were inoculated onto
blood agar plates (BA).When the MGIT culture showed growth Ziehl Nielsen staining was performed. If micro-organisms
other then acid fast bacilli were seen, a BA was inoculated and subsequent colonies identified using biochemical tools. Results
Time between sample submission and processing varied from 1-50 days with a median of 9 days. Increased contamination in MGIT was found when the time between sample submission and processing was extended; 18.6% for 1-7 days,
27.4% for 8-14 days and 37.5% for more than 15 days. The overall contamination rate was 27.6% (95% CI 24.0-31.4). Of
584 decontaminated sputum 197 (33.7%) showed growth on BA. Out of the 197 samples showing growth 86 (43.6%)
were also contaminated in MGIT whereas for samples that showed no growth on BA, 70 (18%) out of 387 were contaminated. Contaminated organism from the sputum and from the contaminated MGIT culture were identified for 35
out of the 86. Identical species were identified in only sixteen (45.7%) of these 35 samples, whereas in 19 (54.3%) cases
a different contaminanting species was found in the MGIT culture compared to the decontaminated sample.
Conclusion
High contamination on MGIT is a problem in our setting. Delayed processing of samples increases the chance of samples
being contaminated. The contamination of samples that did not show any growth on BA from decontaminated sputum
as well as different species isolated from the contaminated MGIT compared to the decontaminated sputum suggest that
a major part of the contamination may be due to laboratory factors. Oral Communication
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PROGRAMMATIC COMMUNITY-BASED MANAGEMENT OF
MDR-TB: EXPERIENCE IN KARACHI, PAKISTAN
Ahmed, Altaf; Qazi, Fahad; Khan, Amir Javed
The Indus Hospital, Karachi, Pakistan
Introduction
Multidrug Resistant Tuberculosis (MDR-TB) is defined as TB resistant to the two most powerful anti-TB drugs, Isoniazid (H)
and Rifampicin (R). Pakistan is ranked 8th among the high TB burden countries.In November 2007, the Karachi DOTS-Plus
program was established with an objective to provide free, comprehensive, community-based management of MDR-TB patients in Karachi and Hyderabad based on Partners in Health (PIH) and World Health Organization (WHO) guidelines.The
purpose of this paper is to share our initial experience at programmatic management of MDR-TB in a low income setting.
Methodology
At baseline registration, smears, cultures, DST, radiology, and ancillary tests were performed on each patient and each
patient house was mapped using GPS devices. Inclusion criteria were as follows:
1) Acid Fast Bacilli Culture and Sensitivity (AFBCS) showing MDR-TB or PDR-TB 2) Clinical, Radiological or Bacteriological (smear and culture) evidence of active disease
Enrollment was based on availability of funds, clinical judgment and proximity to center. All patients were managed in the
community. Each enrolled patient received monthly consultation, uninterrupted and monitored second line drug supply,
laboratory tests as per program guidelines. Social support was also provided to all patients in the form of:
1) Monthly Food Baskets
2) Professional Counseling
3) Treatment Supporters (TS)
Results
As of May 2009, 106 MDR-TB patients were registered in the program (49 males, 57 females). The mean age was 29.58
(+/-12.8). 73 patients were put on treatment. Their classification according to previous treatments was as follows:
1) After failure of retreatment ……30 (41%)
2) Relapse ………………………...14 (19%)
3) After failure of first treatment …12 (16%)
4) New …………………………….6 (8%)
5) Registered after default…………5 (7%)
6) Other…………………………….1 (1%)
7) Transfer in ………………………1 (1%)
68 (90%) patients had received first line treatment previously, 1 (2%) had received second line treatment and 6 (8%) were
new patients. HIV testing on 68 of 73 enrolled patients came negative for all. Adverse events recorded were recorded.
Comorbid conditions included DM,Tobacco use, substance abuse, chronic lung disease, hepatitis, respiratory failure, pregnancy, hypertension, gastritis, renal failure and neuropathy. Although treatment duration for the first pool of p
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EVALUATION OF CAPILIA (TAUNS) FOR RAPID IDENTIFICATION OF
MYCOBACTERIUM TUBERCULOSIS COMPLEX FROM CULTURES
C Muchwa2,3, J Akol2,3, F Mumbowa5, P Orikiriza2,3, K Morgan2,3, K Eisenach4 , M Joloba1,3, A Etwom2,3, P Mugyenyi2, R Mugerwa3
1 - Department of Medical Microbiology, Makerere University Medical School, Kampala, Uganda
2 - Joint Clinical Research Centre, Kampala, Uganda
3 - Uganda-CASE Research Collaboration, Kampala, Uganda
4 - University of Arkansas for Medical Sciences and Central Arkansas Veterans Healthcare System, Little Rock, USA;. 5Infectious Diseases Institute (IDI), Kampala, Uganda
Introduction
The Capilia TB assay is a simple immunochromatographic assay which uses anti-MPB64 monoclonal antibodies to discriminate MTB from non-tuberculosis mycobacteria. Evaluation of Capilia to determine its costs, performance and turn
around time (TAT) was done using PCR IS6110 assay (PCR) as a gold standard.
Methods
Respiratory and blood samples specimens were digested and decontaminated using 1.5% NAOH/NALC, concentrated
by centrifugation and inoculated into BACTEC MGIT 960 culture tubes for incubation. Blood was aseptically inoculated
and incubated in the BACTEC 9120 instrument. All BACTEC positive cultures were screened for acid fast bacilli by the
Ziehl-Neelsen method before testing for MTB. Blood cultures were then inoculated on 7H10 agar and incubated for
MTB isolation.The Capilia test was performed according to the manufacturer’s instructions while PCR was done according to laboratory protocol. The test included 155 respiratory and 70 blood samples were tested.
Results
Overall agreement between Capilia and PCR IS6110 methods was 98.2%. Capilia achieved a sensitivity of 98.4% and
specificity of 97.9%. Initial PCR comparison for respiratory cultures resulted in sensitivity and specificity of 97.4% and
98.7% respectively. Blood achieved specificity of 27.4% only; this may be due to false negative PCR results caused by PCR
inhibitors in blood cultures. PCR testing was performed from colonial growth on 7H10 accuracy and reliability of PCR
as a gold standard and the resulting Capilia test sensitivity increased to 100% and 94.4% specificity.
Conclusion
The Capilia has an overall sensitivity of 98.4% and specificity of 97.9% and is far more accurate method of identifying
MTB directly in blood cultures. It is less expensive (≈ $5) compared to PCR (≈ $45). It is easier to perform with TAT of
20 minutes while PCR has TAT of 8 hours for respiratory cultures.
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COMPARISON OF CAPILIA (TAUNS) AND IS6110 PCR FOR
RAPID IDENTIFICATION OF Mycobacterium tuberculosis
COMPLEX FROM CULTURES IN KAMPALA, UGANDA.
C Muchwa2,3, J Akol2,3, P Orikiriza2,3, K Morgan2,3, F Mumbowa5, K
Eisenach4, A Etwom2,3, M Joloba13
1 - Department of Medical Microbiology, Makerere University Medical School, Kampala, Uganda
2 - Joint Clinical Research Centre, Kampala, Uganda
3 - Uganda-CASE Research Collaboration, Kampala, Uganda
4 - University of Arkansas for Medical Sciences and Central Arkansas Veterans Healthcare System, Little Rock, USA
5 - Infectious Diseases Institute (IDI), Kampala, Uganda
Introduction
The Capilia TB assay, a simple immunochromatographic method which uses anti-MPB64 monoclonal antibodies to discriminate MTB from non-tuberculous mycobacteria has been successfully evaluated in other settings. Before adopting
capilia in our laboratory, an evaluation to determine its costs, performance and turn around time (TAT) compared to the
existing molecular identification method using PCR IS6110 assay (PCR) was done.
Methods
Sputum/Gastric samples were processed using the 1.5%NAOH/NALC decontamination method and the sediment
cultured using the BACTEC MGIT 960 system.. Blood/Pleural fluids were aseptically inoculated in the BACTEC 9120
instrument. All BACTEC positive cultures, (sputum and body fluids) were screened for acid fast bacilli by the ZiehlNeelsen method before testing for MTB. The Capilia test was performed on all ZN positive MGIT 960 tubes according
to the manufacturer’s (TAUNS ) instructions while the PCR was done according to laboratory protocol.The ZN positive
blood and other body fluid cultures were inoculated on 7H10 agar for MTB isolation. Capillia was performed on ZN
positive isolates.The test included 155 sputum/gastrics and 70 body fluid samples.The Capilia and PCR assays were done
by separate technicians who were blinded to the other test results.
Results
There was an overall agreement of 98.2% between the Capilia and PCR IS6110 methods. Capilia achieved a sensitivity
of 98.4% and specificity of 97.9%. These results concurred with the finding of Jann Wang et al publication of 98.6% &
97.9% respectively. The initial PCR comparison for sputum/gastric cultures resulted in a 97.4% and 98.7% sensitivity and
specificity respectively. When the PCR test was performed from colonial growth on 7H10, the accuracy and reliability of
PCR as a gold standard increased and the resulting Capilia test sensitivity was 100% and 94.4% specificity. The sensitivity
and specificity of Capilia on contaminated cultures was 97.0% and 98.7% respectively while that on pure cultures was
99.0% and 94.4%. There was no significant difference in the test performance between contaminated and pure cultures
(p values of 0.12405 and 0.786 respectively). The recurrent cost for capilia was ~$5 and that of PCR ~$45. The average
TAT for Capilia was 20 minutes while that of PCR 8 hours for sputum/gastric cultures and 22 days for blood cultures.
Overall Capilia was much easier to learn, had fewer steps and requires no instruments
Conclusion/Discussion
• The sensitivity and specificity of Capilia is comparable to PCR for both pure and contaminated MTB cultures.
• Capilia is less expensive, faster and easier to perform than PCR.
• Capilia is capable of identifying MTB directly in blood cultures.
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DIRECT TESTING FOR MULTI DRUG RESISTANT TUBERCULOSIS WITH
FOUR ASSAYS EVALUATED AT KAMPALA, UGANDA
Freddie Bwanga1,2,3, Sven Hoffner2,3, Melles Haile2,3, Moses L Joloba1
1 - Department of Medical Microbiology Makerere University College of Health Sciences Kampala, Uganda
2 - Department of Bacteriology, Swedish Institute for Infectious Diseases Control, Solna Sweden
3 - Department of Microbiology, Tumour and Cell Biology (MTC), Karolinska Institute, Stockholm, Sweden
Background
Multi drug resistant tuberculosis (MDR TB) is on the rise worldwide. Early detection of MDR TB is important for effective
control of MDR TB transmission. In most TB high burden countries this remains a challenge due to lack of rapid tests.
This study evaluated four rapid tests for MDR TB detection.
Methods
Smear positive re-treatment TB patients were consecutively recruited at Mulago – Uganda’s National referral Hospital.
Samples were processed using a final concentration of 1.5% NAOH-NALC method. Sediments were used directly to
set susceptibility to isoniazid and rifampicin with four rapid tests at the National TB Reference Laboratory Kampala.
The four tests included the Nitrate Reductase Assay (NRA), Microscopic Observation Drug Susceptibility (MODS),
Mycobacterium Growth Indicator Tube (MGIT 960) and Genotype® MTBDRplus (Hain Life Sciences, Nehren, Germany).
Results of the four tests were compared to those of the conventional indirect Lowenstein-Jensen proportion method (L-J PM).
Results
A total of 66 patients were recruited. Interpretable results were obtained for all the samples with the LJ PM and MODS
assay, 64 (97%) with the NRA and MGIT 960, and 62 (94%) with the Genotype® MTBDRplus. Interpretable results across
all the five tests were available for 56 samples and results obtained on initial testing were 100%, 98%, 91%, 82% and 68%
with the Genotype® MTBDRplus, MODS, LJ PM, NRA, and MGIT 960, respectively. Repeat testing with the MGIT 960 was
due to power failure -13 samples, contamination - 4 samples and undergrowth - one sample.
Sensitivity and specificity for detection of resistance to isoniazid was 100% and 100% for NRA, 100% and 95% for MODS,
93% and 98% with the Genotype® MTBDRplus, and 93% and 100% with the MGIT 960, respectively. For rifampicin it was
100% and 100% with NRA, 91% and 93% with MODS, 100% and 96% with Genotype MTBDR®plus, and 73% and 100%
with the MGIT 960, respectively.
The average time to results was 2 days (range 1-3 days) for Genotype® MTBDRplus, 8 days for MGIT 960 (range 5-13
days), 8 days for MODS (range 7-18 days) and 11 days for NRA (range 10-21 days). Results obtained within 10 days were
91%, 88%, and 75% for the MODS, MGIT 960, and NRA, respectively.
Conclusion
Findings show excellent performance of the direct NRA, MODS, and Genotype® MTBDRplus for MDR TB detection, with
most of the interpretable results obtained on initial testing in <14 day.
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PEA PRODUCTION BY MYCOBACTERIA AND ITS
APPLICATION IN A RAPID DRUG SUSCEPTIBILITY TEST.
McNerney, Ruth 1, Turner, Claire 2, Mallard, Kim 1, O’Sullivan, Denise 1
1 - London School of Hygiene & Tropical Medicine
2 - Cranfield University
Metabolites produced during bacterial growth may be used to monitor the impact of drugs on bacteria. We have investigated volatile organic compounds emitted by mycobacteria growing on Lowenstein Jensen (LJ). Mass spectrometry
determined one of the major volatile compounds from M. bovis BCG to be phenylethyl alcohol (PEA), a bacteriostatic
compound that is a reversible inhibitor of DNA synthesis. PEA production was also observed in mycobacteria other than
tuberculosis (MOTT).That such a compound is produced in quantity by mycobacteria growing on LJ is surprising and may
explain the limited growth of mycobacteria on this media. PEA is only produced during bacterial growth and monitoring
production during exposure may provide a means of determining susceptibility to antimicrobial compounds. To test this
hypothesis, bacteria were placed on LJ slopes containing drug and incubated at 37C. Headspace vapors from the culture
tubes were analysed using the zNose, an ultra-rapid capillary gas chromatograph coupled to a SAW detector. The test
required no sample preparation and each slope was tested in less than 2 minutes.To minimize the risk of creating infected
aerosols culture tube caps incorporated a PTFE/silicone septum enabling air to be drawn from the tube without removing the cap. Samples collected were heat sterilized during testing. Headspace samples from drug containing slopes were
compared to control slopes without drug. The incubation time required for differentiation between positive and negative
samples or ‘growth’ and ‘no growth’ depended on the size of the inoculum and the species of mycobacteria and ranged
from hours to a few days. MOTT could be differentiated from M. tuberculosis complex bacilli by continued production of
PEA in the presence of 500ug/ml para-nitrobenozic acid. We conclude that detection of volatile metabolites emitted by
mycobacteria growing on Lowenstein Jensen offers a simple, rapid alternative to assess their drug susceptibility.
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PP-99
VARIOUS STRATEGIES TO DECONTAMINATE ACID FAST BACILLI
POSITIVE LIQUID CULTURES FROM BACTEC MGIT 960
Balmoi, Faith
Joint Clinical Research Centre, Kampala, Uganda
Introduction
Though liquid culture is enriched and more sensitive in the recovery of MTB, it yields a greater percentage of contaminants as well. It is essential that MTB cultures be isolated pure to permit drug susceptibility testing which is vital for prognosis of patients besides determining the presence of MDR-TB and XDR-TB.
In this study we evaluated various approaches to reduce or eliminate contamination in Bactec MGIT 960 cultures. Method
Contaminated AFB PCR positive MGIT cultures were subjected to decontamination methods of:
i) Double PANTA Polymyxin B (750µg/ml); Amphotericin (75µg/ml); Nalidixixic (300µg/ml); Trimethoprim (75µg/ml) and
Azlocillin (75µg/ml)
ii) VAN vancomycin (30.5µg/ml), Amphotericin (106µg/ml) and Nalidixic acid (226.7µg/ml) iii) 2% NaOH each contaminated culture was divided and subjected to all three decontaminating procedures. The decontaminated cultures were then inoculated in MGIT tubes and 7H10 selective whole plates and incubated in Bactec MGIT 960 and a 5%-10% CO2 at 37°C respectively.
The plates were read weekly until a sufficient colonial growth was achieved. The positive MGIT was subjected to ZiehlNeelsen (ZN) smear and blood agar culture for purity check. Results
A total of 50 specimens had analyzable results for each method. Double PANTA was able to recover 62.0%,VAN 60.0%
and 2% NaOH 24.4% while 16.0%, 14.0% and 6.7% respectively remained contaminated. However, 22.0%, 26.0% and
68.9% were non-viable after decontamination with double PANTA, VAN and 2% NaOH respectively. Further testing is
being done so as to generate more reliable results.
Conclusion
• VAN and double PANTA were comparable as far as giving more specimens (p value = 0.614). With pure cultures
when they are used for decontamination.
• More specimens were unrecoverable when 2% NaOH was used than the other two methods.
• The cost of decontaminating a sample using VAN was ~$1 while double PANTA was ~$5.70
European Society of Mycobacteriolog y | 30 th Annual Congress | July 2009 | Por to - Por tugal
171
PP-100
LOW COST ISOLATION OF Mycobacterium tuberculosis (MTB) FROM BLOOD
Orikiriza, Patrick
Uganda-CASE Research Collaboration, Kampala
Introduction
Routinely, identification of MTB from body fluids requires a PCR based technique which has been adopted as a gold
standard. This technique however has been unsuccessful against blood cultures which are often affected by inhibitors. In
this study, we evaluated a simple but effective way of isolating and identifying MTB in blood cultures. Method
A total of 91 blood cultures positive for acid fast bacilli were sub-cultured onto 7H10 medium and Lowenstein Jensen
slants and incubated at 37ºc in a 5% carbon dioxide incubator. The isolates that grew on solid media were further tested
by PCR to confirm MTB. The original blood cultures were tested by diluting an aliquot of blood culture in PCR waters
and repeating the IS6110 PCR and also by Capilia for identification of MTB.
Results
The study found 15 cultures PCR positive when diluted in PCR water and 56 positive with Capilia. On the other hand,
cultures grown on the solid media before performing PCR yielded 53 PCR positives from 7H10, 44 PCR positives from
LJ and 28 did not grow on either medium. The time it took for the cultures to turn positive on the different media was also noted
Conclusion
Identification of MTB by PCR was more efficient blood cultures were sub cultured to solid media for identification assay
Capilia tests performed directly from blood culture media gave comparable results (95%) with MTB identification by
PCR from solid growth.
There was an overall better turn around time using the 7H10 media compared to LJ medium
172
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PP-101
COMPARISON OF RAPID COLORIMETRIC METHOD,
PROPORTION METHOD AND BACTEC460 TB SYSTEM FOR TESTING
.
SUSCEPTIBILITY OF M.tuberculosis TO RIFAMPINE AND ISONIASIDE
. .
o
Begum KAYAR, Ülkü ORAL ZEYTINLI, Ayse
, KARACALI, Ayben SOYAL, Togrul Nagiyev, Fatih KÖKSAL
Cukurova University, Medical Faculity
Introduction
Multidrug-resistant (MDR) M. tuberculosis (MTB) strains founded resistant to at least isoniaside [INH] and rifampin [RIF],
the two most important first-line drugs pose a serious threat to the control of tuberculosis (TB) and the spread of these
strains has become a major public health problem. In this study, the performance of antimycobacterial susceptibility testing for the first line drugs (RIF and INH) with colorimetric nitrate reductase-based antibiotic susceptibility (CONRAS)
and conventional proportion method depend on solid agar culture (LJ) were compared to BACTEC 460 TB system as
the gold standard.
Methods
Of the total of 187 MTB strains, when 24 isolates were found as resistant to RIF and INH by Bactec 460 TB other 163
strains were susceptible for these antibiotics. All of strains were isolated in sputum from patient with pulmonary disease and identified as MTB by NAP test in Bactec 460 system. The CONRAS test was performed with 0.1 mg/mL of
INH and 1 mg/mL of RIF on LJ as modification of standart methods described by H.Syre et al . Antibiotic susceptibility
testing with proportion method was performed on LJ medium according to the standard protocol laid down by WHO. Results:The sensitivity, specificity and overall agreement of the CONRAS test were 95.83% (23/24), 99.38%(162/163) and
97.60% for RIF and 83.33% (20/24), 99.38% (162/163) and 91.35% for INH, respectively. Results for proportion tests were
found as; 91.66% (22/24), 93.25% (152/163) and 92.45% for RIF and 75% (18/24), 97.54% (159/163) and 86.27% for
INH, respectively by Bactec 460TB system as the gold standart.
Interpretation & conclusion
CONRAS test showed good agreement with Bactec 460 (the overall agreement of this test was 94.47%) for each of the
antimicrobial tested. CONRAS test is simple, easy to perform, less expensive, reliable and may be used as a preliminary
screen for susceptibility testing of MTB in particularly for resource-poor countries. European Society of Mycobacteriolog y | 30 th Annual Congress | July 2009 | Por to - Por tugal
173
PP-102
EVALUATION OF THE MGIT TBc ID TEST VS TWO COMMERCIALLY
AVAILABLE RAPID IMMUNOASSAYS FOR M. tuberculosis
COMPLEX ORGANISM DETECTION FROM LIQUID AND SOLID CULTURE
Crews, Virginia 1, Warns, Matthew 1, Pfeltz, Richard 1, Beaty, P. Shawn 1, Rosales, Julie 1, Kopher, Ken 1, Joshi, Sudhaunshu 1,
Hoosen, Anwar 2, Said, Halima 2
1 - BD Diagnostic Systems, Sparks, Maryland, United States of America
2 - Department of Medical Microbiology, Faculty of Health Sciences, University of Pretoria and National Health
Laboratory Service, Tshwane Academic Division, Pretoria, South Africa
Introduction
Chromatographic lateral flow immunoassays for the MPT64 protein antigen can provide a rapid and cost-effective method to qualitatively detect Mycobacterium tuberculosis complex (Mtbc) organisms from acid-fast bacillus (AFB) positive
cultures.The purpose of this study was to compare the sensitivity and specificity of three such devices with mycobacteria
from liquid and solid media cultures.
Methods
Positive liquid (BACTEC MGIT 960 7 mL tubes) and solid (BBL Löwenstein-Jensen slants) culture media seeded with mycobacteria provided inocula for side-by-side evaluation of two commercially available tests, Capilia
TB (“Capilia”, Tauns Laboratories, Numazu, Japan) and SD Bioline TB Ag MPT64 Rapid Test (“Bioline”, Standard
Diagnostics, Inc, Kyonggi-do, Korea), and the BD MGIT TBc Identification Test (“TBc ID”, BD Diagnostic Systems,
Sparks, MD, USA). The MGIT TBc ID test is currently in clinical trials. Devices were inoculated per manufacturer’s instructions and visually assessed as positive for detection (visible test line) and reagent function (visible control line).
RESULTS. All three devices detected each of the 20 Mtbc organisms tested (100% sensitivity), which included 15 M.
tuberculosis strains. Specificities for the 18 non-tuberculous mycobacteria (NTM) tested were as follows: 100% for the
TBc ID device, 94% for the Capilia device (due to cross-reactivity with M. marinum), and 94% for the Bioline device (due
to cross-reactivity with M. gastri). Additional testing with the two cross-reactive NTM species confirmed these results.
Sensitivity and specificity results for a given device were identical between solid and liquid media. However, flow issues
after inoculation from solid medium as well as background discoloration after inoculation from liquid medium occurred
regularly with the Bioline device.
Conclusions
The three rapid Mtbc organism identification immunoassay products exhibited very good sensitivity.The specificity demonstrated by the TBc ID device was also very good, but specificity was lower for the Capilia and Bioline devices due to
cross-reactivity with specific NTMs.
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PP-103
NITRATE REDUCTASE ASSAY APPLIED TO DIRECT DETECTION OF
DRUG RESISTANCE IN MYCOBACTERIUM TUBERCULOSIS.
Montoro, Ernesto 1, Milián,Yoslaine 1, Lemus, Dihadenys 1, Echemendía, Miguel 1,
Yzquierdo, Sergio 1, Martin, Anandi 2,Van der Stuyft, Patrick 2, Palomino, Juan Carlos 2
1 - Institute of Tropical Medicine Pedro Kourí (IPK), Havana, Cuba
2 - Institute of Tropical Medicine, Antwerp, Belgium
Tuberculosis still represents a major public health problem; especially in low-resource countries were the burden of the
disease is more important. Standard methods for drug susceptibility testing of Mycobacterium tuberculosis, such as the
proportional method (PM), the absolute concentration method, and the resistance ratio method, are used globally but
depend on culture on solid media and are therefore time-consuming. The time lag is a significant threat to the patient,
the community, and health care workers. To reduce this period, we have evaluated the nitrate reductase assay (NRA)
on smear-positive sputa for the direct detection of drugs resistance in Mycobacterium tuberculosis. A total of 63 smearpositive sputum were used in this study. The samples were decontaminated using the modified Petroff method, a portion was used to carried out the NRA as susceptibility test to isoniazid (INH, 0,2 µg/mL), streptomycin (SM, 4 µg/mL),
ethambutol (EMB, 2 µg/mL) and rifampicin (RIF, 40 µg/mL). The resulting sample was used to perform the indirect PM
on Löwenstein-Jensen which was used as gold standard method. The NRA results were obtained between 14 and 28
days. However, were necessary 28 or 42 days to obtain PM results.The sensitivity of MNR to INH, SM, EMB and RIF was
88,9%, 75%, 0% and 71,4% respectively. The low sensitivity obtained for all drug evaluated was due to a small amount of
resistance strains founded. On the other hand, the specificity of MNR to each drugs was 96,3% (INH), 96,1% (SM), 95,2%
(EMB) and 98,2% (RIF). In general, the concordance between MNR and PM was 95,2% to INH, 92,1% to SM, 93,7 to EMB
and 95,2% to RMP. In conclusion, the MNR applied in sputum samples is more rapid than any conventional method. Two
drugs that define multidrug-resistance and are the most potent in the treatment of tuberculosis, INH and RIF, showed
the higher values of concordance wit PM. The MNR results are easy to performance, use each drug with identical critical
concentration in the same solid media than PM and could be a useful tool for detection of tuberculosis drug resistance
in low-resource countries with limited laboratory facilities.
European Society of Mycobacteriolog y | 30 th Annual Congress | July 2009 | Por to - Por tugal
175
PP-104
USE OF NICOTINAMIDE IN COLORIMETRIC METHODS FOR RAPID
DETECTION OF PYRAZINAMIDE RESISTANCE IN
MYCOBACTERIUM TUBERCULOSIS
Montoro, Ernesto 1, Lemus, Dihadenys 1, Madruga, Mariela 1, Mirabal, Niuris 1, Milián Yoslaine 1, Yzquierdo, Sergio 1,
Echemendía, Miguel 1, Martín, Anandi 2,Van der Stuyft, Patrick 2, Palomino, Juan Carlos 2
1 - Institute of Tropical Medicine Pedro Kourí (IPK), Havana, Cuba
2 - Institute of Tropical Medicine, Antwerp, Belgium
Pyrazinamide (PZA) is one of the most effective anti-tuberculosis drugs. It is also bactericidal to semidormant
Mycobacterium tuberculosis and it reduces the total treatment time. The current susceptibility testing methods for this
drug are difficult due to the poor growth of the bacteria in acid medium which is required for drug activity. One alternative has been the use of nicotinamide (NIC), an analogue of PZA that can be converted by PZAase into active form in
a physiological pH that does not hinder bacterial growth. Recently, the NIC has been applied successfully in inexpensive
susceptibility testing such as nitrate reductase assay (NRA) for rapid detection of PZA resistance. The purpose of this
study was to develop the NRA and malachite green indicator (MGI), both with NIC, as rapid susceptibility testing to PZA
in M. tuberculosis. The NRA and MGI were carried out on 120 M. tuberculosis strains from the collection at Tuberculosis
National Reference Laboratory. The concentration of NIC applied in both methods was 1 000 µg/mL and all results
were compared with Wayne method which was used as gold standard, employing 100 µg/mL of PZA.The Wayne method
results were obtained in 4-7 days whereas MGI and NRA results required 7-14 days. A total of 17 and 85 strains were
reported as resistant and susceptible respectively by the three methods but MGI had 16 discordant results and NRA only
4 discrepancies.The MGM showed a sensitivity and specificity of 80,95% and 87,88% respectively whereas NRA provided
a sensitivity of 90,48% and specificity of 97,98%. In general, the concordance of MGI and NRA were 86,67% and 96,67%
respectively.The MGI employing NIC showed a low sensitivity to detect resistance to PZA. In contrast, the NRA showed
a high concordance with Wayne method.This assay is rapid, accurate and could be an attractive option for rapid detection
of PZA resistance, especially in limited-resource countries with high levels of resistance.
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ESM 2009
PP-105
Implementation of liquid culture for tuberculosis
diagnosis in a remote setting: lessons learned
Pamela Hepple1, Jonathan Novoa-Cain2, Chris Cheruiyot2, Elvira Richter3, Koert Ritmeijer4
1 - Manson Unit, Médecins sans Frontières, London, UK
2 - Médecins sans Frontières OCA South Sudan, Lokichoggio, Kenya
3 - Forschungszentrum Borstel, Nationales Referenzzentrum für Mykobakterien, D-23845 Borstel, Germany
4 - Médecins sans Frontières OCA, Amsterdam, the Netherlands
Issues
The diagnosis of tuberculosis (TB) in Médecins sans Frontières (MSF) projects is based on sputum smear microscopy, which has low sensitivity. Following WHO recommendations, MSF established a TB liquid culture laboratory in
Lokichoggio, Kenya, processing samples from 4 South Sudan projects, for diagnosis of smear-negative and extra-pulmonary (EP) TB and follow-up of patients.
Description
The manual MGIT (mycobacterial growth indicator tube, Becton Dickinson) system was used with Lowenstein-Jensen
media. One positive culture per patient was sent to Borstel Supranational Reference Laboratory, Germany for speciation
using the Hain Genotype Mycobacteria series and sequencing techniques.
From March 2007 to December 2008, sputum culture was performed for 64 diagnostic and 24 follow-up patients. Ten
EP samples were also cultured.
For diagnostic patients, of two smear-positives, one was culture-positive for both Mycobacterium tuberculosis (MTB) and
M. fortuitum, and one for M. fortuitum.
Eight of 62 (13%) smear-negatives were culture-positive for MTB complex (3 for MTB and 5 for MTBC) with nine (14%)
culture-positive for other identified mycobacteria; six (9%) grew unknown, not validly-described mycobacteria, and 39
(61%) were culture negative.
For follow-up patients, of seven smear-positives, one was culture-positive for MTB, one for M. intracellulare and one for
M. fortuitum complex, and four (57%) were culture-negative. Among the smear-negatives, eight of 17 (47%) were culturepositive, four of which were unknown mycobacterial species, and four non-tuberculous mycobacteria (NTM). Nine (53%)
were culture-negative.
Samples from three EP patients of 10 grew mycobacteria, species unidentifiable.
In total, only 10 of 36 (28%) culture-positive patients grew mycobacteria from sputum which could be identified as MTB or MTBC.
Lessons learned
Due to the long turn-around time between sample production and species identification due to shipment issues (approximately 4-6 weeks), clinicians often did not wait for results before initiating or adjusting therapy.The high proportion
of NTM was difficult to interpret. The culture results had little clinical impact, and culture lab activities were suspended
in February 2009.
Recommendations
Future TB culture programmes require facilities for on-site speciation of mycobacteria or, if working with reference labs,
should minimize turn-around time to results by ensuring timely shipment of samples.
European Society of Mycobacteriolog y | 30 th Annual Congress | July 2009 | Por to - Por tugal
177
PP-106
RAPID DETECTION OF RESPIRATORY ACTIVE
MYCOBACTERIA BY AURAMINE O-CTC DOUBLE STAINING
Ichijo, Tomoaki; Izumi,Yoko;Yamaguchi, Nobuyasu; Nasu, Masao
Osaka University
Introduction
Clarifying the dynamics of nontuberculosis mycobacterial (NTM) and determining their hot spots in aquatic environments are important to prevent their infection to human beings. For this purpose, methods for the detection of active
mycobacterial cells are required. Culture methods are widely used for detection of mycobacterial cells, but these methods are rather difficult to detect mycobacteria quantitatively and rapidly. Mycobacterial cells are quantifiable under a
fluorescent microscope within several hours with acid-fast staining. In addition, several fluorochromes are available to
determine the bacterial activities. In this study, we attempted to detect mycobacteria with physiological activity rapidly
by combining Auramine O acid-fast staining with 5-cyano-2, 3-ditoryl tetrazolium (CTC) as an indicator of respiratory
activity (Auramine O-CTC).
Methods
Mycobacterium smegmatis was stained with CTC and fixed with formaldehyde. Fixed cells were stained with Auramine
O and observed under an epifluorescent microscope with blue excitation.
Results And Discussion
We firstly optimized the condition of the Auramine O-CTC double staining method to address the following problems;
(i) CTC formazan dissolved in the decolorization step and (ii) fluorescent intensity of Auramine O was decreased by
fluorescent resonant energy transfer (FRET). Specificity of this method was confirmed by staining non-targeted bacteria.
Results can be obtained within 90 min by the optimized procedure, while more than one week is required for the culture-dependent approach. In conclusion, the Auramine O-CTC double staining method was useful for rapid detection of
respiratory active mycobacteria. This method may contribute to identifying dynamics of NTM in aquatic environments.
Acknowledgement
This work was supported by the JSPS Grant-in-Aid for Scientific Research (A)(20249007).
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PP-107
SYNERGISTIC ACTIVITY OF TWO ANTITUBERCULOUS
DRUG COMBINATIONS AGAINST CLINICAL ISOLATES OF
Mycobacterium tuberculosis RESISTANT TO ISONIAZID.
Emma Rey, Griselda Tudó and Julià González-Martín
Servei de Microbiologia. Hospital Clínic-IDIBAPS. Dept. Microbiologia i Parasitologia Sanitaria, Universitat de Barcelona.
Spanish Network for the Research in Infectius Diseases (REIPI, RD06/0008).
Objective
To determine the synergistic activity of 2 drug combinations: isoniazid (H)+rifampicin (R)+ethambutol (E) and ofloxacin
(O)+R against M. tuberculosis clinical isolates resistant to H versus drug susceptible isolates.
Methods
Individual MICs of the strains were studied. Both combinations were studied crossing 7 concentrations of each antibiotic
(including their MIC). The inoculum was of 104CFU/ml. All cultures were performed with the proportional method in
Middlebrook 7H11 medium and incubated 4 weeks. On analysing the results of the combinations, the fractional inhibitory
concentration (FIC) was interpreted: FIC≤0.5, synergistic activity; FIC from 1-4 indifference and FIC>4 antagonistic activity.
Results
H+R+E Combination: 11 H resistant isolates were studied: 4 (36%) isolates had MIC=52µg/ml and 7 (64%) had MIC=0.8µg/
ml. 9 drug-susceptible isolates were also studied. Among the 20 isolates the individual MIC for R ranged from 1-2 µg/ml,
from 2.5-5 µg/ml for E, being 0.05µg/ml for H in susceptible isolates. In H-resistant isolates, the MIC in combination for
H and R decreased up to 3 dilutions (average) respect to their individual MIC. A lesser decrease (2-3 dilutions) was observed for E. In H-resistant isolates, 9/11 (81.1%) showed synergistic activity while 2/11 (18.18%) of the resistant isolates
indicated indifference (FIC index =0.7). MICs in combination of susceptible strains decreased an average of 2 dilutions
compared to their individual MIC. The susceptible strains had FIC indices ≥ 0.748. O+R Combination: 21 isolates were
studied:11 had MIC≤1µg/ml, 4 MIC=1-6.4 µg/ml and 6 MIC>6.4. 9 drug-susceptible isolates were studied. Individual MICs
for R ranged from 1-2, with 1 for O. MICs in combination of all strains were the same or decreased up to 1 dilution with
regard to their individual MIC. The combination of OR in 21 strains did not show synergism or antagonism in either the
resistant or the susceptible strains being the FIC values mainly 1.5.
Conclusions
1)These results suggest that in strains resistant to H with an MIC of 0.8µg/ml in vitro, the standard antibiotic regimen, including H, would be effective due to the possible compensatory effects of R and E. 2) Although strains resistant to H with
an MIC of 51.2 µg/ml showed synergism, these strains would be within the range of resistance. 3)The combination of HRE
against susceptible strains and that of OR did not show synergism probably because of high individual bactericide action.
European Society of Mycobacteriolog y | 30 th Annual Congress | July 2009 | Por to - Por tugal
179
PP-108
TOBRAMYCIN-CLARITHROMYCIN COMBINATION ON
Mycobacterium tuberculosis CLINICAL ISOLATES
Karolien Stoffels1, Hamidou Traore2, Raymond Van Hoof3 and Maryse Fauville-Dufaux1
1 - National Reference Laboratory of Tuberculosis and Mycobacteria, Scientific Institute of Public Health, Department Institut Pasteur, Rue Engeland 642, 1180 Brussels, Belgium
2 - Laboratoires SMB, Rue de La Pastorale 26-28, 1080 Brussels, Belgium
3 - National Reference Laboratory of Resistance to Aminoglycosides, Scientific Institute of Public Health, Department Institut Pasteur, Rue Engeland 642, 1180 Brussels, Belgium
Objectives
This study investigated the in vitro susceptibility of 25 Mycobacterium tuberculosis clinical isolates to two well-known drugs,
tobramycin (TM) and clarithromycin (CL).The effect of both drugs administered together was also investigated to detect
a possible synergistic effect.
Methods
MIC of isolates with variable resistance profiles was determined by the radiometric BACTEC 460 TB and was defined as
the minimal antibiotic concentration for which at least 99% of the mycobacterial population growth was inhibited. The
influence of TM on the MIC of CL was interpreted by Fractional Inhibitory Concentration (FIC).
Results
The median MIC for both TM and CL was 8 µg/ml (range: 2 to 8 µg/ml and ≤2 to >16 µg/ml respectively). For 36% (9/25)
of the tested isolates a decrease of the MIC of CL by a single or twofold dilution was observed (FIC ≤ 0,5) when a subinhibitory concentration of TM was added. No antagonistic effect (FIC > 4) was observed. Similar results were observed
for isolates susceptible or resistant to first-line antituberculosis drugs.
Conclusion
Although the MICs for CL and TM seem high compared to conventional anti-tuberculosis drugs, these antibiotics should
not immediately be ruled out as clinically insignificant. Indeed, after administration of 300mg aerosolised TM, antibiotic concentrations in the epithelial lining fluid are higher than 10 times the median MIC observed for the TB isolates tested in this
study (8µg/ml). Oral administration of 500 mg CL twice daily leads to a peak concentration of 13,50 +/- 3,3 µg/g in the lungs,
505 ± 293 µg/ml in alveolar cells and 34 ± 5 µg/ml in epithelial lining fluid, thus all exceeding the MIC of 8 µg/ml.
Promising new drug delivery systems such as the Dry Powder Inhaler allow achieving very high local antibiotic concentration, e.g. 7 times higher for TM compared to aerosol administration and thus far beyond the MIC of resistant isolates.
These results suggest that both drugs should be investigated further as potential adjuncts to the difficult treatment of
multi-drug resistant tuberculosis where other alternatives have failed.
180
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PP-109
PREVALENCE OF EFFLUX-MEDIATED RIFAMPICIN RESISTANCE
IN Mycobacterium tuberculosis CLINICAL ISOLATES
Carrie K. W. Au-Yeang, T. K. Au, Edward W. C. Chan, Raphael C.Y. Chan
Department of Microbiology,The Chinese University of Hong Kong,The Prince of Wales Hospital, Shatin, New Territories, Hong Kong
Rifampicin is one major ingredient in the cocktail-regimen used in treatment of Mycobacterium tuberculosis (MTB) infection. Although mutations in the rpoB gene are considered the basis of rifampicin resistance, we noted that a significant
proportion of local resistant cases could not be attributed to mutations. Alternative mechanisms such as drug efflux
have been proposed. In order to evaluate the role of drug efflux mechanisms in mediating rifampicin resistance in MTB,
we examined the prevalence of efflux activities in rifampicin resistant isolates using three efflux inhibitors: reserpine,
carbonyl cyanide chlorophenylhydrazone and verapamil. Forty-two rifampicin resistant and nine drug susceptible MTB
clinical isolates were studied. The minimum inhibitory concentration (MIC) values for rifampicin were determined in the
presence and absence of efflux inhibitors. The magnitude of MIC reduction for each efflux inhibitor and the prevalence
of efflux-mediated resistance were examined. We found that among the three efflux inhibitors tested, significant MIC
reduction, ≥ 2-fold MIC decrease, was observed only for verapamil. 61% (31/51) of the test isolates had an MIC reduction between 2 to 8-fold in the presence of verapamil. This verapamil-sensitive efflux-mediated MIC reduction effect
was much more apparent in rifampicin resistant isolates than the drug susceptible controls: 71% (30/42) versus 11%
(1/9). Likewise, this phenomenon was more prevalent in isolates resistant to 3 - 5 anti-tuberculosis drugs than isolates
resistant to 1 - 2 drugs: 77% (24/31) versus 55% (6/11). This data support the notion that drug efflux systems contribute
significantly to rifampicin resistance in MTB clinical isolates and highlight the need for determining the extent by which
these systems contribute to resistance to other anti-tuberculosis drugs.
This study is supported by a grant (08070292) from Research Fund for the Control of Infectious Diseases.
European Society of Mycobacteriolog y | 30 th Annual Congress | July 2009 | Por to - Por tugal
181
PP-110
INTRA AND EXTRACELLULAR ACTIVITY OF RUTHENIUM COMPLEXES
AGAINST Mycobacterium tuberculosis AND THEIR CYTOTOXICITY
Leite, Clarice Queico Fujimura 1, Pavan, Fernando Rogério 1, Maia, Pedro I da S 2, Deflon,Victor M 2, Sato, Daisy Nakamura 3,
Azevedo, Alzir A 4, Poelhsitz, Gustavo V 4, Leite, Sérgio Roberto Andrade 1, Franzblau, Scott G 5
1 - São Paulo State University
2 - University of São Paulo
3 - Adolfo Lutz Institute
4 - Federal University of São Carlos
5 - University of Illinois
Introduction
Worldwide, tuberculosis (TB) remains the most frequent and important infectious disease causing morbidity and death.
One-third of the world’s population is infected with Mycobacterium tuberculosis (MTB), the etiologic agent of TB. The
World Health Organization estimates that about eight million new TB cases occur annually. The pulmonary TB (most
common form of TB), is highly contagious and has been increasing in incidence in many areas, not only in developing countries but also in industrialized countries. The global resurgence of TB and the rapid emergence of MDR-TB, underscore
the importance of the development of new antituberculous drugs.
Objectives
Objecting to find new compounds with high activity against TB, we determined the cytotoxicity, and intra/extracellular
anti-M. tuberculosis activity of 29 new compounds involving different class of ligands such as diimines, phosphines and
Schiff bases with ruthenium.
Material and Methods
As analytical methods, three standardized techniques were used: 1- in vitro Microplate Rezarurin Assay (REMA) for detection of minimal concentration of compounds necessary to inhibit 90% of bacillary growth (PALOMINO et al., 2002),
2- Cytotoxicity (IC50) of compounds against mamarian cells (AHMED et al., 1994) and 3- Determination of compounds
activity against M. tuberculosis internalized in a macrophage cells (SNEWIN et al., 1999).
Results and Conclusion
From the 29 compounds analyzed, 7 of them containing the ruthenium, were qualified as promising anti-TB agents.These
complexes presented inhibitory activity ranging of 0.25 – 3.9 μg/mL, whose values are better than some drugs commonly
used in the TB treatment, low cytotoxicity (IS > 10) and intracellular inhibitory activity high than 70%.
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PP-111
ANTI-MYCOBACTERIUM TUBERCULOSIS ACTIVITY OF
THIOSEMICARBAZONES, SEMICARBAZONES AND HYDRAZONES
Leite; Sergio 1, Pavan; Fernando 2, Maia; Pedro 3, Deflon;Victor 3, Batista; Alzir 4, Sato; Daisy 5, Franzblau; Scott 6, Leite; Clarice 2
1 - Universidade Estadual Paulista, Instituto de Química
2 - Universidade Estadual Paulista, Faculdade de Ciências Farmacêuticas
3 - Universidade de São Paulo, Instituto de Química de São Carlos
4 - Universidade Federal de São Carlos, Departamento de Química
5 - Instituto Adolfo Lutz, Unidade de Ribeirão Preto
6 - University of Illinois at Chicago, College of Pharmacy
The aim of this study was to identify a candidate drug for anti-tuberculosis therapy development from previously synthesized thiosemicarbazones, semicarbazones and hydrazones, comprising a total of 17 compounds. The Minimal Inhibitory
Concentration (MIC) of these compounds, determined by the resazurin reduction method, was investigated in order to
determine their in vitro antimycobacterial activity against Mycobacterium tuberculosis. In vitro cytotoxicity values (IC50)
of the same compounds were determined on J774 cells to establish a selectivity index (SI = IC50/MIC). Lower values of
MIC were found for four thiosemicarbazones and four hydrazones, namely: 2-acetylpyridine N4 (etil) thiosemicarbazone;
2-acetylpyridine N4 (cyclohexyl) thiosemicarbazone; di-2-pyridyl ketone N4 (phenyl) thiosemicarbazone; 2-acetylpyridine
morpholyl thiosemicarbazone; mono benzoylacetone isonicotinoyl hydrazone; mono-acetylacetone isonicotinoyl hydrazone; di-2-pyridyl ketone isonicotinoyl hidrazone; di-2-pyridyl ketone thiophene hidrazone (MIC values ranging from 0.78
to 6.25 μg/mL). All the compounds presented very low cytotoxicity, with the exception of 2-acetylpyridine morpholyl
thiosemicarbazone (IC50 ≤ 3.9 μg/mL and SI ≤ 5).The results obtained with the other 7 compounds (SI ranging from 100
to 800) qualify them as candidates for anti-TB drugs, once their in vitro results are comparable to some of “first line” and
“second line” drugs commonly used in the TB treatment.
European Society of Mycobacteriolog y | 30 th Annual Congress | July 2009 | Por to - Por tugal
183
PP-112
METHODS FOR ASSESSMENT OF ETHIDIUM BROMIDE
TRANSPORT ACROSS Mycobacterium smegmatis CELL WALL
Jorge Ramos1*, Liliana Rodrigues1,2, Isabel Couto1,3, Leonard Amaral1,2 and Miguel Viveiros1
1 - Unit of Mycobacteriology, Instituto de Higiene e Medicina Tropical, Universidade Nova de Lisboa (IHMT/UNL), Lisboa, Portugal
2 - UPMM, IHMT/UNL, Lisboa, Portugal
3 - Centro de Recursos Microbiológicos (CREM), Faculdade de Ciências e Tecnologia, UNL, Caparica, Portugal
*Correspondig author: Jorge Ramos, Unit of Mycobacteriology, Instituto Higiene e Medicina Tropical, Universidade Nova
de Lisboa (IHMT/UNL), Rua da Junqueira 96, 1349-008 Lisboa, Portugal.
Tel: +351 21 3652600; Fax: +351 21 3632105; E-mail: [email protected]
Active efflux systems and reduced cell wall permeability are considered to be the main causes of mycobacterial intrinsic
resistance to many antimicrobials. Although several mycobacterial efflux pumps have already been described, their role in
drug resistance is not yet fully understood. Recent studies showed that both LfrA and MspA, the main efflux pump and
porin in M. smegmatis, respectively, are involved in reduced susceptibility to several antimicrobials.
We have compared the M. smegmatis wild-type strain mc2155 with LfrA and MspA M. smegmatis deleted mutants, for
their ability to extrude ethidium bromide (EtBr), a known efflux pump substrate, under different energy conditions
and in the presence or absence of efflux pumps inhibitors (EPIs), by (i) a 96 well microplate screening assay with the
mycobacterial cells grown in Middlebrook 7H9 with 10% of OADC in presence of increasing concentrations of EtBr
and different concentrations of the EPIs and the plates examined with a UV transilluminator and photographed after
24 hours of incubation; and (ii) a semi-automated fluorimetric method that detects efflux on a real time basis during a
period of 30 minutes.The EPIs employed were chlorpromazine, thioridazine, carbonyl cyanide m-chlorophenylhydrazone
and verapamil.
The efflux activity detected for each strain by these two methods was then correlated with resistance to several antibiotics (ATBs), by the determination of their minimal inhibitory concentrations in the presence or absence of the EPIs.
The ATBs tested were streptomycin, isoniazid (INH), rifampicin (RIF), ethambutol (ETB), amikacin, ciprofloxacin (CIP)
and clarithromycin (CLR).
In the absence of the major porin of M. smegmatis, MspA, it was observed that accumulation of EtBr decreased and the
cells became more resistant to several ATBs. On the other hand, the mutant for the major efflux pump LfrA showed
increased accumulation of EtBr. This strain also presented increased susceptibility to EtBr, INH, RIF, ETB, CIP and CLR.
These results show that MspA is an important channel for entrance of quaternary ammonium compounds and ATBs and
that the pump LfrA is involved in low-level resistance to several ATBs and quaternary ammonium compounds in M. smegmatis.
184
ESM 2009
PP-113
the human macrophage as a model to select
compounds active against MDR/XDR-TB
Marta Martins1,2,*, Miguel Viveiros1,3, Isabel Couto1,4 and Leonard Amaral1,2,3.
1 - Unit of Mycobacteriology, Instituto de Higiene e Medicina Tropical, Universidade Nova de Lisboa (IHMT/UNL), Lisbon, Portugal
2 - UPMM, IHMT/UNL, Lisbon, Portugal
3 - COST ACTION BM0701 (ATENS)
4 - Centro de Recursos Microbiológicos (CREM), Faculdade de Ciências e Tecnologia, UNL, Caparica, Portugal
* Corresponding author: Unit of Mycobacteriology and UPMM, Instituto de Higiene e Medicina Tropical, Universidade
Nova de Lisboa (IHMT/UNL), Rua da Junqueira, 96, 1349-008, Lisbon, Portugal;Telf: +351213652600; Fax: +351213632105;
e-mail: [email protected]
The emergence of Multi- and Extensively-Drug Resistant Mycobacterium tuberculosis (MDR/XDR-TB) represents a major
threat to public health worldwide. Both infections result in high mortality, especially if the patient is co-infected with
HIV. The selection of therapy for these multi-drug resistant infections is limited, and for most situations, ineffective as
many of these strains are untreatable with the available drugs. Thus, there is an urgent need to design and develop new
compounds against drug resistant M. tuberculosis that are effective within the main target of this infection, the human
macrophage. We have recently demonstrated that efflux pumps inhibitors are active against mycobacteria, by enhancing
the killing activity of the human macrophage and may represent an alternative to the conventional antibiotherapy for the
treatment of the MDR/XDR-TB infections.
From previous studies we have demonstrated that thioridazine (TZ) enhances the killing of MDR-TB phagocytosed by
human macrophages. However, the mechanism of action of TZ on these cells is not fully understood. We have studied
the activity of TZ, several of its derivatives, organosilicon (SILA) compounds and other known inhibitors of K+ and Ca2+
transport (ouabain, reserpine and verapamil) on macrophages infected with MDR-TB and XDR-TB. After phagocytosis,
the compounds were added to the macrophage cultures. Following incubation cells were lysed and the intracellular bacterial concentration determined. Our results demonstrate that TZ, three of its derivatives and one SILA compound (SILA
421) enhanced substantially the macrophage killing activity.
The killing activity of neutrophils is correlated with the K+ availability, which is dependent upon transport processes
affected by agents that inhibit Ca2+-activated K+ pumps. Based on this and on our results, we postulate that the enhancement of the macrophage killing activity by these compounds could be due to the inhibition of Ca2+ and K+ transport that
promotes the activation of hydrolases and the killing of intracellular bacteria. A model describing the sequence of events
that lead to the killing of intracellular bacteria will be presented. Moreover, the
������������������������������������������
ex-vivo testing of compounds using patient’s own macrophages in the clinical TB laboratory might allow screening the most effective compounds against MDR/
XDR-TB providing the basis for the intelligent selection of drugs to be used in the therapy of these infections.
European Society of Mycobacteriolog y | 30 th Annual Congress | July 2009 | Por to - Por tugal
185
PP-114
IN VITRO ACTIVITIES OF JPC 2067 ALONE AND IN
COMBINATION WITH SMX AGAINST NOCARDIA SPECIES
Michael Cynamon, Swagatam Mookherjee, Carolyn Shoen
Veterans Affairs Medical Center, Syracuse, New York, USA
E-mail: [email protected] FAX: 315-425-4871
Background
JPC 2056, a biguanide prodrug of JPC 2067, a dihydrotriazine DHFR inhibitor, is being developed as an antimalarial therapeutic. Previously we demonstrated that earlier compounds related to JPC 2067 had promising activities in vitro alone
and in combination with sulfamethoxazole (SMX) against nocardia species. JPC 2067 is active against M. tuberculosis, M.
kansasii, and M. marinum at ≤ 1µg/ml. The purpose of the present study was to evaluate the in vitro activities of JPC 2067
alone and in combination SMX against a group of clinical nocardia isolates.
Methods
JPC 2067 was provided by Jacobus Pharmaceutical Co., Princeton, NJ. SMX was purchased from Sigma Chemical Co, St.
Louis, MO. Each drug was dissolved in DMSO at a final concentration of 1 mg/ml. Aliquots were frozen at -200C. Drugs
were thawed prior to testing and diluted in modified 7H10 broth (pH 6.6; 7H10 agar formulation with agar and malachite
green omitted) with 10% OADC enrichment and 0.05% Tween 80. JPC 2067 and SMX were tested alone from 64µg/ml
– 0.06µg/ml.When tested together SMX was evaluated at fixed concentrations of 1 µg/ml and JPC at 16µg/ml – 0.015µg/
ml. Twenty eight nocardia isolates (from the ATCC and clinical isolates provided by B. Body and B. Forbes) were used in
the study. An in vitro broth dilution method similar to that defined by CLSI was utilized.
Results:
The MIC50 and MIC90 for JPC 2067, SMX and the combination (SMX fixed at 1µg/ml) were 0.125 µg/ml and 4 µg/ml, 16
µg/ml and 32 µg/ml, and 0.03 µg/ml and 2 µg/ml respectively.
Conclusions
JPC 2067 was more active than the previously tested dihydrotriazine analogs against nocardia. The addition of SMX had a
modest but consistent effect on lowering the JPC 2067 MIC. It is likely that this effect would be more pronounced if the
concentration of SMX was increased to perhaps10 µg/ml (a readily achieved serum level for this agent). JPC 2067 alone
and in combination should be evaluated in animal models of both nocardial and mycobacterial infection to understand
the clinical potential of these agents.
186
ESM 2009
PP-115
Nosocomial TB in a laboratory setting
Jaime M S Nina1,2,3
1 - Instituto Nacional de Saúde Doutor Ricardo Jorge
2 - Universidade Nova de Lisboa
3 - Hospital Egas Moniz
Abstract
TB is recognized as a major cause of morbidity and mortality worldwide. Its easily transmissibility is also generally recognized, both at the family level, in the household, at the place of work and inside health care facilities. This last way of
transmission, properly called nosocomial transmission, has been suspected for long time, and was formally demonstrated
in the ward, both among patients, and health care workers. Several professional bodies and other institutions, both at
the national and international level, produced guidelines trying to minimize TB transmission to health care workers inside wards and emergency services. Furthermore several countries produced legislation to protect health care workers
against nosocomial TB, and/or included TB in the list of professional diseases or hazards to health care workers.
However, much less attention has been given to the TB transmission potential to laboratory workers. Even if several
countries moved laboratory work with live TB samples to LSB-3 facilities, the evidence on which to base this decision is
thin, and no systematic study has been published.
Herein are presented three cases of nosocomial transmission inside laboratory settings, in Lisbon. These cases cover all
spectra of professional differentiation, from basic level auxiliary personnel, to a laboratory technician, and to a microbiologist physician. In one case the way of transmission was a very common kind of laboratory accident, in another a
common inattention, and in the last one no specific way of transmission was found. One of the cases was found to be a
MDX TB.
In conclusion, the trend to carry out all routine work with live TB samples only inside a BSL-3 facility seems a right one,
and so it is fully justified. Also justified would be the inclusion of health laboratory workers in the legislation that provide
safety measures and insurance cover to health care workers.
European Society of Mycobacteriolog y | 30 th Annual Congress | July 2009 | Por to - Por tugal
187
Author Index
Abadia E
Abreu C
Afonso A
Aguilar D
Ahmadi M
Ahmed A
Ahmed I
Åkerström M
Akol J
Akpaka PE
Al Balushi L
Al Busaidi S
Al- Mahruqi S
Albarral MIP
Alcaide F
Aldashev A
Algamdi S
Al-Hajoj S
Ali AB
Ali MS
Ali Veleyati A
Allix-Béguec C
Al-Maniri AA
Almeida da Silva P
Almeida EA
Al-Omari R
Alonso C
Al-Rawas O
Alves A
Amado J,
Amaral L
Amondi Ouma N
Amorim A
Andersson E
Ängeby K
Anoosheh S
Anthony R
Arnold C
Au TK
Au-Yeang CKW
Awad C
Ayles H
Azevedo AA
Baboolal S
Babu Okatch F
Bachiiska E
Balacó I
Balbin JA
Baldan R
Balmoi F
Barbe V
Baritaki M
Baritaki S
Barrera L
Barry III CE
Bartu V
Batista A
Baumanis V
Bauskenieks M
Bazigos S
Beaty PS
Beltrán M
Biet F
Boehme C
Boeree M
Bonte L
Borile C
Borroni E
Boschiroli ML
Bourdon E
Braga JE
BragaR
Brankova N
Brisse Smangenot S
Brosch R
Brown T
Brum L
Brzostek A
Bwanga F
Cabral J
Cacho J
Caldas C
Cambau E
Cañete C
Cano I
Cardoso N
Cardoso RF
Cardoso S
Carmona JA
Carvalho C
Carvalho F
Carvalho MA
Carvalho T
Casal M
Cassone A
Castro AG
Causse M
Chan Chiu Y
Chan CYR
Chan EWC
Chan RCY
Chan WCE
Chau CH
Cheruiyot C
Cho E-J
Cho SN
Chryssanthou E
Cirillo D
Cirillo DM
Clivillé R
Cochard T
Codina G
Coelho R
Coll P
Conceição EC
Correa N
Correia-Neves M
Costa ARF
Couto I
Couto S
Crews V
Cruz A
Cubillos A
Cvetnic Z
Cynamon M
Dafae F
David S
De Bock A
De Cruz K
De Gispert FX
De Haas P
De la Hoz F
De Sousa MS
Deflon V
Deflon VM
Dekhuijzen R
Del Portillo P
Del Val-Romero B
Dementieva A
Den Hertog A
Di Giulio B
Dias A
Diogo J
Diwan V
Docx S
Drobniewski F
DuarteR
Duvnjak S
Dziadek B
Dziadek J
Echemendía M
Ehricht R
Eisenach K
Elmoula IF
Etwom A
Eum SY
Fajfar N
Falkinham, III, JO
Farnia P
Fattorini L
Fauville-Dufaux M
Felix C
Ferme D
Fernandes P
Fernandes SJ
Ferreira A
Ferreira C
Ferreira D
Ferreira S
Ferro RS
Feuerriegel S
Fey F
Figueira R
Filippini P
Fissette K
Fiuza de Melo F
Frade R
Fraga AG
Frangopoulos F
Franz S
Franzblau S
Franzblau SG
Fung SL
Furlaneto IP
Furtado C
Gagneux S
Gaile I
Gama JB
Gao Q
Garcia MJ
García-Cañas A
Gavin P
Gegia M
George AG
Gharbia S
Giampaglia CS
Gicquel B
Gil MJ
Gilpin CM
Giske C
Gitti Z
Goguet de la Salmonière YOL
Goldfeder LC
Golec M
Gomes H
Gomes HM
Gomes MH
Gonçalves H
González Torralba A
González-Martín J
Grce M
Greib C
Guillard B
Gutiérrez C
Gutierrez J
Gutierrez MC
Gutierrez-Aroca JB
Hadadi M
Haenn S
Haile M
Haroun RZ
Havelkova M
Hepple P
Herbawi M
Hernández J
Hernández-Pando R
Hillemann D
Hirata MH
Hirata RDC
Hoffner S
Homolka S
Honisch C
Honscha G
Hoosen A
HubansC
Hwang SH
Ibrahim TA
Ichijo T
Imperiale B
Ingham C
Ioannidis P
Isakova J
Ivanov A
Izumi Y
European Society of Mycobacteriolog y | 30 th Annual Congress | July 2009 | Por to - Por tugal
Jahromi NS
Jansone I
Janssen HG
João I
Joloba M
Joloba ML
Jordão L
Joshi S
Julián E
Julián EG
Jureen P
Kaal E
Kahlmeter G
Kam KM
Kanavaki S
Kang HS
Kantor I
Kapata N
Karabela S
Karacali A
Kargar M
Karoui C
Katalinic-Jankovic V
Katalinic-Jankovic V
Kayar B
Kayar Mb
Kazempour M
Kern WV
Khan AJ
Khechinashvili G
Kim JH
Klatser P
Koeleman M
Köksal F
Kolk A
Konstantinidou E
Kontos F
Kopher K
Kosmadakis G
Kritski A
Krt B
Kuan HO
Kubin M
Kuijper S
Labarre M
Lai WMR
Langerak E
Lazaro E
Leão SC
Lee H
Lee J
Lee J-I
Leimane V
Leite C
Leite CQF
Leite S
Leite SRA
Leite SRA
Lemos S
Lemus D
Levina K
189
Levterova V
Lezcano MA
Lima EJC
Lima KVB
Locht C
Lockwood D
Logarinho E
Lopes ML
López AG
López B
Lopez-Calleja AI
Loureiro C
Lucas F
Luo T
Luquin M
Lyashchenko K
Lyberopoulos P
Macedo R
Machado D
Madruga M
Maia P
Maia PIS
Maio JN
Mak KX
Malagari AI
Malaquias A
Malaspina AC
Mallard K
Marceau M
Markova N
Marques M
Martín A
Martin C
Martín-Casabona N
Martinez-Martinez L
Martins M
Martins MC
Martins TG
Masjedi M
Masjedi MR
Matic I
Matos G
Maugein J
Mazarrasa CF
Mbulo G
McNerney R
Mdivani N
Médigue C
Mei J
Mello FAF
Mendes AC
Mendes NH
Mendes NH
Menendez MC
Mestre O
Meyers WM
Mézard M
Michel G
Mihailelis E
Milho C
Milián Y
Millan I
Millet J
Min JH
Miotto P
Mirabal N
Miranda A
Miyagi-Shiohira C
Miyata M
Moilleron R
Mokrousov I
190
Monecke S
Monge I
Moniz-Pereira J
Monteiro G
Montemayor M
Montoro E
Mookherjee S
Morcillo N
Morgan K
Mosko M
Mota M
Moulin L
Moure R
Moyoyeta M
Muchwa C
Mugerwa R
Mugyenyi P
Müllerova M
Mumbowa F
Munga Waweru P
Murcia M
Musunsa A
Muvwimi M
Mwamba P
Mwanza W
Nagiyev T
Nascimento I
Nasu M
Navarro A
Neo ZY
Neonakis IK
Niemann S
Nikolaou S
Nina J
Nogueira C
Nogutia EN
Noroozi J
Noruzi J
Novoa-Cain J
Nowroozi J
Nuak J
Nyirenda CN
Oberhauser B
Obrovac M
Obrovac M
Ocepek M
Oelemann MC
Ojeda P
Oliveira P
Omar AR
Oral Zeytinli U
Orikiriza P
Orozco H
O’Sullivan D
Paasch F
Palaci M
Palomino JC
Panaiotov S
Pando RH
Pandolfi JRC
Papaventsis D
Papiris S
Pardini M
Park SK
Pate M
Paulo C
Pavan F
Pavan FR
Pawelczyk J
Pedrosa J
Peeling R
Pellegrin JL
Perdigão J
Pereira DR
Pereira E
Pereira Miguel J
Pérez Meixeira A
Perkins M
Pfeltz R
Pfyffer G
Pimentel M
Pinheiro MD
Poelhsitz GV
Pole I
Portaels F
Portugal C
Portugal I
Possuelo L
Post E
Prata P
Proença F
Qazi F
Quieng MD
Racic I
Radomski N
Ramos J
Ramos Martos
Ramos MH
Ramoutar D
Raposo A
Rasolofo V
Rastogi N
Rauzier J
Refrégier G
Régis M
Reniero A
Rey E
Reyes A
Ribeiro JN
Richter E
Ridell M
Riley LW
Ritacco V
Ritmeijer K
Robledo J
Rocha G
Rodrigues A
Rodrigues F
Rodrigues L
Rodrigues S
Rodríguez-Güell E
Rohde KH
Rosales J
Roura-Mir C
Ruimy R
Ruiz P
Rumijowska-Galewicz A
Rüsch-Gerdes S
Russell DG
Saaed NS
Sabino A
Şahan Kipalev A
Said H
Sainti A
Salas S
Saluotsa M
Salvadó M
Salvignol G
Sampaio D
Samper S
Samutela M
Sánchez-Concheiro M
Sancho L
Sandoval A
Santos ACB
Santos C
Santos R
Saraiva M
Sardinha E
Sardinha T
Sarmento A
Sato D
Sato DN
Schön T
Secanella SP
Seif S
Shamputa IC
Sharaf-Eldin GS
Shikama M-L
Shinnick T
Shinnick TM
Shoen C
Siddiqi S
Silva A
Silva C
Silva F
Silva K
Silva M
Silva P
Silva S
Simões MF
Sing LH
Singh JPN
Skenders G
Slickers P
Sola C
Somoskövy A
Sousa AS
Sousa C
Sousa G
Sousa JG
Sovhozova N
Soyal A
Spandidos DA
Spicic S
Stoffels K
Sturegård E
Suffys PN
Supply P
Svensson E
Tabarsei P
Tajeddin E
Takiff H
Tancredo L
Tang YW
Tap J
Tavares M
Tavares Magalhães A
Teles JMM
Telles MAS
Thibault V
Tonjum T
Torrado E
Tortoli E
Traore H
Tudó G
Turner C
Ueki SYM
Valcheva V
Valdés I
Valente A
Valente F
Valente I
Van der Stuyft P
van der Wel N
Van Hoof R
van Ingen J
Van Soolingen D
Varaine F
Varghese B
Vasconcelos O
Velayati A
Velayati AA
Via LE
Viallard JF
Viana BHJ
Viana-Niero C
Villar M
Villela G
Viveiros M
Vladimirov K
Von Groll A
Vultos TD
Wang S
Warns M
Watson C
Weizenegger M
Werngren J
Westman L
Willery E
Winkler S
Wong AP
Yamaguchi N
Yamane N
Yan SW
Yates M
Yew WW
Yip CW
Yubero J
Yula E
Yzquierdo S
Zaitseva E
Zaldumbide MA
Zambrano MM
Zdelar-Tuk M
Zellweger J-P
Zenhorst R
Zerolo FJ
Zerva L
Zhang J
Zmak L
Zolnir - Dovc M
Žolnir Dov- M
Zozio T
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European Society of Mycobacteriolog y | 30 th Annual Congress | July 2009 | Por to - Por tugal
191
Vol. XVI Suplementno 1 A
REVISTA PORTUGUESA DE
pneumologia
P O RT U G U E S E J O U R N A L O F P U L M O N O L O G Y
Volume XVI
Suplemento 1 A
Janeiro 2010
REVISTA PORTUGUESA DE PNEUMOLOGIA
Janeiro 2010
30.º CONGRESSO ANUAL DA SOCIEDADE EUROPEIA
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th
30 ANNUAL CONGRESS OF THE EUROPEAN SOCIETY
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Dr.ª Fátima Rodrigues
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Referenciada na Embase, Excerpta
Medica Database desde Janeiro
de 2001 (Vol. VII), no Index Medicus,
MEDLINE, PubMED desde Jan/Fev
de 2003 (Vol. IX), na SciElo desde
Jan/Fev de 2006 (Vol. XII) e no Thomson
Reuters desde Jan/Fev 2008 (Vol. XIV).
All issues referred in Embase,
Excerpta Medica Database
since January 2001 (Vol. VII), Index
Medicus, MEDLINE, PubMED since
Jan/Feb 2003 (Vol. IX), SciElo since
Jan/Feb 2006 (Vol. XII) and Thomson
Reuters since Jan/Feb 2008 (Vol. XIV).
Pneumologia 16 Supl 1A - Miolo - 4ª PROVA.indd 1
Conselho Científico
Dr.ª Margarida Cancela de Abreu
Lisboa
Dr. Abel Afonso
Vila Real
Prof. A. Bugalho de Almeida
Lisboa
Dr. João Almeida
Porto
Prof. Manuel J. Antunes
Coimbra
Prof. M. Fontes Baganha
Coimbra
Prof.ª Cristina Bárbara
Lisboa
Dr.ª Celeste Barreto
Lisboa
Dr. A.M. Sousa Barros
Porto
Dr. Ulisses Brito
Faro
Dr.ª Gabriela Brum
Lisboa
Dr.ª Paula Campos
Lisboa
Prof. J. H. Paiva de Carvalho
Coimbra
Prof.ª Lina Carvalho
Coimbra
Prof. Carlos Robalo Cordeiro
Coimbra
Dr. J. Duro da Costa
Lisboa
Prof. Melo Cristino
Lisboa
Dr. João Cunha
Braga
Dr. J. Roque Dias
Santarém
Dr. António Domingos
Torres Vedras
Prof.ª M.ª Teresa Magalhães Godinho
Lisboa
Dr. Júlio Gomes
Guarda
Prof.ª M.ª João Marques Gomes
Lisboa
Prof. Venceslau Hespanhol
Porto
Prof. J. Agostinho Marques
Porto
Prof. José António Badinni Martinez
Ribeirão Preto/S. Paulo
Dr. J. Pontes da Mata
Lisboa
Dr. Ibraímo Maulide
Lisboa
Dr.ª Conceição Souto Moura
Porto
Dr. Ricardo C. Nascimento
Funchal
Prof.ª Denise Duprat Neves
Rio de Janeiro
Dr.ª Bárbara Parente
Vila Nova de Gaia
Dr. Rui Pato
Coimbra
Dr. J. M. Dias Pereira
Ponta Delgada
Dr. Jaime Pina
Lisboa
Dr. Jorge Pires
Coimbra
Prof. Henrique Queiroga
Porto
Prof. A. Bensabat Rendas
Lisboa
Dr. Fernando Rodrigues
Amadora
Prof. Henrique Luz Rodrigues
Lisboa
Dr. J. Moura e Sá
Vila Nova de Gaia
Dr. R. César Sá
Vila Nova de Gaia
Dra. Maria João Valente
Lisboa
Prof. Fernando Ventura
Lisboa
Dr. Jorge Roldão Vieira
Almada
Dr. Miguel Villar
Lisboa
Prof. João Carlos Winck
Porto
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R E V I S T A
P O R T U G U E S A
D E
P N E U M O L O G I A
SOCIEDADE PORTUGUESA DE PNEUMOLOGIA
Direção Comissões de Trabalho
Presidente: Prof. António Segorbe Luís
Vice-Presidentes: Dr. Renato Sotto-Mayor
Prof. Henrique Queiroga
Prof.ª Lina Carvalho
Secretário-Geral: Dr. José Manuel Rosal Gonçalves
Secretário-Adjunto: Dr. José Miguel Carvalho
Tesoureiro: Dr. Jorge Roldão Vieira
Alergologia Respiratória
Coordenador: Dra. Luísa Semedo
Secretário: Dr. Carlos Lopes
Tuberculose
Coordenador: Dra. Aurora Carvalho
Secretário: Dra. Sandra André
Pneumologia Oncológica
Coordenador: Dra. Ana Figueiredo
Secretário: Maria de la Salete Valente
Técnicas Endoscópicas
Coordenador: Dra. Yvette Martins
Secretário: Dr. Júlio Semedo
Reabilitação Respiratória
Coordenador: Dra. Ana Paula Simão de Oliveira
Secretário: Dra. Paula Teresa Rodrigues de Almeida
Mesa da Assembleia Geral
Presidente: Prof. A. Bugalho de Almeida
Secretário: Dr. José Manuel Dias Pereira
Vogal: Prof.ª Cristina Bárbara
Fisiopatologia Respiratória
Coordenador: Dr. Nuno Cortesão
Secretário: Dra. Maria João Matos
Doenças do Interstício Pulmonar
e Doenças Ocupacionais
Coordenador: Dr. António Morais
Secretário: Dra. Cristina Cristóvão
Cirurgia Torácica
Coordenador: Dra. Isilda Mendes
Secretário: Dr. João Bernardo
Tabagismo
Coordenador: Dra. Ivone Pascoal
Secretário: Dra. Sofia Ravara
Conselho Fiscal
Presidente: Dr. Jorge Branco Pires
1.º Vogal: Dr. Júlio Gomes
2.º Vogal: Dr. Ulisses de Brito
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Infecciologia Respiratória
Coordenador: Dr. Filipe Froes
Secretário: Dra. Cecília Pardal
Patologia do Sono
Coordenador: Dra. Marta Drummond
Secretário: Dra. Paula Pinto
10-05-2010 10:48:01
ÍNDICE
INDEX
30.º CONGRESSO ANUAL DA SOCIEDADE EUROPEIA
DE MICOBACTERIOLOGIA
30th ANNUAL CONGRESS OF THE EUROPEAN SOCIETY
OF MYCOBACTERIOLOGY
Susana David Introdução
Introduction
5
Miguel Villar A tuberculose em Portugal
Tuberculosis in Portugal
7
Fernando AF de Melo A experiência brasileira de controlo da multidroga-resistência
Brazilian experience in the management of multidrug-resistance
11
Sebastien Gagneux Forças evolutivas do Mycobacterium tuberculosis
Evolutionary forces in Mycobacterium tuberculosis
21
Joseph O Falkinham III Epidemiologia e ecologia de micobactérias não tuberculosas
Epidemiology and ecology of nontuberculous mycobacteria
27
Jean-Pierre Zellweger O uso da análise de libertação de gama interferão como auxiliar no controlo
da tuberculose
The use of interferon gamma release assays as an aid in the control
of tuberculosis
31
Lee W Riley Regulação da composição lipídica da parede celular do Mycobacterium
tuberculosis e o seu efeito na persistência bacteriana in vitro
Regulation of Mycobacterium tuberculosis cell wall lipid composition and
its effects on in vitro bacterial persistence
37
Jesus Gonzalo Asensio Uma nova vacina viva contra a tuberculose com base na inativação do phoP
Ainhoa Arbues A new live tuberculosis vaccine based on phoP inactivation
Dessi Marinova
Carlos Martín
43
Ruth NcNerney Simpósio: Testes rápidos para o rastreio preliminar da tuberculose
Symposium: Point-of-care tests for tuberculosis
49
Thomas M Shinnick Criar capacidade laboratorial para a tuberculose: Necessidades e estratégias
Building tuberculosis laboratory capacity: needs and strategies
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Afranio Kritski Experiência da Rede Brasileira de Pesquisa em Tuberculose no desenvolvimento
e avaliação de novos métodos de diagnóstico da tuberculose
The experience of the Brazilian Tuberculosis Research Network in the
development and evaluation of new methods of diagnosing tuberculosis
Moisés Palaci Ensaios clínicos de novas drogas e testes diagnósticos em tuberculose: Desafios
micobacteriológicos
Clinical trials of new tuberculosis drugs and diagnostic tests: mycobacteriological
challenges
Karina de Prince Avaliação das moléculas com atividade antiTB das plantas do cerrado brasileiro
Fernando R Pavan Screening of molecules with anti-TB activity from the brazilian cerrado plants
Daisy N Sato
Wagner Villegas
Sergio RA Leite
Clarice QF Leite
Caroline Allix-Béguec
Christine Hubans
Stéphanie Ferreira
Philip Supply
Novas ferramentas de fácil utilização para genotipagem padronizada e com qualidade
controlada de estirpes do complexo Mycobacterium tuberculosis
New, easy-to-use tools for standardised and quality-controlled genotyping
of Mycobacterium tuberculosis complex strains
Elvira Richter Simpósio: Avaliação externa da qualidade
Symposium: External quality assurance
67
77
83
89
95
Editora convidada/Guest editor: Susana David
A edição deste suplemento foi patrocinada com fins educacionais pelo
Instituto Nacional de Saúde Doutor Ricardo Jorge e pela Fundação Luso-Americana para o Desenvolvimento/
This supplement is made possíble thanks to an unrestricted educational grant from
Instituto Nacional de Saúde Doutor Ricardo Jorge and Luso-American Development Foundation
Este suplemento foi escrito ao abrigo do Novo Acordo Ortográfico
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Introdução
Introduction
Susana David
A Sociedade Europeia de Micobacteriologia
(ESM, http://www.esmycobacteriology.eu)
é considerada uma das sociedades científicas
internacionais mais ativas na área da micobacteriologia e doenças relacionadas. As
reuniões da ESM, que têm lugar cada ano
num país europeu diferente, promovem a
comunicação de especialistas internacionais
de renome, criando oportunidades para atualização do conhecimento dos últimos avanços científicos, compartilhar experiências e
ideias e participar ativamente no desenvolvimento da micobacteriologia.
Este número especial da Revista Portuguesa
de Pneumologia é dedicado ao 30.º Congresso Anual da Sociedade Europeia de Micobacteriologia (ESM2009), que se realizou
no Porto, Portugal, de 5 a 8 de julho de
2009. A organização deste congresso deveu-se à ESM e ao Instituto Nacional de Saúde
Doutor Ricardo Jorge (INSA), o braço laboratorial do sistema português de saúde
(http://www.insa.pt).
O Congresso ESM2009 deu particular atenção ao esforço global e multidisciplinar em
micobacteriologia necessário na luta contra
a tuberculose. As sessões científicas e minissimpósios versaram os seguintes temas:
• Programas de controlo da tuberculose;
• Epidemiologia molecular e vigilância da
resistência a fármacos;
• Deteção da resistência a fármacos por
genética molecular;
The European Society of Mycobacteriology
(ESM, http://www.esmycobacteriology.eu)
is considered one of the most active international scientific societies in the area of mycobacteriology and related diseases. The
ESM meetings, held each year in a different
country of Europe, promote the exchange
of distinguished experts from all around the
world creating the opportunity to update
information in the front-line of scientific
achievement, share experience and ideas,
and actively participate in contribution to
the field of mycobacteriology.
This special issue of the Revista Portuguesa de
Pneumologia (RPP) is dedicated to the 30th
Annual Congress of the European Society of
Mycobacteriology (ESM2009) hosted in Porto, Portugal, from July 5-8, 2009. This congress was co-organized by the ESM and the
National Health Institute Doutor Ricardo
Jorge (INSA), the laboratory arm of the Portuguese health system (http://www.insa.pt).
The ESM2009 congress gave particular attention to the multi-disciplinary effort
needed, from mycobacteriologists on a
world wide basis, in the fight against tuberculosis. Scientific sessions and mini-symposia covered the following themes:
• Tuberculosis control programs;
• Molecular epidemiology and drug resistance surveillance;
• Molecular genetic detection of drug resistance;
Laboratório Nacional de Referência das Infecções Respiratórias para as Micobactérias da Unidade de Referência e Vigilância Epidemiológica do Departamento de
Doenças Infecciosas do Instituto Nacional de Saúde Dr. Ricardo Jorge, Lisboa, Portugal
E-mail: [email protected]
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introdução
Susana David
•
•
•
•
•
•
•
•
•
Testes de susceptibilidade e tratamento
da tuberculose;
Micobactérias não tuberculosas;
Novas questões no papel do laboratório
de tuberculose;
Desenvolvimento de vacinas e patogenia;
Testes point-of-care para tuberculose;
Fortalecimento do laboratório;
Desenvolvimento farmacológico;
Aspetos práticos e controlo de qualidade na epidemiologia molecular;
Controlo de qualidade externo.
Este número é constituído por uma compilação de breves comunicações baseadas
nos temas de algumas das principais conferências. O programa completo pode ser
consultado nos websites da ESM e do INSA
ou em http://www.esm2009.org.
Gostaríamos de manifestar o nosso especial
agradecimento ao Dr. Renato Sotto-Mayor e
à Revista Portuguesa de Pneumologia pelo seu
interesse na publicação deste número e a todos os que amavelmente deram a sua contribuição científica. A realização do Congresso
ESM 2009 não teria sido possível sem o apoio
das instituições: Fundação Calouste Gulbenkian
(http://www.gulbenkian.pt); Fundação Luso-Americana de Desenvolvimento (http://www.
flad.pt); STOP-TB Working Group on New
Diagnostics, Point of Care sub group (www.
stop tb.org) e patrocinadores: HAIN LifeSciences (http://www.hain-lifescience.com); Becton
Dickinson (http://www.bd.com); Quilaban
(http://www.quilaban.pt); BioMerieux (http://
www.biomerieux.com); Cepheid (http://www.
cepheid.com); Microsens (http://www.microsens.co.uk); Genoscreen (http://www.genoscreen.com).
Susana David
Presidente do Congresso ESM2009
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•
•
•
•
•
•
•
•
•
Susceptibility testing and treatment of
tuberculosis;
Non tuberculous mycobacteria;
Issues in the modern tuberculosis laboratory;
Vaccine development and pathogenesis;
Point-of-care tests for tuberculosis;
Laboratory strengthening;
Drug development;
Practical aspects and quality assurance
in molecular epidemiology;
External quality assurance.
This issue is comprised of a compilation of
short communications based on the themes
of some of the main conferences. The full
program may be consulted on the ESM and
INSA websites or at http://www.esm2009.
org.
We would like to give special thanks to Dr.
Renato Sotto-Mayor and to the RPP for their
interest in putting together this issue and to all
those who kindly accepted to give their scientific contribution. The ESM2009 congress
would not have been possible without the
support of the partners: Fundação Calouste
Gulbenkian (http://www.gulbenkian.pt); Luso-American Foundation (http://www.flad.
pt); STOP-TB Working Group on New Diagnostics, Point of Care sub group (www.
stoptb.org) and sponsors: HAIN LifeSciences
(http://www.hain-lifescience.com); Becton
Dickinson (http://www.bd.com); Quilaban
(http://www.quilaban.pt); BioMerieux (http://
www.biomerieux.com); Cepheid (http://
www.cepheid.com); Microsens (http://www.
microsens.co.uk); Genoscreen (http://www.
genoscreen.com).
Susana David
Chairman of the ESM2009 congress
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30.º Congresso Anual da Sociedade Europeia de Micobacteriologia
30th Annual Congress of the European Society of Mycobacteriology
Miguel Villar1
A tuberculose em Portugal
Tuberculosis in Portugal
A tuberculose (TB) é um problema global com um número estimado de 9 milhões de novos casos por ano, 83% dos
quais se situam na África subsariana e no
Sudeste Asiático, onde se encontram muitos dos países com a maior carga de TB
(Fig. 1). A Organização Mundial de Saúde (OMS) estima a emergência global de
cerca de meio milhão de novos casos de
tuberculose multirresistente (MDR-TB),
por ano, incluindo 50 000 casos de tuberculose extensamente resistente a fármacos (XDR-TB).
Em 2007, a União Europeia (UE) teve uma
incidência média de 17/100 000, com Portugal a registar um dos mais elevados índices
na UE (27/100 000). Nas últimas duas décadas, a incidência em Portugal tem vindo a
diminuir consistentemente, e em mais de
7% anualmente nos últimos 5 anos (Fig. 2).
Esta diminuição é mais notória no grupo
etário dos 25 aos 44 anos, o que resulta numa
alteração da média de idades, tanto nos doentes nacionais como nos imigrantes (Fig. 3).
1
Tuberculosis is a global problem with an estimated 9 million new cases per year, 83%
of which occur in Sub-Saharan Africa and
South-East Asia, where many of the high
burden countries are found (Fig. 1). The
World Health Organisation (WHO) estimates an emergence of about half a million
new cases of multidrug-resistant tuberculosis (MDR-TB), globally, each year, including 50 000 of extensively drug-resistant tuberculosis (XDR-TB).
In 2007, the European Union (EU) had an
incidence rate of 17/100 000, with Portugal
registering one of the highest rates in the
EU (27/100 000).
Over the last two decades, the incidence in
Portugal has decreased consistently, and
more than 7% per year, in the last five years
(Fig. 2). This reduction is mainly in the age
group between 25 and 44 years old, leading
to a shift to the right of the median age,
both in national citizens and in immigrants
(Fig. 3). The foreign-born have represented
about 12% of the TB cases, and the preva-
Em representação do Director-Geral de Saúde/On behalf of the General-Director of Health
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a tuberculose em portugal
Miguel Villar
0-24
25-49
50-99
100 ou mais/100 or more
Não reportado/No report
Casos de tuberculose notificados (novos
e relapsos) por cada 100 000 habitantes/
Notified TB cases (new and relapse) per
100 000 population
Fig. 1 – Taxas de incidência da tuberculose por país, 2006. Fonte: OMS
Fig. 1 – Tuberculosis incidence rates, by country, 2006. Source: WHO
Fig. 2 – Evolução das taxas de incidência de tuberculose notificada no continente e regiões autónomas (todas as formas,
10–5 habitantes). Fonte: DGS
Fig. 2 – Evolution of the tuberculosis incidence rates notified in Mainland Portugal and Autonomous Regions (all forms/10-5
inhabitants). Source: DGS
Os estrangeiros têm representado cerca de
12% dos casos, e a prevalência de VIH tem
sido de aproximadamente de 14%, com uma
redução de 34% nos últimos cinco anos.
Entre 2003 e 2007, a maioria dos casos de
TB foram das formas pulmonares (74,1%),
dos quais 67,5% SS+ e 75,3% confirmados
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lence of HIV has been around 14%, with a
34% reduction in the last five years.
Between 2003 and 2007, most TB cases
were pulmonary forms (74.1%), 67.5% of
which were SS+ and 75.3% were confirmed
by culture. MDR-TB and XDR-TB, during
the same period, represents 1.9% (154) of
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a tuberculose em portugal
Miguel Villar
Fig. 3 – Evolução da taxa de incidência de tuberculose notificada, por grupos etários (105 habitantes). Fonte: DGS
Fig. 3 – Evolution of the tuberculosis incidence rates, according to age
groups (105 inhabitants). Source: DGS
por cultura. Neste mesmo período, os casos
de MDR-TB e XDR-TB representam 1,9%
(154) de todos os casos de tuberculose no
início do tratamento, variando de 1,3%
(22), em 2006, e de 2,4% (38), em 2004,
com uma média de 31 casos por ano (1,9%).
Estes números são representativos, já que a
cobertura dos testes de sensibilidade a fármacos (TSF) é superior a 80%.
No que respeita ao tema deste congresso, a
Rede de Laboratórios de Micobacteriologia
é uma componente importante do Programa Nacional para a Tuberculose (PNT),
com colaboração na deteção e definição de
casos, diagnóstico precoce de MDR-TB,
identificação de Mycobacterium tuberculosis
e realização de TSF de 1.ª e 2.ª linha. Para
além da confirmação dos casos de TB,
permite-nos, também, acompanhar o índice
de negatividade, especialmente até aos dois
meses de tratamento, o que é importante
para o controlo da transmissão e da eficácia
do tratamento. No mesmo período,
verificou-se um índice de negatividade documentada de 34% aos dois meses.
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the TB cases at the beginning of treatment,
varying from 1.3% (22), in 2006, and 2.4%
(38), in 2004, with an average of 31 cases
per year (1.9%). These proportions are representative, since the coverage of drug sensibility tests (DST) is over 80%.
As regards the theme of this Congress, the
Mycobacteriology Laboratory Network is
an important component in the National
Tuberculosis Programme (NTP), with collaboration in case detection, case definition, early diagnosis of MDR-TB, identification of Mycobacterium tuberculosis and
performance of 1st and 2nd line DST. Besides confirmation of TB cases, it also allows us to accompany the negativity rate,
especially up to two months of treatment.
This is important for the control of transmission and treatment efficacy. In the same
period mentioned above, we had 34% documented negativity at two months.
The Laboratory is also very useful in the
screening strategy of the tuberculosis surveys, mainly through the use of nucleic-acid
amplification tests (NAAT). Analysing the
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a tuberculose em portugal
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O laboratório é também muito útil na estratégia de monitorização dos rastreios da tuberculose, principalmente pelos testes de ampliação do ácido nucleico (TAAN). Ao
analisar os resultados, como um complemento importante da estratégia DOTS, obtivemos um índice de sucesso de 85,6% na população em geral, de acordo com os objetivos
da OMS. No que se refere aos grupos de risco, o índice de sucesso no grupo sem risco foi
de 90%, mas, nos outros grupos, e de acordo
com os objetivos da OMS, foi nomeadamente de 69% no de TB/VIH, de 53% no de
MDR-TB e de 82% no de imigrantes.
Finalmente, terminámos a nossa apresentação abordando a estratégia para controlo de
MDR/XDR-TB em Portugal, através da
criação, em junho de 2007, do Centro Nacional de Referência da Tuberculose Multi-Resistente (Circular Informativa 14-DT,
da Direcção-Geral de Saúde). Os seus principais objetivos são os de reduzir a prevalência e evitar a transmissão da MDR-TB,
apoiar os médicos na escolha dos regimes
terapêuticos, colaborar com eles no ajuste
desses regimes por efeitos adversos e, se necessário, na determinação do internamento
dos doentes e nas condições de isolamento.
Em 2009, uma das grandes prioridades do
PNT foi a implementação de uma rede de
centros regionais de referência para tratamento de casos de MDR/XDR-TB.
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outcomes is an important complement of
the DOTS Strategy; we had an 85.6% success rate in the general population, in accordance to the WHO goals. As to risk
groups, the success rate in the risk-free
group was of 90%, but under WHO goals,
in the other risk groups, it was namely 69%
in TB/HIV, 53% in MDR TB and 82% in
immigrants.
Finally, we finished our presentation addressing the strategy for MDR/XDR-TB
control in Portugal, through the creation of
the National Reference Centre for Multidrug-resistant Tuberculosis, in June 2007
(policy document 14-DT, General-Directorate of Health). Its main objectives are to
reduce prevalence and prevent transmission
of MDR-TB, support clinicians in choosing
the therapeutic regimens, collaborate with
them in the management of adverse effects
and, if needed, in the hospitalization and
isolation of patients in the best possible
conditions.
In 2009, one of the high priorities of NTP
was the implementation of a network of regional reference centres for the treatment of
MDR/XDR TB cases.
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A experiência brasileira de controlo da multidroga-resistência
Fernando Augusto Fiuza de Melo1
Brazilian experience in the management of multidrugresistance
1
Resumo
Neste artigo de revisão, o autor faz uma revisão de
como evoluiu a abordagem da tuberculose multirresistente (MDR) no Brasil, desde a introdução da rifampicina associada a isoniazida e a pirazinamida (RHZ).
Mostra que o país foi um dos primeiros no mundo a
aplicar o esquema RHZ dentro de um sistema de tratamento com um esquema de primeira linha, outro
específico para as formas meningoencefálicas, para retratamento para recidivas ou retorno com tuberculose
ativa após abandono, e um esquema de reserva. O sistema era de aplicação nacional com garantia de fornecimento gratuito das drogas e autoadministrado. Avalia
a evolução da resistência aos medicamentos, a emergência da resistência múltipla e como foi organizado o
controlo desta forma da doença.
Abstract
In this article the author reviews the evolution of the
approach to multidrug-resistant tuberculosis (MDRTB) in Brazil following the introduction of rifampicin
associated to isoniazid and pyrazinamide (RHZ). It
shows Brazil was one of the world’s first countries to
use the RHZ regimen within a treatment system, with
a first line regimen, another one specific for meningoencephalic forms, for re-treatment of recurrences or of
patients who returned with active tuberculosis after
abandoning treatment, and a reserve regimen. The system was applied nationwide with guaranteed cost-free
provision of medication, and self-administered. The
author evaluates the growth of drug resistance, the
emergence of multidrug-resistance and how management of this form of the disease has been organised.
Palavras-chave: Tuberculose multirresistente a múltiplos medicamentos (TB-MDR), tuberculose superresistente (TB-XDR).
Key-words: Multidrug-resistant tuberculosis (MDRTB), extensively drug-resistant tuberculosis (XDR-TB).
Médico, Diretor do Instituto Clemente Ferreira – Coordenadoria de Controle de Doenças da Secretaria de Estado da Saúde de São Paulo – ICF/CCD/SES-SP/Physician,
Director, Instituto Clemente Ferreira, Coordinator of Disease Control of the Secretary of State for Health of São Paulo – ICF/CCD/SES-SP
Doutorado em Medicina, área de pneumologia, pela Escola Paulista de Medicina da Universidade Federal de São Paulo – EPM/UNIFESP/PhD, Pulmonology, Escola
Paulista de Medicina da Universidade Federal de São Paulo – EPM/UNIFESP
Membro do Comitê de Assessoria Técnico-Científico do Programa Nacional de Controle da Tuberculose do Ministério da Saúde (PNCT/MS)/Member of the Technical-Scientific Committee of the Ministry of Health’s National Tuberculosis Control Programme (PNCT/MS)
Correspondência/Correspondence to:
Rua Santo Estácio, 248
Cidade Vargas, CEP 04319-010 – São Paulo, SP, Brasil
Tele-fax: 0055 11 3218 8653
Telemóvel: 0055 11 8469 4330
e-mail: [email protected]
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Introdução
Embora seja um fenômeno mundial, a tuberculose (TB) apresenta características variáveis de acordo com a região do mundo,
dependente de fatores raciais, ecológicos,
socioeconômicos, interrelações com outras
endemias, como a da VIH/SIDA, perfil de
resistência às drogas em uso e do desenvolvimento do controlo da doença.
Assim como a TB, também a tuberculose
multidroga-resistente (MDR-TB), que espantou o mundo, apresenta um desenvolvimento próprio no Brasil1,2,3.
Introduction
Although a worldwide phenomenon, the
characteristics of tuberculosis (TB) vary in
line with the world region, depending on
racial, ecological and socioeconomic factors,
inter-relationships with other endemics,
such as HIV/AIDS, the resistance profile of
the drugs in use and the development of
management of the disease.
Similarly to TB, multidrug-resistant tuberculosis (MDR-TB), which has alarmed the
world, has had its own development in
Brazil1-3.
O sistema brasileiro
de tratamento da tuberculose
O Brasil foi o primeiro país não desenvolvido a usar esquema de curta duração com
participação da rifampicina (R) associada a
hidrazida (H), além da pirazinamida (Z), ao
reorganizar em todo o país o Programa Nacional de Controle da Tuberculose, do Ministério da Saúde (PNCT-MS), em 1979.
Essa mudança estabelecida, após um ensaio
dirigido comparando o esquema RHZ com
esquema de longa duração (12 meses) com a
H associada à estreptomicina (S) e ao etambutol (E)4, estabelece, não somente alterações de medicamentos, mas normatiza um
sistema de tratamento.
Esse sistema (Fig. 1) conta com um esquema de primeira linha, de curta duração, o
E-1 (2RHZ/4RH), indicado para todas as
formas de TB sem tratamento anterior, menos para a forma meningoencefálica, com
uso de corticosteroides na fase de ataque e
prolongando a medicação para nove meses,
o E-2 (2RHZCort/7RH). Para os recidivantes após cura (RC) ou reingresso após abandono com doença ativa (RA), a indicação
The Brazilian tuberculosis
treatment system
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Brazil was the first developing country to use
a short-course regimen involving rifampicin
(R) associated to isoniazid (H) and pyrazinamide (Z), in reorganising a nationwide Tuberculosis Control Programme of the Ministry of Health (TBCP-MH), in 1979.
After a trial comparing the RHZ regimen
with a 12-month long combination regimen with the association of H streptomycin
(S) and ethambutol (E)4, this change, once
instituted, established changes in medication and harmonised a treatment system.
This system (Fig. 1) includes a short-course
first line regimen, the E-1 (2RHZ/4RH),
suitable for all forms of TB without prior
treatment, except the meningoencephalic
form, using corticosteroids during the attack
stage and prolonging the medication for
nine months, the E-2 (2RHZCort/7RH).
The same E-1 was prescribed for recurrences
after cure (RC), or for patients who relapsed
with active disease (RA) after abandoning
treatment, reinforced with ethambutol E
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CURE
CR
E-1
NT
E-1R
2RHZ/4RH
F
E-2(m)
2RHZ*/7RH
*steroid
AR
E-3
2RHZE/4RH
ABANDON
F
3SZEEt/
9EEt
DEATH
TBMR
F FAILURE
Fig. 1 – O sistema de tratamento da tuberculose no Brasil, 1999
Fig. 1 – Tuberculosis treatment system in Brazil, 1999
do mesmo E-1, reforçado com o etambutol
(E) na fase de ataque, o E-1R. Para os que
apresentassem falência com o E-1, foi previsto um esquema de segunda linha, com
duração de 12 meses, onde além da S, Z e E,
se associava a etionamida (Et), o E-3
(3SZEEt/9EEt)5 (Fig. 1).
A dose média da H no país é de 10 mg/kg/
/dia, bem maior do que a usada internacionalmente. As características operacionais
básicas do sistema eram a aplicação nacional, a garantia de fármacos, gratuita, com
R+H em um único comprimido e de uso
autoadministrado.
Para os fracassos com o E-3, a orientação era
encaminhar o doente para referências, a fim
de avaliar drogas alternativas, se acessíveis,
ou condutas cirúrgicas, se possíveis5.
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during the attack stage, the E-1R. In those
for whom the E-1 failed, there was a
12-month long second line regimen, which
associated ethionamide (Et) to S, Z and E;
the E-3 (3SZEEt/9EEt)5 (Fig. 1).
The mean dosage of H used in Brazil is 10
mg/kg/day, substantially higher than that
used internationally. The system’s basic logistical characteristics were: cost-free application
nationwide, guaranteed medication, R+H in
a single tablet to be self-administered.
Patients whom E-3 failed were referred to
reference units for evaluation of alternative
medication, if available, or surgical procedures, if possible5.
Evolution of the system’s
resistance and success
A reference centre cohort study evaluated the
growth of resistance in three-yearly cohorts in
the 1960s, 1970s and 1980s. It showed that
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Fig. 2 – Evolução da resistência primária durante três décadas: 1960, 1970 e 1980 – ICF/SP
Fig. 2 – Evolution of primary resistance over three decades: 1960’s, 1970’s and 1980’s – ICF/SP
Evolução da resistência
e do rendimento do sistema
Um estudo de coortes, numa referência,
avaliou evolutivamente a resistência em coortes trienais nas décadas de 1960, 1970 e
1980, mostrando que o PNCT organizado
na década de 1960 e a introdução da R na
sua reorganização, na década seguinte, possibilitaram uma excepcional proteção das
drogas clássicas com significativa redução da
resistência à H e à S. A redução entre 1960
e 1970 (p=0,002) foi mais significativa que
entre 1970 e 1980 (p=0,032), sugerindo
que um bom programa protege melhor a resistência do que a introdução de uma droga
potente, como a R (Fig. 2)6.
A avaliação na rotina do rendimento do E-1
na década de 1980, excluindo as transferências e considerando as mudanças de drogas
por toxicidade nos estudos de coortes, apresentou uma taxa de eficácia/efetividade de
94,6/77,8; com taxa de abandono de 13,7%,
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the TBCP organised in the 1960s and the introduction of R in its reorganisation in the
1970s provided exceptional protection of the
classic drugs with significant reduction in resistance to H and S. The reduction between 1960
and 1970 (p = 0.002) was more significant
than that between 1970 and 1980 (p = 0.032),
suggesting that a good programme offers better
protection against resistance than the introduction of a potent drug such as R (Fig. 2)6.
The routine evaluation of the success of E-1
in the 1980s, excluding transfers and taking
into consideration medication changes due
to toxicity in the cohort studies, showed a
94.6/77.8 rate of efficacy. It had a 13.7%
rate of abandonment, a 1.5% rate of failure,
a 3.1% rate of change due to adverse effects
and a 3.9% mortality rate7. In the 1990s
and early 2000s (1990-2002) these rates
were 93.9, 77.1, 13.1, 1.7, 3.3 and 4.8%,
respectively8. The success rate of the reserve
E-3 was low, with a rate of efficacy ranging
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de falência de 1,5%, troca por efeitos adversos
de 3,1% e de mortalidade de 3,9%7. Na década de 1990 e início do novo milênio (1990-2002), estas taxas foram de 93,9, de 77,1, de
13,1, de 1,7, de 3,3 e de 4,8%, respetivamente8. Quanto ao E-3 de reserva, o rendimento
foi baixo, com a taxa de eficácia/efetividade
variando entre 57,5/85,2 e 66,7/84,7, respetivamente9.
A piora da efetividade do sistema foi relacionada com o abandono, certamente resultante do regime autoadministrado e a
emergência da coinfecção TB-VIH/SIDA,
apresentando uma tendência de melhoria
pela introdução e expansão do tratamento
supervisionado e a boa qualidade do programa de controlo do VIH no país.
A evolução e a abordagem
resistência múltipla no Brasil
Com base nestes números, acrescentando as
taxas de RC e RA, em 1993 estimou-se que
cerca de 4 a 5% dos notificados acabariam
por apresentar resistência associada a R+H ou
impossibilidade de uso destas duas drogas por
toxicidade, com indicação do E-3. Seriam
portadores de MDR-TB, chamados entre nós
resistentes ao E-1. Como o rendimento do
E-3 é baixo, um número de doentes acaba por
sair do sistema sem perspetivas de tratamento
com as drogas programáticas (R, H, Z, E, S e
Et), estimado entre 0,3 e 0,4% dos notificados anuais, denominados portadores de tuberculose multirresistente (TBMR)10.
Para esses doentes, teoricamente sem perspectivas terapêuticas com as drogas programáticas, a
orientação das normas ministeriais no passado
era que fossem encaminhados para unidades
de referências sem lhes oferecer recursos e condições para sua atenção e abordagem.
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from 57.5/85.2 to 66.7/84.7, respectively9.
The system’s worsening rate of efficacy was
related to abandonment, almost certainly a
result of the self-administration regimen
and the emergence of the TB-HIV/AIDS
co-infection, tending towards improvement
with the introduction and expansion of supervised treatment and the good quality of
Brazil’s HIV management programme.
The progress and management
of multidrug-resistance in Brazil
Based on those numbers, to which are added the rates of RC and RA, in 1993 it was
estimated that around 4 – 5% of those notified as having the disease presented resistance associated to R+H or the impossibility
of the use of these drugs due to toxicity,
with indication for E-3. These patients had
MDR-TB, and were classified as resistant to
E-1. As E-3 has a low success rate, a number
of patients ended up leaving the system with
no prospect of treatment with the programmed drugs (R, H, Z, E, S and Et).
These were estimated as ranging from 0.3
– 0.4% of the annual number of those notified and known to carry multidrug-resistant
tuberculosis (MDR-TB)10. For these patients, in theory with no perspectives of
treatment with the programmed drugs, earlier Ministry norms referred them to reference units without offering the latter the
resources and possibilities to treat and manage them.
The first attempts at treatment were put
together by state programmes (provincial)
with bigger and better conditions. In the
later 1980s and early 1990s there were several trials of alternative regimens. All the
programmed drugs, lung resection surgery
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As primeiras tentativas de tratamento acabaram sendo realizadas por programas estaduais
(provinciais), com maiores e melhores condições. Na segunda metade dos anos de 1980
e início da década de 1990, surgiram diversas experiências de esquemas alternativos.
Associava-se de uma só vez todas as drogas
programáticas, cirurgias de ressecção pulmonares e uso de antigos fármacos não normatizados pelo PNCT, como a canamicina
(KM), o ácido para-aminossalicílico (PAS),
tiossemicarbazona (TSCZ) e outros. Foram
testadas a clofazenina (CFZ) usada no tratamento da hanseníase, a amicacina (AM) e,
mais recentemente, as novas quinolonas. A
efetividade dos esquemas tomava como base
a cura ou mesmo a sobrevida maior encontrada entre os que evoluíam sem tratamento
alternativo11,12,13.
Em 1995, o Centro de Referência Professor Hélio Fraga, do MS (CRPHF/MS)/
Rio de Janeiro, organiza um protocolo nacional com esquema associando AM, levofloxacino (LFX), terizidona (TRZ), CFZ
e E (se ainda sensível), testado em três
centros de referências entre 1995 a 1997,
com taxas de eficácia/efetividade razoáveis
(n=187-56/48), quando confrontados
com estudos internacionais14.
Em 2000, inicia no CRPHF/MS o Programa de Vigilância Epidemiológica de TBMR
e em 2004 um convénio com a associação
civil brasileira sem fins lucrativos, “Projeto
MSH” (Management Sciences for Health), e
com recursos financiados pela USAID (United States Agency for International Development), garante o financiamento das drogas
alternativas. Um guia de vigilância epidemiológica de TBMR foi elaborado condensando o conhecimento acumulado no país,
estabelecendo normas para o diagnóstico,
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and use of earlier drugs non-standardized
by the TBCP, such as kanamycin (KM), paminosalicylic acid (PAS), thiosemicarbazone (TSCZ) and others were simultaneously contemplated. Clofazimine (CFZ)
used to treat hanseniasis, amikacin (AM)
and, more recently, the new quinolones
were tested. Cure or even the greater survival seen in those who progressed without
alternative treatment was the basis for assessing the regimens’ efficacy11-13.
In 1995, the Centro de Referência Professor
Hélio Fraga do MS (CRPHF/MS)/Rio de
Janeiro organised a national protocol with a
regimen associating AM, levofloxacin
(LFX), terizidone (TRZ), CFZ and E (if
still sensitive), tested in three reference centres, from 1995 to 1997, with fair efficacy
rates (n = 187– 56/48) in comparison with
international studies14.
In 2000, the CRPHF/MS initiated an
MDR-TB epidemiological surveillance programme and, in 2004, had an agreement
with a non-profit making Brazilian civil association “Project MSH” (Management
Sciences for Health) financed by the USAID
(United States Agency for International Development), guaranteeing the financing of
the alternative drugs. A guide to MDR-TB
epidemiological surveillance was drawn up,
summarising the country’s collected knowledge, establishing diagnostic, treatment,
prevention and biosafety norms, epidemiological surveillance guidelines, human resources training and financial support for
carrying out the programme. A nationwide
notification system specifically for MDRTB was instituted15.
Subsequent studies and routine review of
notifications showed much smaller numbers
than predicted, with regional variations dif-
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tratamento, prevenção e biossegurança,
orientações para a vigilância epidemiológica, formação de recursos humanos e provisão de recursos materiais para execução do
programa. Foi instituído um sistema de notificação específico para a TBMR, aplicado
nacionalmente15.
Estudos posteriores e revisão de notificados
na rotina acabaram por apresentar números
bem menores do que os estimados, com variações regionais diferenciadas pela situação
sócioeconômica, pela qualidade do programa local e por uma provável subnotificação
devido à baixa oferta da cultura e testes de
sensibilidade15 (Fig. 3).
O esquema alternativo atual para os portadores de TBMR usado no país dentro desse
programa é: AM (ou S se sensível) por 12
meses; OFX, TRZ e E (se sensível) por 18
meses e Z (se sensível) por seis meses. Alguns serviços usam, ao invés da Z, o metronidazol (MTZ), por 18 meses. As taxas de
cura variam entre 62 a 85%, com o abando-
ferentiated by local socioeconomic situations, the quality of the local program and
by a probable under-notification due to the
meagre amount of culture and sensitivity
tests available15 (Fig. 3).
The current alternative regimen for MDRTB patients used in Brazil within the programme is AM (or S if sensitive) for 12
months; OFX, TRZ and E (if sensitive) for
18 months and Z (if sensitive) for six
months. Some Units use metronidazol
(MTZ) instead of Z for 18 months. Cure
rates range from 62% to 85%, the rate of
abandonment from 5-7%, failure 10-15%
and the mortality rate dropped from 33%
to 11%, depending on the greater or lesser
degree of organisation and quality of treatment15,16.
The MDR-TB cases were mostly post-primary (74-80%), with the primaries around
6-8%, especially between contacts and risk
groups (conscripts, the homeless, health
care professionals and others with high ex-
Fig. 3 – Casos de TBMR no Brasil (1994-2006) (n = 2632 casos – incidência anual média: 75 000 casos/ano)
Fig. 3 – MDR-TB cases in Brazil (1994-2006) (n=2632 cases, mean annual rate 75 000 cases/year)
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no entre 5 e 7%, a falência entre 10 e 15%
e o óbito decresceu de 33 para 11%, dependendo do maior ou menor grau de organização e qualidade do atendimento15,16.
Os casos de TBMR são maioritariamente pós-primários de 74 a 80%, sendo os primários
em torno de 6 a 8%, especialmente entre contatos e grupos de risco (conscritos, sem-abrigo
e profissionais de saúde e outros com alta exposição) e, 11 a 20%, indeterminados (ausência de informações capazes de estabelecer o
grupo). A ocorrência de TBMR entre coinfectados pelo VIH no país é baixa, entre 1,6
e 3%. Não se pode afirmar se há uma tendência a manter esse número ou se deve aumentar
a ocorrência, ou mesmo se não existe uma
subnotificação15,16,17. A grande maioria dos casos apresenta lesões pulmonares bilaterais, cerca de 80%, o que inviabiliza condutas cirúrgicas de rotina, seja associado ao tratamento ou
higiénicas (?), para prevenir recidiva, como
propõem alguns cirurgiões no país18.
Casos de TB superresistentes (TB-XDR),
com resistência a duas drogas usuais e três alternativas, observados a partir de 2000, vêm
sendo encontrados no Brasil. As baixas ofertas de testes de sensibilidade, em especial para
drogas alternativas, dificultam a seleção dessas formas da doença. No Instituto Clemente
Ferreira, que conta com TS automatizados e
tem convénios com serviços que avaliam a
resistência pelo MIC em meio líquido e leitura pela técnica de Alamar-Blue, um levantamento recente documentou 34 casos resistentes a quinolonas, sendo 16 resistentes a
AM e 18 a S. Tratados com o esquema alternativo indicado para TBMR, o resultado foi
nove curas e 25 falências, entre estes 17 óbitos. Um dado alarmante foi o encontro de
três casos de TB-XDR primários, um indício
de que bacilos superresistentes podem estar
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posure) and 11-20% undetermined (lack
of information capable of establishing a
group). There is a low incidence of MDRTB in patients co-infected with HIV in
Brazil, between 1.6% and 3%. We cannot
state if there is a trend to maintain this
number or if it will increase, or even if
there was under-notification15-17. The vast
majority of cases, around 80%, present bilateral pulmonary lesions, which rules out
routine surgical procedures, be it associated to treatment or hygiene (?) to prevent
recurrences, as some Brazilian surgeons
propose18.
Cases of extensively drug-resistant tuberculosis (XDR-TB) with resistance to two usual
and three alternative drugs, seen after 2000,
have been observed in Brazil. The scarcity of
drug sensitivity testing, in particular to alternative drugs, makes it hard to select for
these forms of disease. In the Instituto Clemente Ferreira, which has automated susceptibility testing and agreements with units
which evaluate resistance using minimal inhibitory concentration (MIC) in liquid medium and reading using the Alamar-Blue
technique, 34 quinolone-resistant cases
were recently documented with 16 resistant
to AM and 18 to S. Treatment with the alternative regimen indicated for MDR-TB
resulted in nine cures and 25 failures, with
17 deaths among the latter. A cause for
alarm was finding three cases of primary
XDR-TB, indicating that super-resistant
bacilli could be in our midst and not only
occasioned by treatment errors19,20.
Note: Brazil, based on a review of the progress of primary and post-primary resistance,
decided to change the initial E-1 with association of E as the fourth drug in the attack
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circulando no nosso meio, e não apenas produzidos por erros terapêuticos19,20.
Nota: O Brasil, com base em revisão da evolução da resistência primária e pós-primária,
decidiu e está alterando o E-1 inicial com
associação do E como quarta droga na fase
de ataque, com implantação gradativa no
início de 2010. Foram considerados os aumentos da taxa de resistência a H da TB-MDR em coinfetados TB-HIV/AIDS e da
longevidade populacional com estoque de
TB-latente selecionado nos 30 anos de uso
de R+H. Essa mudança faz-se com o uso de
associação dos fármacos na forma de comprimidos com dose fixa combinada (4 em
1), altera também o E-3 de reserva agora
para doentes portadores de TB-MDR e passa a considerar os que não conseguem a cura
dentro do sistema como TB-XDR21.
stage, phased in from 2010 on. The increased rates of resistance to H, MDR-TB
in patients co-infected with TB-HIV/AIDS
and longevity of the population with a stock
of latent TB selected in the 30 years of use
of R+H were considered. This change is in
the use of the association of drugs in tablet
form with fixed combined dosage (4 in 1)
and also changes the reserve E-3 now for
MDR-TB patients and considers those who
cannot find a cure within the system as
XDR-TB cases21.
Bibliografia/Bibliography
1. Melo FAF. Evolução dos conhecimentos, o controle e
algumas questões pendentes na tuberculose pulmonar
multirresistente, Pneumologia: atualização e reciclagem.
Sociedade Paulista de Pneumologia e Tisiologia 2003;
5:285-292.
2. CDC/USA. National action plan to combat of
multidrug-resistant tuberculosis. Meting the challenge
of multidrug-resistant tuberculosis: summary of a conference. Management of persons exposed to multidrug-resistant tuberculosis. MMWR 1992; 41(RR-11).
3. Snider DE Jr, Roper WL. The news tuberculosis
(Eds.). N Engl J Med 1992; 267:703-705.
4. Gerhardt G, Teixeira GM, Hijjar MA, Feitosa JVP,
Penna MLF. Resultados iniciales del tratamiento de
corta duración en condiciones de rutina en los servicios
de salud del Brasil. Bol UICT 1982; 57:87-92.
5. Ministério da Saúde/Fundação Nacional de Saúde/
Centro de Referência Prof. Hélio Fraga-Rio de Janeiro
– Sociedade Brasileira de Pneumologia e Tisiologia.
Controle da tuberculose: uma proposta de integração
ensino-serviço. 5.ª ed. 2000.
R e v i s t a
6. Melo FAF, Afiune JB, Ribeiro LHG, Almeida, EA,
Castelo A. Resistência primária do M. tuberculosis num
serviço ambulatorial de referência em São Paulo:
evolução por três décadas e comparação com outros estudos nacionais. J Pneumol 1996; 22:3-8.
7. Ministério da Saúde/Fundação Nacional de Saúde/
Centro de Referência Prof. Hélio Fraga-Rio de Janeiro
Documento Básico da Reunião de Avaliação operacional
e epidemiológica do PNCT na década de 80. Bol Pneumol Sanit 1993, número especial.
8. Ministério da Saúde/Secretaria de Vigilância em
Saúde/Centro de Referência Prof. Hélio Fraga-Rio de
Janeiro. Análise da situação da tuberculose no Brasil nos
anos 90 e início da década atual. Bol Pneumol Sanit
2005; 13:133-179.
9. Campos HS, Melo FAF. Efetividade do esquema 3
(3sSZEEt/9EEt) no retratamento da tuberculose na rotina
das unidades de saúde. Bol Pneumol Sanit 2000; 8:7-14.
10. Melo FAF, Ide Neto J, Seiscento M, Pinto JA, Afiune
JB. Tuberculose multirresistente. J Pneumol 1993; 19:73-82.
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a experiência brasileira de controlo da multi-droga-resistência
Fernando Augusto Fiuza de Melo
11. Picon PD, Pereira AN, Dias CA. Tuberculose pulmonar: análise terapêutica de 62 casos crônicos. Rev
AMERIGS 1980; 24:36-8.
12 Comissão de terceira linha do Hospital Sanatório
Partenon: Eficácia terapêutica do esquema de terceira
linha ofloxacina–amicacina–tiacetazona-hidrazida para
tuberculose multirresistente. J Pneumol 1995; 21:215-224.
13. Seiscento M, Fiuza de Melo FA, Ide Neto J, Noronha AML, Afiune JB, Inomata T, Cruz ML. Tuberculose multirresistente (TBMR): aspectos clínico-laboratoriais, epidemiológicos e terapêuticos. J Pneumol 1997;
23:237-244.
14. Dalcolmo MP, Fortes A, Melo FAF, Motta R, Ide
Neto J, Cardoso N, Andrade M, Barreto AW, Gerhardt
G. Estudo de efetividade de esquemas alternativos para
o tratamento da tuberculose multirresistente no Brasil. J
Pneumol 1999; 25:70-77.
15. Ministério da Saúde/ Secretaria de Vigilância em
Saúde/Centro de Referência Prof. Hélio Fraga-Rio de
Janeiro. Tuberculose multirresistente: guia de vigilância
epidemiológica; Rio de Janeiro, 2007.
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16. Ministério da Saúde/Secretaria de Vigilância em
Saúde/Centro de Referência Prof. Hélio Fraga-Rio de
Janeiro. Dados coletados no Sistema de Informação da
Tuberculose Multirresistente (Sistema TBMR), mediante uso de senha. 2008.
17. Melo FAF, Afiune JB, Ide Neto J, Almeida EA, Spada DTA, Antel ANL, Cruz ML. Aspectos epidemiológicos da tuberculose multirresistente em serviço de referência na cidade de São Paulo. Rev da Soc Brasil Med
Trop 2003; 36:733-740.
18. Leite LPS, Costa ALP, Andrade RNS, Galvão T.
Tratamento cirúrgico adjuvante de tuberculose pulmonar
multirresistente. Jornal de Pneumologia 1997; 23:11-14.
19 Emergence of Mycobacterium tuberculosis with extensive resistance to second line drugs worldwide 2000 –
2004. MMWR 2006; 55(11).
20. Savioli MTG, Melo FAF, Morrone N e Rodrigues
DS. Tuberculosis with extensive resistance to drugs in a
TB reference center in Sao Paulo, Brazil. Poster accepted
CHEST, San Diego, California 2009.
21. III Diretrizes para a tuberculose da Sociedade Brasileira de Pneumologia e Tisiologia. J Bras Pneumol
2009; 35:1018-1048.
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Forças evolutivas do Mycobacterium tuberculosis
Sebastien Gagneux1
Evolutionary forces in Mycobacterium tuberculosis
A análise da diversidade genética de patogénios bacterianos ajuda a compreender melhor a epidemiologia e a evolução destes micróbios. A compreensão da evolução de
patogénios é particularmente importante
nos dias de hoje, com a crescente resistência
antimicrobiana1. Porém, o estudo da diversidade genética do complexo Mycobacterium
tuberculosis (CMTB) é um desafio, porque a
variação da sequência de ADN nestes organismos é baixa2 e os métodos-padrão de genotipagem, como a tipagem de sequência
multiloco (TSML), já bem estabelecida noutras bactérias3, proporcionam pouca informação4. Além disso, deveriam usar-se diferentes métodos de genotipagem quando se
investigam questões diferentes5. A investigação epidemiológica molecular clássica da
transmissão de doenças e a diferenciação entre recidiva e reinfeção exógena requer marcadores de genotipagem com grande força
discriminatória6, enquanto as análises evolutivas deveriam apoiar-se em marcadores filogeneticamente robustos7. Infelizmente,
ocorre frequentemente trade-off entre essas
Analyzing the genetic diversity of bacterial
pathogens helps to better understand the
epidemiology and evolution of these microbes. Understanding the evolution of
pathogens is particularly important in today’s era of increasing antimicrobial resistance1. However, studying the genetic diversity of the Mycobacterium tuberculosis
complex (MTBC) is challenging because
the DNA sequence variation in these organisms is low2, and standard genotyping
tools such as multilocus sequence typing
(MLST), which have been well established
in other bacteria3, provide little information4. Moreover, different genotyping
tools should be used when addressing different research questions5. Classical molecular epidemiological investigation of
disease transmission and differentiating
between relapse and exogenous re-infection requires genotyping markers with a
high discriminatory power6, whereas evolutionary analyses should rely on phylogenetically robust markers7. Unfortunately,
there is often a trade-off between these
1 Division of Mycobacterial Research, MRC National Institute for Medical Research, London, UK
e-mail: [email protected]
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propriedades nos marcadores individuais
usados na genotipagem, particularmente
quando se estudam bactérias geneticamente
monomórficas, como CMTB8,9. Com o
CMTB, a spoligotipagem e a tipagem por
MIRU-VNTR têm sido usadas com grande
sucesso para tipagem e investigação epidemiológica molecular, mas essas técnicas são
limitadas quando se inferem relações filogenéticas entre estirpes10. Isto deve-se ao facto
de a spoligotipagem e a tipagem por MIRU-VNTR dependerem de marcadores moleculares repetitivos que se alteram rapidamente e, por consequência, esses marcadores têm
um elevado índice de evolução convergente10. Em contraste, as deleções genómicas ou
polimorfismos de sequência (LSP) e os polimorfismos de um nucleótido único (SNP)
aglomeram-se de modo relativamente lento
no genoma de CMTB, podendo assim serem usados para definir linhagens filogeneticamente robustas11.
Já mostrámos que os LSP definem seis linhagens de estirpes principais no CMTB
humano12, que incluem as duas linhagens
geralmente referidas como M. africanum
África Ocidental 1 e 2. Mostrámos também
que estas linhagens eram correspondentes a
agrupamentos observados na tipagem de
SNP11. No entanto, devido a limitações inerentes a esses estudos anteriores, a real distância genética entre linhagens e nelas próprias é ainda desconhecida. Para definir
mais quantitativamente a diversidade genética em CMTB, realizámos uma análise de
sequências multilocos em larga escala
(MLSA) de 108 estirpes globalmente representativas13, que incluíram membros de
CMTB adaptado a animais, como M. bovis,
M. microti, M. caprae e M. pinnipedii. Para
todas estas 108 estirpes, gerámos a sequên-
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properties in individual markers used for
genotyping, and this is particularly true
when studying genetically monomorphic
bacteria such as MTBC8,9. In MTBC, spoligotyping and MIRU-VNTR typing have
been used very successfully for fine typing
and molecular epidemiological investigations, but these techniques are limited
when inferring phylogenetic relationships
between strains10. This is because spoligotyping and MIRU-VNTR typing rely on
repetitive molecular markers that change
rapidly, and as a consequence these markers exhibit a high rate of convergent evolution10. By contrast, genomic deletions,
also known as large sequence polymorphisms (LSPs), and single nucleotide polymorphisms (SNPs) accumulate relatively
slowly in the MTBC genome and can thus
be used to define phylogenetically robust
strain lineages11.
We have previously shown that LSPs define six main strain lineages within the
human MTBC12. These include the two
lineages generally referred to as M. africanum West-Africa 1 and 2. We have also
shown that these lineages were congruent
with groupings found based on SNP-typing11. However, because of the inherent
limitations of those previous studies, the
actual genetic distance within and between strain lineages remained unknown.
To define more quantitatively the genetic
diversity in MTBC, we performed a largescale multilocus sequence analysis (MLSA)
of 108 globally representative strains13.
These included members of the animaladapted MTBC like M. bovis, M. microti,
M. caprae, and M. pinnipedii. For all of
these 108 strains, we generated the complete DNA sequence of 89 genes, which
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cia de ADN completa de 89 genes, que
compreenderam genes de housekeeping, genes de virulência e genes antigénicos. Usámos os genes concatenatos completos para
definir uma nova filogenia do CMTB com
base na sequência do ADN13. Esta é a mais
abrangente e robusta filogenia do CMTB
até à data14 e é também altamente congruente com a nossa anterior classificação do
CMTB, baseada na análise de LSP, em seis
linhagens principais15. A natureza quantitativa dos novos dados das sequências de ADN
proporcionou um novo discernimento. Em
particular, enquanto todas as formas de
CMTB adaptadas de animais se agrupavam,
representavam apenas um subgrupo da diversidade genética observada em toda a filogenia, sugerindo que a diversidade genética
do CMTB humano é mais pronunciada do
que anteriormente se pensava13. Além disso,
as duas linhagens M. africanum, quase exclusivamente observadas na África Ocidental, são as mais ancestrais. Conjuntamente
com o facto de ser a África o único continente onde se encontram as seis mais importantes linhagens do CMTB humano,
estes achados apoiam a teoria de que o
CMTB terá sido originado em África12.
Os dados do nosso novo estudo por análise de
sequências multilocos permitiram-nos estudar
a história evolutiva do CMTB humano em
maior detalhe. Quando comparámos os dados
da diversidade genética com as distâncias geográficas que separam os locais de origem das
estirpes incluídas no estudo, encontrámos correlações estatisticamente significativas entre
estas duas medidas13. Os presentes resultados
apoiaram um cenário ‘Fora-de-e-de-volta-a-África’ para a evolução do CMTB humano13.
De acordo com este cenário, o CMTB originou-se na África e espalhou-se originalmente
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comprised housekeeping genes, virulence
genes, and antigenic genes. We used the
complete gene concatenates to define a
new DNA sequence-based phylogeny of
MTBC13. This phylogeny is the most
comprehensive and most robust phylogeny of MTBC to date14, and is also highly
congruent with our previous LSP-based
classification of MTBC into six main lineages15. The quantitative nature of the
new DNA sequence data offered some
new insights. In particular, while all animal-adapted forms of MTBC clustered
together, they represented only a sub-set
of all the genetic diversity observed across
the whole phylogeny, suggesting that the
genetic diversity of human MTBC is more
pronounced than previously thought13.
Furthermore, the two M. africanum lineages, which are almost exclusively observed in West Africa or the most ancestral lineages. Together with the fact that
Africa is the only continent that harbours
all six main lineages of human MTBC,
these findings support to view that MTBC
originated in Africa12.
Our new MLSA data allowed us to study
the evolutionary history of human MTBC
in more detail. When we compared the genetic diversity data to the geographic distances separating the places of origin of the
strains included in the study, we found statistically significant correlations between
these two measures13. Our results supported
an ‘Out-of-and-back-to-Africa’ scenario for
the evolution of human MTBC13. According to this scenario, MTBC originated in
Africa and spread originally out of Africa accompanying ancient human migrations
which occurred approximately 50 000 years
ago. Through these ancient migrations,
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Sebastien Gagneux
para fora desse continente, acompanhando
antigas migrações humanas que ocorreram há
aproximadamente 50 000 anos. Através dessas
antigas migrações, três ‘modernas’ linhagens
evolutivas de CMTB espalharam-se por áreas
da Europa, da Índia e da China, respectivamente. Essas regiões passaram por grande aumento da população humana durante as últimas centenas de anos, o que levou a uma
expansão destas linhagens de CMTB. Concomitantemente, estas linhagens modernas começaram a disseminar-se globalmente e regressaram a África, através de recentes ondas
de colonização, comércio e conquista.
Outra observação interessante que decorreu
da nossa análise de sequências multilocos foi
que o rácio SNP não sinónimos: SNP sinónimos (uma medida conhecida como dN/dS) é
muito maior no MTBC do que na maioria
das outras bactérias13. Um dN/dS elevado em
patogénios bacterianos tem normalmente
sido associado à ancestralidade recente16. Por
outras palavras, ainda não houve tempo suficiente para a purificação da seleção eliminar
SNP não sinónimos, a maioria dos quais tendem a ser ligeiramente nocivos à competência
bacteriana. Porque os SNP sinónimos se acumulam ao longo do tempo sem serem eliminados pela seleção natural, um elevado dN/dS
pode ser indicador de ocorrência recente. Porém, as nossas análises apoiam a noção de que
a razão dN/dS é elevada no CMTB porque a
purificação da seleção deste organismo é reduzida, provavelmente pela consequência do aumento aleatório da deslocação genética associada às acumulações de populações durante a
transmissão doente a doente13. Isto sugere que
o ‘acaso’, e não apenas a seleção natural, tem
conduzido a evolução do CMTB.
Em resumo, o presente trabalho de análise de
sequências multilocos confirmou que a estru-
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three evolutionarily “modern” lineages of
MTBC seeded areas in Europe, India, and
China, respectively. These regions experienced strong human population increase
during the last few hundred years, leading
to an expansion of these MTBC lineages.
Concomitantly, these modern lineages started to spread globally and back to Africa,
through recent waves of colonization, trade,
and conquest.
Another interesting observation coming
out of our MLSA work was that the ratio
of nonsynonymous SNPs to synonymous
SNPs (a measure known as dN/dS) is
much higher in MTBC than in most other
bacteria13. A high dN/dS in bacterial
pathogens has generally been associated
with recent ancestry16. In other words,
there has not been enough time of purifying selection to remove nonsynonymous
SNPs, the majority of which tend to be
slightly deleterious to bacterial fitness.
Because synonymous SNPs accumulate
over time without being removed by natural selection a high dN/dS can be indicative of recent emergence. However,
our analyses supported the view that the
reason dN/dS is high in MTBC is because
purifying selection in this organism is reduced, most likely as consequence of increased random genetic drift associated
with the serial population bottlenecks
during patient-to-patient transmission13.
This suggest that ‘chance’, and not just
natural selection has been driving the evolution of MTBC.
In summary, our MLSA work confirmed
that the genetic population structure of
human MTBC consists of six main strain
lineages. Our findings also show that the
human MTBC lineages are more geneti-
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tura genética da população humana de
CMTB consiste em seis linhagens de estirpes
principais. Os achados mostram também que
as estirpes do CMTB humano são mais geneticamente diversas do que se pensava anteriormente. Há evidência crescente de que a linhagem de CMTB pode afetar a evolução da
tuberculose infeção e doença17-20. O presente
trabalho de sequenciação identificou muitos
SNPS filogeneticamente informativos, o que
permitirá aos investigadores classificar rapidamente estirpes em agrupamentos robustos10 e
continuar a explorar diferenças específicas das
linhagens nos contextos experimental e clínico. As nossas análises de genética de populações revelaram que a diversidade genética do
CMTB foi moldada por migrações humanas
antigas e mais recentes e que a deslocação genética aleatória pode ser uma importante força condutora na evolução do CMTB. À medida que os custos da sequenciação de ADN
continuam a baixar, a sequenciação do genoma completo tem potencial para se tornar o
instrumento ‘final’ para genotipagem de bactérias21. A sequenciação genómica comparativa de grandes coleções de estirpes de CMTB
melhorará a nossa compreensão das forças
evolutivas que moldam a diversidade genética
deste importante patogénio15.
cally diverse than previously thought.
There is mounting evidence that MTBC
lineage can affect the outcome of tuberculosis infection and disease17,18,19,20. Our
sequencing work identified many phylogenetically informative SNPs, which will
allow researchers to rapidly classify
strains into robust groupings10, and to
further explore lineage-specific differences in experimental and clinical contexts. Our population genetic analyses
revealed that the genetic diversity in
MTBC has been shaped by both ancient
and more recent human migrations, and
that random genetic drift might be an
important driving force in the evolution
of MTBC. As DNA sequencing costs
continue to decrease, full genome-sequencing has the potential to become
the “ultimate” genotyping tools for bacteria21. Comparative genome sequencing
of large strain collections of MTBC will
improve our understanding of the evolutionary forces shaping the genetic diversity in this important pathogen 15.
Bibliografia/Bibliography
1. Borrell S, Gagneux S. Infectiousness, reproductive fitness and evolution of drug-resistant Mycobacterium tuberculosis. Int J Tuberc Lung Dis 2009; 13:1456-1466.
2. Sreevatsan S, Pan X, Stockbauer KE, Connell ND,
Kreiswirth BN, et al. Restricted structural gene polymorphism in the Mycobacterium tuberculosis complex
indicates evolutionarily recent global dissemination.
Proc Natl Acad Sci USA 1997; 94: 9869-9874.
3. Maiden MC. Multilocus sequence typing of bacteria.
Annu Rev Microbiol 2006; 60: 561-588.
R e v i s t a
4. Baker L, Brown T, Maiden MC, Drobniewski F. Silent nucleotide polymorphisms and a phylogeny for Mycobacterium tuberculosis. Emerg Infect Dis 2004; 10:
1568-1577.
5. Feil EJ. Small change: keeping pace with microevolution. Nat Rev Microbiol 2004; 2: 483-495.
6. Mathema B, Kurepina NE, Bifani PJ, Kreiswirth
BN. Molecular epidemiology of tuberculosis: current insights. Clin Microbiol Rev 2006; 19: 658-685.
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Sebastien Gagneux
7. Achtman M, Wagner M. Microbial diversity and the
genetic nature of microbial species. Nat Rev Microbiol
2008; 6: 431-440.
8. Achtman M. Evolution, population structure, and
phylogeography of genetically monomorphic bacterial
pathogens. Annu Rev Microbiol 2008; 62: 53-70.
9. Pearson T, Okinaka RT, Foster JT, Keim P. Phylogenetic understanding of clonal populations in an era of
whole genome sequencing. Infect Genet Evol 2009; 9:
1010-1019.
10. Comas I, Homolka S, Niemann S, Gagneux S. Genotyping of genetically monomorphic bacteria: ADNsequencing in Mycobacterium tuberculosis highlights the
limitations of current methodologies. PLoS One 2009;
4: e7815.
11. Gagneux S, Small PM. Global phylogeography of
Mycobacterium tuberculosis and implications for tuberculosis product development. Lancet Infect Dis 2007; 7:
328-337.
12. Gagneux S, Deriemer K, Van T, Kato-Maeda M, de
Jong BC, et al. Variable host-pathogen compatibility in
Mycobacterium tuberculosis. Proc Natl Acad Sci USA
2006; 103: 2869-2873.
13. Hershberg R, Lipatov M, Small PM, Sheffer H, Niemann S, et al. High functional diversity in Mycobacterium tuberculosis driven by genetic drift and human demography. PLoS Biol 2008; 6:e311.
14. Smith NH, Hewinson RG, Kremer K, Brosch R,
Gordon SV. Myths and misconceptions: the origin and
S 26
R e v i s t a
P o r t u g u e s a
evolution of Mycobacterium tuberculosis. Nat Rev
Microbiol 2009; 7: 537-544.
15. Comas I, Gagneux S. The past and future of tuberculosis research. PLoS Pathog 2009; 5: e1000600.
16. Rocha EP, Smith JM, Hurst LD, Holden MT,
Cooper JE, et al. Comparisons of dN/dS are time dependent for closely related bacterial genomes. J Theor
Biol 2006; 239: 226-235.
17. Caws M, Thwaites G, Dunstan S, Hawn TR, Thi Ngoc
Lan N, et al. The influence of host and bacterial genotype
on the development of disseminated disease with Mycobacterium tuberculosis. PLoS Pathog 2008; 4: e1000034.
18. de Jong BC, Hill PC, Aiken A, Awine T, Antonio M,
et al. Progression to active tuberculosis, but not transmission, varies by Mycobacterium tuberculosis lineage in
the Gambia. J Infect Dis 2008; 198: 1037-1043.
19. de Jong BC, Hill PC, Brookes RH, Gagneux S, Jeffries DJ, et al. Mycobacterium africanum elicits an attenuated T cell response to early secreted antigenic target, 6
kDa, in patients with tuberculosis and their household
contacts. J Infect Dis 2006; 193: 1279-1286.
20. Thwaites G, Caws M, Chau TT, D’Sa A, Lan NT, et
al. The relationship between Mycobacterium tuberculosis genotype and the clinical phenotype of pulmonary
and meningeal tuberculosis. J Clin Microbiol 2008; 46:
1363-1368.
21. Medini D, Serruto D, Parkhill J, Relman DA, Donati C, et al. Microbiology in the post-genomic era. Nat
Rev Microbiol 2008; 6: 419-430.
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Epidemiologia e ecologia de micobactérias não
tuberculosas
Joseph O Falkinham III1
Epidemiology and ecology of nontuberculous
mycobacteria
As micobactérias não tuberculosas (MNT) são
patogénios oportunistas dos humanos e animais
encontrados no meio ambiente1,2. Nos Estados
Unidos, a prevalência de doenças por MNT
vem aumentando 8% ao ano, atingindo atualmente quase 35 casos por cada 100 0003. Em
Ontário, Canadá, a prevalência de doença pulmonar por MNT aumentou de 1,5 para 9,0
por 100 000 (6 vezes) no período 1997-20034.
A evidência de que o ambiente é a fonte das
doenças por MNT em seres humanos foi
conseguida a partir do ADN de isolados de
MNT de doentes com SIDA, de água de beber5 e de doentes imunocompetentes e de
isolados de MNT de terra de vasos6 ou de um
chuveiro doméstico7. Os seres humanos estão
continuamente expostos a MNT, uma vez
que estes vivem e crescem normalmente nos
sistemas de distribuição da água de beber8, e
20% das amostras recolhidas em biofilmes
nas cabeças dos chuveiros domésticos nos Estados Unidos tinham M. avium9.
1
Nontuberculous mycobacteria (NTM) are
opportunistic human and animal environmental pathogens1,2. In the United States,
the prevalence of NTM disease is increasing
by 8% per year and now stands at almost 35
cases per 100 0003. In Ontario, Canada the
prevalence of NTM pulmonary disease increased from 1.5 to 9.0 per 100 000 (6-fold
increase) over the period 1997-20034.
Evidence that the environment was the
source of NTM disease in humans was gained
from the identity of DNA fingerprints of
NTM isolates from AIDS patients and drinking water5 and from immunocompetent patients and NTM isolates from potting soils6
or a home shower7. Humans are continually
exposed to nontuberculous mycobacteria
(NTM) as they are normal inhabitants and
grow in drinking water distribution systems8
and 20 % of showerhead biofilms (swab samples) collected from households in the United
States had M. avium9.
Department of Biological Sciences, Virginia Tech,
Blacksburg, Virginia 24061-0406
Phone 1-540-231-5931
Fax 1-540-231-9307
e-mail: [email protected]
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Epidemiologia e ecologia de micobactérias não tuberculosas (MNT)
Joseph O Falkinham III
Os fatores de risco para doenças por MNT
incluem: imunocompetência diminuída devido a infeção por VIH, cancro, quimioterapia,
ou imunossupressão associada a transplantação, doença pulmonar preexistente, como
pneumoconiose, silicose e doença do pulmão
negro, alterações na arquitetura normal do
tronco, alcoolismo e hábitos tabágicos10. Recentemente, mutações no regulador da condutância transmembranária na fibrose quística (RCTFQ) e genes de α-1-antitripsina têm
sido associados ao risco aumentado de doença
pulmonar por MNT11. É preocupante o facto
de a doença pulmonar por MNT ter aumentado drasticamente entre homens e mulheres
idosos magros que, não tendo os fatores de
risco clássicos para doença micobacteriana,
parecem ser substancialmente mais suscetíveis
a doenças por MNT12. Com o envelhecimento das populações, é expectável que o número
de idosos com doença pulmonar por MNT
aumente, com o consequente peso para os
prestadores de cuidados de saúde. A terapêutica recomendada para infeções por MNT
inclui múltiplos antibióticos (e.g., claritromicina, etambutol e rifampin) por períodos de
24 meses13.
As micobactérias não tuberculosas sobrevivem, crescem e persistem em habitats partilhados por seres humanos e animais. São oligotróficos, podendo crescer em água com
mais de 50 μg de carbono orgânico assimilável (COA/)L14. A presença de grandes quantidades de MNT em pântanos de águas escuras e ácidas15 e no solo de pinhais16 deve-se,
em parte, ao crescimento estimulado pela
matéria orgânica do solo raramente metabolizada por outros microrganismos, nomeadamente ácidos húmico e fúlvico17.
As micobactérias têm uma verdadeira membrana externa18, cujos ácidos micólicos de
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Risk factors for NTM disease include: reduced immune competence as a result of
HIV infection, cancer, chemotherapy, or
immunosuppression associated with transplantation, preexisting lung disease such as
pneumoconiosis, silicosis, and black lung
disease, altered normal chest architecture,
alcoholism, and smoking10. Recently, mutations in the cystic fibrosis transmembrane conductance regulator (CFTR) and
α-1-antitrypsin genes have been associated
with increased risk of NTM pulmonary
disease11. Quite alarming is the fact that
pulmonary NTM disease has increased
dramatically amongst elderly slender men
and women, who lack the classic risk factors for mycobacterial disease, yet appear
to be substantially more susceptible to
NTM disease12. As the human population
ages, it would be expected that the number
of elderly with NTM pulmonary disease
will increase, placing further demands on
healthcare providers. Recommended therapy for NTM infections include multiple
antibiotics (e.g., clarithromycin, ethambutol, and rifampin) for periods as long as 24
months13.
NTM survive, grow, and persist in a number of habitats that are shared with humans
and animals. NTM are oligotrophs; able to
grow in water containing greater than 50
μg assimilable organic carbon (AOC/)L14.
The presence of high numbers of NTM in
coastal acidic, brown water swamps15 and
pine forest soils16 is due, in part, to the
growth stimulation by soil organic matter
material rarely metabolized by other microorganisms; namely humic and fulvic
acids17.
Mycobacteria have a true outer membrane18, whose long chain mycolic acids
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cadeia longa contribuem para a hidrofobicidade, impermeabilidade e crescimento lento
das células de MNT19. A hidrofobicidade da
superfície das células de MNT é a mais elevada de todas as bactérias e é uma importante
determinante da fagocitose dos macrófagos20.
Embora a parede hidrofóbica reduza o índice
de transferência de nutrientes hidrofílicos,
contribui para a resistência a desinfetantes
(e.g., cloro e biócidos) e a antibióticos21,22.
As MNT penetram nos sistemas de tratamento de águas aderentes a partículas, sobrevivem à desinfeção e crescem em biofilmes na
ausência de competidores mortos pela disinfeção8. A formação de biofilme também aumenta a resistência das MNT a desinfetantes23 e a antibióticos24. A hidrofobicidade
também promove a aerossolização de micobactérias da água para o ar em ambientes
como chuveiros e banheiras domésticos e nas
profissões onde se manuseiam aerossóis25.
contribute to the hydrophobicity, impermeability, and slow growth of NTM cells19.
The surface hydrophobicity of NTM cells
is the highest amongst bacteria and is a
major determinant of phagocytosis by
macrophages20. Although the hydrophobic
wall reduces the rate of transfer of hydrophilic nutrients, it contributes to disinfectant- (e.g., chlorine and biocides) and antibiotic-resistance21,22.
NTM enter a water treatment system on
particulates, survive disinfection, and
grow in biofilms in the absence of competitors killed by disinfection8. Biofilm
formation also increases NTM resistance
to disinfectants23 and antibiotics24. Hydrophobicity also promotes the aerosolization
of mycobacteria from water to air in environments such as showers and hot tubs in
the home and occupations where aerosols
are generated25.
Bibliografia/Bibliography
1. Wallace RJ, Brown BA, Griffith DE. Nosocomial
outbreaks pseudo-outbreaks caused by nontuberculous
mycobacteria. Annu Rev Microbiol 1998; 52;453-490.
2. Falkinham JO III. Surrounded by mycobacteria: nontuberculous mycobacteria in the human environment. J
Appl Microbiol 2009; 107;356-367.
3. Iseman MD, Marras TK. The importance of nontuberculous mycobacterial lung disease. Am J Respir Crit
Care Med 2008; 178:999-1000.
4. Marras TK, et al. Isolation prevalence of pulmonary
non-tuberculous mycobacteria in Ontario, 1997-2003.
Thorax 2007; 62;661-666.
5. von Reyn CF, et al. Persistent colonisation of potable
water as a source of Mycobacterium avium infection in
AIDS. Lancet 1994;343;1137-1141.
6. De Groote MA, et al. Relationships between Mycobacterium isolates from patients with pulmonary mycobacterial infection and potting soils. 2006; Appl Environ Microbiol 2006;72;7602-7606.
R e v i s t a
7. Falkinham JO III, et al. Mycobacterium avium in a
shower linked to pulmonary disease. J Water Health
2008; 6;209-213.
8. Falkinham JO III, Norton CD, LeChevallier MW.
Factors influencing numbers of Mycobacterium avium,
Mycobacterium intracellulare, and other Mycobacteria
in drinking water distribution systems. Appl Environ
Microbiol 2001; 67;1225-1231.
9. Feazel LM, et al. Opportunistic pathogens enriched
in showerhead biofilms. Proc Natl Acad Sci USA
2009;106;16393-16399.
10. Marras TK, Daley CL. Epidemiology of human pulmonary infection with nontuberculous mycobacteria.
Clin Chest Med 2002; 23;553-567.
11. Kim JS, et al. Nontuberculous mycobacterial infection: CT scan findings, genotype, and treatment responsiveness. Chest 2005; 128;3863-3869.
12. Kennedy TP, Weber DJ. Nontuberculous mycobacteria – an underappreciated cause of geriatric lung di-
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Joseph O Falkinham III
sease. Am Rev Respir Crit Care Med 1994;149;1654-1658.
13. Griffith DE, et al. An official ATS/IDSA statement:
Diagnosis, treatment, and prevention of nontuberculous mycobacterial diseases. Am J Respir Crit Care Med
2007; 175;367-416.
14. Norton CD, LeChevallier MW, Falkinham JO III.
Survival of Mycobacterium avium in a model distribution system. Water Res 2005; 38;1457-1466.
15. Kirschner RA Jr, Parker BC, Falkinham JO III. Epidemiology of infection by nontuberculous mycobacteria.
Mycobacterium avium, Mycobacterium intracellulare, and
Mycobacterium scrofulaceum in acid, brown-water swamps
of the southeastern United States and their association
with environmental variables. Am Rev Respir Dis 1992;
145;271-275.
16. Iivanainen EK, et al. Mycobacteria in boreal coniferous
forest soils. FEMS Microbiol Ecol 1997;23;325-332.
17. Kirschner RA, Parker BC, Falkinham JO III. Humic and fulvic acids stimulate the growth of Mycobacterium avium. FEMS Microbiol Ecol 1995; 30;327-332.
18. Hoffmann C, et al. Disclosure of the mycobacterial
outer membrane: Cryo-electron tomography and vitreous sections reveal the lipid bilayer structure. Proc Natl
Acad Sci USA 2008; 105;3963-3967.
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19. Brennan PJ, Nikaido H. The envelope of mycobacteria. Annu Rev Biochem 1995; 64;29-63.
20. van Oss CJ, Gillman CF, Neumann AW. Phagocytic
engulfment and cell adhesiveness. 1975; Marcel Dekker,
New York.
21. Rastogi N, et al. Multiple drug resistance in Mycobacterium avium – Is the wall architecture responsible
for the exclusion of antimicrobial agents. Antimicrob
Agents Chemother 1981; 20;666-677.
22. Taylor R, et al. Chlorine, chloramine, chlorine dioxide, and ozone susceptibility of Mycobacterium avium.
Appl Environ Microbiol 2000; 66;1702-1705.
23. Steed KA Falkinham JO III. Effect of growth in biofilms on chlorine susceptibility of Mycobacterium avium
and Mycobacterium intracellulare. Appl Environ Microbiol 2006; 72;4007-4011.
24. Falkinham, JO III. Growth in catheter biofilms and
antibiotic resistance of Mycobacterium avium. J Med
Microbiol 2007; 56;250-254.
25. Parker BC, George KL, Falkinham JO III. Epidemiology of infection by nontuberculous mycobacteria. IV.
Preferential aerosolization of Mycobacterium intracellulare from natural waters. Am Rev Respir Dis 1984;
123;652-656.
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O uso da análise de libertação de gama interferão como
auxiliar no controlo da tuberculose
Jean-Pierre Zellweger1
The use of interferon gamma release assays as an aid in
the control of tuberculosis
As análises de libertação de gama interferão
(IGRA) são testes in vitro que detetam a
presença de infeção por tuberculose latente
(ITBL) em pessoas assintomáticas que podem ter sido infetadas por Mycobacterium
tuberculosis num passado recente ou remoto
e que podem beneficiar dum tratamento
preventivo para diminuir o risco de reativação posterior da tuberculose1, 2.
A investigação dos contactos é a busca de casos
secundários de tuberculose entre os contactos
dum caso primário, e a busca de contactos com
uma infeção latente de tuberculose que possam
beneficiar dum tratamento preventivo de
modo a evitar a futura progressão para a doença ativa. Esta é a segunda prioridade no controlo da tuberculose, depois da deteção e tratamento dos casos ativos. Se feito de modo
sistemático, e em países com entidades bem
organizadas no controlo da TB, esta atividade
pode ser eficaz em termos de custo.
A deteção de casos secundários assenta essencialmente nos exames clínico, radiológico e
bacteriológico dos contactos com queixas. A
Interferon Gamma Release Assays (IGRAs)
are in vitro tests detecting the presence of
latent tuberculosis infection (LTBI) in asymptomatic persons who may have been
infected by Mycobacterium tuberculosis in a
recent or remote past and who may benefit
from a preventive treatment to decrease the
risk of later reactivation of tuberculosis1, 2.
Contact investigation is the search for secondary cases of tuberculosis among the
contacts of a primary (index) case and the
search for contacts with a latent tuberculosis
infection who may benefit from a preventive treatment in order to avoid the future
progression to active disease. This is the second priority for the control of tuberculosis,
after the detection and treatment of active
cases. If conducted in a systematic way and
in countries with a well-organized TB management team, this activity may be very
cost-effective.
The detection of secondary cases relies
mainly on the clinical, radiological and bacteriological examination of contacts with
1 Swiss Lung Association, Berne, Switzerland
e-mail: [email protected]
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Jean-Pierre Zellweger
deteção de contactos com infeção latente de
tuberculose, por definição assintomática,
apoia-se no uso de testes indiretos capazes de
detetar a sensibilização anterior de linfócitos
T por antigénios micobacterianos. O tradicional teste cutâneo de tuberculina (TCT),
com que se faz a maioria dos estudos epidemiológicos, enferma de graves deficiências,
entre as quais a baixa especificidade. As novas
análises de libertação de gama interferão,
com a sua alta especificidade, são muito mais
promissoras e permitem uma melhor seleção
dos contactos que podem beneficiar duma
terapêutica preventiva, evitando a prescrição
dum tratamento desnecessário para contactos não infetados que podem ter um TCT
falso positivo, devido, por exemplo, ao BCG,
ao efeito booster ou à sensibilização com micobactérias não tuberculosas3.
Basicamente, os testes IGRA assentam no
mesmo fenómeno imunológico dos testes
cutâneos de tuberculina, mas duma maneira
muito mais específica, porque os testes não
são influenciados por uma anterior vacinação com BGC ou por uma infeção com a
maioria das micobactérias não tuberculosas
presentes no ambiente. Assim, as indicações
e o uso de testes IGRA são fundamentalmente as mesmas dos testes cutâneos de tuberculina4:
1. Deteção de ITBL em pessoas em contacto com um caso primário de tuberculose;
2. Deteção de ITBL em pessoas com alto
risco de tuberculose, se infetadas (doentes imunossuprimidos, doentes a receber ou programados para receber terapia
imunossupressora, crianças);
3. Vigilância de trabalhadores dos serviços
de saúde expostos (uma vez que o teste
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complaints. The detection of contacts with
a latent tuberculosis infection, by definition
asymptomatic, relies on the use of indirect
tests able to detect the prior sensitization of
T lymphocytes by mycobacterial antigens.
The time-honoured tuberculin skin test
(TST), with which most of the epidemiological studies are performed, suffers from
severe deficiencies, among which a low
specificity. The new Interferon-Gamma Release Assays, with their high specificity, are
much more promizing and allow a better
targeting of the contacts who may benefit
from a preventive therapy, avoiding the
prescription of an unnecessary treatment to
uninfected contacts who may have a falsepositive TST, for instance due to BCG,
booster effect or sensitization with non-tuberculous mycobacteria3.
Basically, the IGRA tests rely on the same
immunological phenomenon as the tuberculin skin tests, but they do it in a much
more specific way, because the tests are not
influenced by a prior vaccination with BGC
or by an infection with most of the non-tuberculous mycobacteria present in the environment. Therefore, the indications and the
use of the IGRA tests are fundamentally the
same as for the tuberculin skin tests4:
1. Detection of LTBI in persons in contact
with an index case of tuberculosis;
2. Detection of LTBI in persons with a
high risk of tuberculosis, if infected
(immunosuppressed patients, patients
receiveing or due to receive immunosuppressive therapy, small children);
3. Surveillance of exposed health care
workers (as the test can be repeated
without risk of inducing a booster effect);
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Jean-Pierre Zellweger
pode ser repetido sem risco de indução
do efeito booster);
4. Auxiliar no diagnóstico de tuberculose
em casos em que o exame bacteriológico não é viável ou fiável (TB extrapulmonar grave, TB em crianças).
O nível de resposta ao IGRA é proporcional
à intensidade da exposição à tuberculose e
pode mudar sob tratamento preventivo ou
curativo, possivelmente indicando que o número de micobactérias vivas também diminuiu. Assim, é possível que a alteração no
nível de IGRA reflita o efeito do tratamento,
mas infelizmente, até agora, não foi possível
provar que a diminuição do nível esteja claramente relacionada com a erradicação de micobactérias e possa ser usada para documentar a cura5. Além disso, não está provado que
um teste positivo evidencie sempre a persistência de micobactérias vivas. É possível que
o teste detete apenas uma resposta imune a
um anterior contacto com micobactérias que,
entretanto, tenham desaparecido6
Os testes IGRA podem contribuir para o
diagnóstico de casos difíceis de TB. Na
tuberculose pulmonar com um esfregaço
negativo, e na tuberculose extrapulmonar
pode ser muito difícil obter evidência bacteriológica da presença de micobactérias.
A libertação de gama interferão de linfócitos isolados a partir de órgãos potencialmente envolvidos em casos suspeitos de
tuberculose (lavagem broncoalveolar na
tuberculose pulmonar com um esfregaço
negativo, líquido cerebrospinal na meningite tuberculosa, líquido peritoneal na tuberculose abdominal) pode ser medida e
está elevada em casos com um diagnóstico
final de tuberculose. Nesses casos, a determinação do nível de libertação de gama
R e v i s t a
4. Aid to the diagnosis of tuberculosis in
cases where a bacteriological examination is not feasible or not reliable (severe
extrapulmonary TB, TB in children).
The level of IGRA response is proportional
with the intensity of exposure to tuberculosis, and may change under preventive or curative treatment, possibly indicating that
the number of living mycobacteria has also
decreased. It is therefore possible that the
change in the level of IGRA reflects the effect of treatment, but there is unfortunately
up to now no solid proof that a decrease in
the level is clearly correlated with the eradication of mycobacteria and could be used as
a documentation of cure5. Furthermore,
there is no proof that a positive test result
always documents the persistence of living
mycobacteria. It may be that the test only
detects a lasting immune response to a prior
contact with mycobacteria that have disappeared in between 6
IGRAs may contribute to the diagnosis
of difficult TB cases. In smear-negative
pulmonary tuberculosis and in extrapulmonary tuberculosis, it may be very difficult to obtain the bacteriological documentation of the presence of mycobacteria.
The release of interferon-gamma from
lymphocytes isolated from the organs potentially involved in cases of suspect tuberculosis (BAL in smear-negative pulmonary tuberculosis, CSF in tuberculous
meningitis, peritoneal fluid in abdominal
tuberculosis) can be measured and is elevated in cases with a final diagnosis of
tuberculosis. In such cases, the determination of the level of Interferon-Gamma
release can be used as an aid to the diagnosis of tuberculosis7.
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Jean-Pierre Zellweger
interferão pode ajudar no diagnóstico da
tuberculose7.
Os testes IGRA podem indicar o futuro desenvolvimento da doença. Nem todas as pessoas em contacto com um caso de tuberculose são infetadas e nem todos os contactos
infetados virão a desenvolver tuberculose.
Estima-se o risco de reativação da tuberculose como sendo de 10% para contactos com
um TCT positivo. Estudos recentes demonstraram que o risco de futura reativação é
maior em contactos com um IGRA positivo
do que em contactos negativos, independentemente do resultado do TCT8. Por isso, um
IGRA positivo pode ter maior valor preditivo
para risco de reativação futura do que o TCT.
Como a proporção de contactos com um
IGRA positivo é menor do que a dos contactos com um teste cutâneo de tuberculina positivo, o número de contactos considerados
infetados e que podem beneficiar de um tratamento preventivo é menor se os testes
IGRA forem usados como definição. A utilização de testes IGRA na deteção de contactos
infetados e na seleção de contactos que necessitam de tratamento preventivo tem, assim,
um efeito moderador9.
Apesar da sua superioridade, os testes IGRA
não são totalmente isentos de problemas na
prática e a sua melhor utilização ainda vem sendo debatida. A variabilidade inter e intraobservador é baixa, mas o nível de resposta pode
variar se o teste for repetido, verificando-se conversões e reversões espontâneas na ausência de
exposição ou de tratamento10-12. Observam-se
ainda mais alterações nos casos com respostas
borderline. Assim, têm sido propostos aumentos
no cut-off da positividade e na definição da conversão na repetição dos testes13.
Algumas linhas de orientação recomendam
o seu uso apenas para confirmação dum
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IGRAs may predict the future development
of TB disease. Not all cases in contact with
a case of tuberculosis will be infected and
not all infected contacts will develop tuberculosis. The risk of reactivation of tuberculosis is estimated to be 10% for contacts
with a positive tuberculin skin test. Recent
studies have demonstrated that the risk of
future reactivation is higher in contacts
with a positive IGRA test than in negative
contacts, independently from the result of
the tuberculin skin test8. Therefore, a positive IGRA test result may have a higher
predictive value for the risk of future reactivation than the tuberculin skin test. As the
proportion of contacts with a positive
IGRA test is lower than the proportion of
contacts with a positive tuberculin skin test,
the number of contacts considered as infected and who may benefit from a preventive treatment is lower if IGRA are used as
a definition. Using IGRA for the detection
of infected contacts and selection of contacts in need of a preventive treatment has
therefore a sparing effect9.
In spite of their superiority, the IGRAs are
not totally devoid of problems in practice
and the best use of them is still a matter of
debate. The intra-observer and inter-observer variability are low, but the level of response may vary if the test is repeated and
there are spontaneous conversions and reversions in the absence of exposure or treatment10-12. More of the changes are observed
for cases with borderline responses. Therefore, proposals have been made for an increase in the cut-off for positivity and for
the definition of conversion in repeated
testing13.
Some Guidelines recommend their use only
for the confirmation of positive TST among
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TCT positivo entre contactos (o chamado
teste de dois passos), enquanto outras recomendam a substituição do TCT de rotina
por IGRA. Fazer um só teste é mais fácil e
evita a possível influência de um TCT anterior na resposta do IGRA.
contacts (the so-called two-step testing procedure) whereas others recommend the routine replacement of the TST by IGRAs. Performing only one test is easier, and avoids a
possible influence of a prior TST on the
IGRA response.
Bibliografia/Bibliography
1. Menzies D, Pai M, Comstock G. Meta-analysis: new
tests for the diagnosis of latent tuberculosis infection:
areas of uncertainty and recommendations for research.
Ann Intern Med 2007; 146(5):340-354.
2. Pai M, Zwerling A, Menzies D. Systematic review:
T-cell-based assays for the diagnosis of latent tuberculosis infection: an update. Ann Intern Med 2008;
149(3):177-184.
3. Diel R, Loddenkemper R, Meywald-Walter K,
Gottschalk R, Nienhaus A. Comparative performance
of tuberculin skin test, QuantiFERON-TB-Gold in
tube assay, and T-Spot.TB test in contact investigations
for tuberculosis. Chest 2009; 135(4):1010-1018.
4. Zellweger JP. Latent tuberculosis: which test in which
situation? Swiss Med Wkly 2008; 138(3-4):31-37.
5. Pai M, Menzies D. Interferon-gamma release assays:
what is their role in the diagnosis of active tuberculosis?
Clin Infect Dis 2007; 44(1):74-77.
6. Mack U, Migliori GB, Sester M, et al. LTBI: latent
tuberculosis infection or lasting immune responses to
M. tuberculosis? A TBNET consensus statement. Eur
Respir J 2009; 33(5):956-973.
7. Jafari C, Ernst M, Strassburg A, et al. Local immunodiagnosis of pulmonary tuberculosis by enzyme-linked
immunospot. Eur Respir J 2008; 31(2):261-265.
R e v i s t a
8. Diel R, Loddenkemper R, Meywald-Walter K, Niemann S, Nienhaus A. Predictive value of a whole-blood
IFN-{gamma} assay for the development of active TB
disease. Am J Respir Crit Care Med 2008; 177:1164-1170.
9. Diel R, Nienhaus A, Lange C, Schaberg T. Cost optimization of screening for latent tuberculosis in close
contacts. Eur Respir J 2006; 28:35-44.
10. Pai M, Joshi R, Dogra S, et al. T-cell assay conversions and reversions among household contacts of tuberculosis patients in rural India. Int J Tuberc Lung Dis
2009; 13(1):84-92.
11. van Zyl-Smit RN, Pai M, Peprah K, et al. Within-subject variability and boosting of T-cell interferon-gamma responses after tuberculin skin testing. Am J
Respir Crit Care Med 2009; 180(1):49-58.
12. Detjen AK, Loebenberg L, Grewal HM et al. Short-term reproducibility of a commercial interferon-gamma
release assay. Clin Vaccine Immunol 2009; 16:1170-1175
13. Pai M, Dendukuri N, Wang L, Joshi R, Kalantri S,
Rieder HL. Improving the estimation of tuberculosis
infection prevalence using T-cell-based assay and mixture models. Int J Tuberc Lung Dis 2008;12(8):895-902.
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Regulação da composição lipídica da parede celular do
Mycobacterium tuberculosis e o seu efeito na persistência
bacteriana in vitro
Lee W Riley1
Regulation of Mycobacterium tuberculosis cell wall lipid
composition and its effects on in vitro bacterial
persistence
Introdução
A marca da infeção humana por Mycobacterium tuberculosis é a sua capacidade de permanecer latente durante vários anos, com
posterior reativação para doença ativa (tuberculose). Pouco se sabe sobre como este
organismo permanece em latência e o que
desencadeia a sua reativação. Frequentemente atribui-se aos lípidos do M. tuberculosis um papel na patogénese, porque grande
parte do genoma do M. tuberculosis é dedicado à biossíntese e à degradação dos lípidos1. Só no metabolismo dos ácidos gordos
quase 250 genes estão envolvidos, comparativamente com apenas cerca de 50 em organismos como a Escherichia coli. Mais de
50% do peso seco da parede celular do M.
tuberculosis é composto por lípidos1-3 e, além
disso, o organismo utiliza os lípidos como
fonte de energia durante a sua persistência
num mamífero hospedeiro4. Esta desproporcionada atenção dispensada ao metabolismo lipídico é muito provavelmente uma
estratégia adotada por este organismo como
Introduction
The hallmark of human infection by Mycobacterium tuberculosis is its ability to remain latent for many years, only to reactivate to cause active disease (tuberculosis).
Very little is known about how this organism remains latent and what triggers it to
reactivate. Much attention is often paid to
the lipids of M. tuberculosis as playing a
role in pathogenesis. This is because a large
proportionof the genome of M. tuberculosis is dedicated to the biosynthesis and
degradation of lipids1. Nearly 250 genes
are involved in metabolism of fatty acids
alone, compared to only about 50 in organisms such as Escherichia coli. More than
50% of the dry weight of the cell wall of
M. tuberculosis is composed of lipids1-3,
and furthermore, the organism utilizes
lipids as energy source during its persistence in a mammalian host4. This disproportionate attention paid to lipid metabolism is very likely a major strategy this
organism has adopted to defend against
1 MD, School of Public Health, University of California, Berkeley
e-mail: [email protected]
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defesa contra o ambiente hostil do seu nicho natural – os granulomas humanos.
Os ácidos micólicos são componentes lipídicos importantes e os mais abundantes ácidos
gordos na parede celular do M. tuberculosis5,6.
O envelope celular do M. tuberculosis contém
três classes de micolatos – ácidos alfa, ceto e
metoximicólicos6. Os micolatos estão normalmente ligados à camada arabinogalactana
e ao carboidrato de trealose6,7. As alterações
estruturais dos ácidos micólicos afetam a virulência do M. tuberculosis. A ausência de ácido
micólico oxigenado está associada à alteração
do M. tuberculosis num modelo de rato8. A
cisciclopropanação de ácidos micólicos mediados por ciclopropano sintetase é necessária
à formação do fator corda e à virulência em
ratos9,10. Por outro lado, a transciclopropanação de ácidos micólicos oxigenados é importante para a virulência na direção oposta. Um
mutante do M. tuberculosis sem anéis transciclopropanos nestes ácidos micólicos é hipervirulenta no modelo de rato11.
O conjunto destas observações sugere um
papel importante dos lípidos e, em particular, dos ácidos micólicos, na modificação da
resposta imune do hospedeiro e no posterior resultado clínico. Os autores vêm estudando a regulação dos lípidos do M. tuberculosis, com particular atenção a um
conjunto de operões que parecem servir
como sistemas transportadores de lípidos na
parede celular – mce operões.
the hostile environment of its natural
niche – the human granulomas.
Mycolic acids are the major lipid constitutent and the most abundant fatty acid
found in the cell wall of M. tuberculosis5,6.
The M. tuberculosis cell envelope contains 3
classes of mycolates-alpha, keto- and methoxy-mycolic acid6. Mycolates are normally
attached to the arabinogalactan layer and
carbohydrate trehalose6,7. Structural alterations of mycolic acids affect the virulence
property of M. tuberculosis. Absence of oxygenated mycolic acid is associated with attenuation of M. tuberculosis in a mouse
model8. Cis-cyclopropanation of mycolic
acids mediated by cyclopropane synthase is
needed for cord formation as well as for
virulence in mice9,10. On the other hand,
transcyclopropanation of oxygenated mycolic acids is important for virulence in the
opposite direction. An M. tuberculosis mutant lacking transcyclopropane rings in
these mycolic acids is hypervirulent in the
mouse model11.
Taken together, these observations suggest
an important role for lipids, and in particular, mycolic acids, in modifying host immune response and the subsequent clinical
outcome. We have been studying the regulation of M. tuberculosis lipids focusing on a
set of operons that appear to serve as lipid
transporter systems in the cell wall – mce
operons.
Papel dos mce operões na
modificação da estrutura da
parede celular
O Mycobacterium tuberculosis contém quatro cópias homólogas de um operão designado mce1-41. Já demonstrámos que o M.
Role of the mce operons in cell
wall structural modification
M. tuberculosis contains 4 homologous copies of an operon designated mce1-41. We
showed that M. tuberculosis disrupted in the
mce1 operon is hypervirulent in mice12. The
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tuberculosis danificado no operão mce1 é hipervirulento em ratos12. O mutante não
provoca uma forte resposta imune do tipo
Th1 e causa migração aberrante de células
pró-inflamatórias, resultando em granulomas mal organizados nos pulmões dos ratos12. A falta de resposta imune do tipo Th1
adequada e de formação granulomatosa organizada impede o controlo da proliferação
bacteriana no rato, o que leva à morte prematura neste animal. Esta observação foi
obtida em ratos BALB/c e C57BL/6 imunocompetentes, exibindo ambos resposta
diferencial tipo Th1 à infeção por M. tuberculosis do tipo selvagem12,13.
O mce1 operão é regulado negativamente
no meio intracelular por mce1R, localizado
imediatamente acima14. O mce1R pertence à
subfamília FadR de reguladores transcricionais GntR1,14. Um mutante do gene mce1R
também é hipervirulento em ratos, mas pela
razão oposta, pois causa uma resposta imunopatológica acelerada, com rápida progressão para a morte do animal após maciça formação granulomatosa nos pulmões15. A
morte do animal deve-se, não à proliferação
bacteriana, mas à resposta hiperpró-inflamatória. Descobrimos, assim, que a
ausência de expressão do operão mce1 (como
com o operão mce1 mutante) está associada
aos granulomas mal organizados e migração
aberrante de células pró-inflamatórias, enquanto a sua expressão constitutiva (como
com o mutante mce1R) causa maciça formação granulomatosa nos pulmões dos ratos.
Ambos os efeitos resultam em consequências clínicas adversas nestes animais, pelo
que achámos que o mce1 operão serve para
regular homeostaticamente a resposta imune do hospedeiro para manter as estruturas
granulomatosas que permitem, não só a so-
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mutant cannot elicit a strong Th1-type immune response and causes aberrant migration of pro-inflammatory cells, resulting in
poorly organized granulomas in the mouse
lungs12. The lack of adequate Th-1 type response and organized granuloma formation
precludes control of bacterial proliferation
in mice, which leads to early death in this
animal. This observation was made in immunocompetent BALB/c as well as in
C57BL/6 mice, both of which exhibit differential Th1 type response to wild type M.
tuberculosis infection12,13.
The mce1 operon is negatively regulated
intracellularly by mce1R, located immediately upstream14. Mce1R belongs to the
FadR subfamily of GntR transcriptional
regulators1,14. A mutant disrupted in mce1R
gene is also hypervirulent in mice but for
an opposite reason. It causes accelerated
immunopathologic response with rapid
progression to death of the animal following massive granuloma formation in their
lungs15. The animal dies, not from bacterial proliferation but from the hyper-proinflammatory response. Therefore, we discovered that the absence of the mce1 operon
expression (as with the mce1 operon mutant) is associated with poorly organized
granulomas and aberrant pro-inflammatory cell migration, while its constitutive expression (as with the mce1R mutant) causes
massive granuloma formation in mouse
lungs. Both of these outcomes result in adverse clinical outcomes in mice. We therefore reasoned that the mce1 operon serves
to homeostatically regulate the host immune response to maintain granuloma
structures that allow not only the host to
survive but also for M. tuberculosis to remain persistent.
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brevivência do hospedeiro, mas também a
persistência do M. tuberculosis.
A análise filogenómica dos operões mce do M.
tuberculosis mostrou que operões relacionados
se encontram entre a maioria dos membros de
Actinomycetales e que estes codificam uma família de transportadores ABC de captação de
lípidos16. Um dos produtos do operão mce1 é
o FadD5, semelhante na sequência (43%) em
E. coli da acil-CoA sintetase FadD17. Além
disso, a proteína Mce1R do M. tuberculosis é
33% idêntica à proteína repressora transcricional FadR encontrada em E. coli, que se liga
à acyl CoA-gorda e induz a expressão dos genes envolvidos na degradação e transporte dos
ácidos gordos18.
O M. tuberculosis danificado em fadD5 é atenuado em ratos e tem crescimento reduzido
in vitro em meio mínimo, com ácido micólico como única fonte de carbono19. Assim,
FadD5 pode servir para reciclar ácidos micólicos que possam ser libertados na morte do
M. tuberculosis durante o desenvolvimento
natural da infeção. Este mecanismo de reciclagem pode contribuir para a sobrevivência
a longo prazo da população viva de M. tuberculosis num ambiente granulomatoso. Deste
modo, o operão mce1 pode incluir um sistema de importação de ácidos micólicos.
Curiosamente, outro grupo propôs que o
operão mce4 pode ser um sistema de importação de colesterol20. Atualmente, a função
dos operões mce2 e mce3 como transportadores de lípidos ainda não está estabelecida.
No entanto, estas observações sugerem que,
durante diferentes estádios da infeção, o M.
tuberculosis utiliza uma variedade de fontes de
carbono para a sua sobrevivência a longo prazo, utilizando vários sistemas distintos de assimilação de lípidos. Estas fontes de carbono
podem tornar-se diferencialmente disponíveis
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A phylogenomic analysis of the M. tuberculosis mce operons showed that related operons are found among most members of
Actinomycetales and that they encode a family of ABC lipid uptake transporters16. One
of the gene products of the mce1 operon is
FadD5, which is similar in sequence (43%)
to the E. coli fatty-acyl-CoA synthetase
FadD17. In addition, the M. tuberculosis Mce1R protein is 33% identical to the FadR
transcriptional repressor protein found in E.
coli, which binds fatty-acyl CoA and induces the expression of genes involved in fatty
acid degradation and transport18.
M. tuberculosis disrupted in fadD5 is attenuated in mice and diminished in growth in
vitro in minimal medium supplied with mycolic acid as the sole carbon source19. Thus,
FadD5 may serve to recycle mycolic acids
that may be released from dying M. tuberculosis during a natural course of infection.
Such a recycling mechanism may contribute
to the long-term survival of live population
of M. tuberculosis in a granuloma environment. Thus, the mce1 operon may comprise
a mycolic acid import system.
Interestingly, another group proposed that
the mce4 operon may serve as a cholesterol
import system20. At this time, the function
of mce2 and mce3 operons as lipid transporters is not established.
Nevertheless, these observations suggest
that during different stages of infection,
M. tuberculosis utilizes a variety of carbon
sources for its long-term survival using
several distinct lipid assimilation systems.
These carbon sources may become differentially available to M. tuberculosis during the turnover of granuloma cells.
Some of the lipids may be released from
these cells turning over, while others may
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ao M. tuberculosis durante o turnover das células granulomatosas, quando alguns dos lípidos
podem ser libertados por estas células, enquanto outros podem advir de um subgrupo de
células de M. tuberculosis mortas pelas moléculas efetoras produzidas por células que constituem os granulomas. Em todas estas situações,
o M. tuberculosis parece ter o fornecimento
contínuo de energia assegurado por um longo
período de tempo. Assim, qualquer interrupção deste equilíbrio entre a população bacteriana e o turnover das células granulomatosas
poderia levar à completa eliminação das bactérias (e.g., por tratamento com fármacos antituberculose) ou à produção da doença ativa
(e.g., por imunossupressão, idade avançada,
inóculo infeccioso inicial elevado e certos fatores relacionados com a estirpe).
Conclusões
Os transportadores ABC semelhantes ao operão
mce conservados nos membros dos Actinomycetales, a maioria dos quais saprófitas de solo,
apoiam a ideia de que o próprio M. tuberculosis
descende de alguma micobactéria de solo. No
reservatório do solo, as saprófitas obtêm os seus
nutrientes e carbono como fonte de energia a
partir de matéria orgânica morta. O M. tuberculosis tem como seu reservatório natural o hospedeiro humano, particularmente a estrutura granulomatosa dos órgãos humanos. Parece que o
M. tuberculosis simplesmente readaptou a função ancestral de sequestração de carbono usada
pelos seus antepassados ao retirar o carbono libertado pelas células granulomatosas mortas e
bactérias no reservatório humano. Infelizmente,
para os humanos, esta readaptação metabólica
levou a que o M. tuberculosis se tornasse uma das
mais frequentes causas de morte por infeção no
adulto em todo o mundo.
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come from a subset of M. tuberculosis cells
that are killed by effector molecules produced by cells that comprise the granulomas. In all of these situations, M. tuberculosis appears to be assured of continued
supply of energy for a long period of time.
Thus, any disruption of this balance between bacterial population and granuloma cell turnover could lead to either complete elimination of the bacteria (e.g., by
treatment with anti-tuberculosis drugs)
or active disease production (e.g., from
immunosuppression, old age, high initial
infectious inoculum, and certain strainrelated factors).
Conclusions
The highly conserved mce-operon-like
ABC transporters across members of the
Actinomycetales, most of which are soil saprophytes, support the idea that M. tuberculosis itself descended from some soil mycobacteria. In the soil reservoir, saprophytes
derive their carbon nutrients as energy
source from dead organic matter. M. tuberculosis has as its natural reservoir the human
host, in particular, the granuloma structure
in human organs. It appears that M. tuberculosis has simply re-adapted its ancestral
carbon-sequestration function its ancestors
used in soil to derive carbon released from
dead granuloma cells and bacteria in the
human reservoir. Unfortunately, for humans, this metabolic readaptation has led
M. tuberculosis to become one of the most
common infectious causes of death in
adults worldwide.
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Bibliografia/Bibliography
1. Cole ST BR, Parkhill J, Garnier T, Churcher C, Harris D, Gordon SV, Eiglmeier KGS, Barry CE 3rd, Tekaia
F, Badcock K, Basham D, Brown D, Chillingworth T,
CR, Davies R, Devlin K, Feltwell T, Gentles S, Hamlin
N, Holroyd SHT, Jagels K, Krogh A, McLean J, Moule
S, Murphy L, Oliver K,, Osborne JQM, Rajandream
MA, Rogers J, Rutter S, Seeger K, Skelton J, Squares
RSS, Sulston JE, Taylor K, Whitehead S, Barrell BG.
Deciphering the biology of Mycobacterium tuberculosis
from the complete genome sequence. Nature 1998;
393:537-544.
2. Goren, M. Biosynthesis and stucture of phospholipids and sulfatides. In The Mycobacteria, part A. L.W. P
Kubica, editor. New York: Marcel Dekker 1984; 379-415.
3. Minnikin, DE. Chemical principles in the organization of lipid components in the mycobacterial cell envelope. Res Microbiol 1991; 142:423-427.
4. McKinney JDBK, Munoz-Elias EJ, Miczak A,
Chen B, Chan WT, Swenson D, Sacchettini JC,
Jacobs WR, Russell DG. Persistence of Mycobacterium tuberculosis in macrophages and mice requires
the glyoxylate shunt enzyme isocitrate lyase. Nature
2000; 406:735-738.
5. Brennan PJ, Nikaido H. The envelope of mycobacteria. Annu Rev Biochem 1995; 64:29-63.
6. Brennan PJ, Nikaido H. Structure of mycobacteria:
recent developments in defining cell wall carbohydrates
and proteins. Rev Infect Dis 1989; 11:S420-430.
7. Takayama KWC, Besra GS. Pathway to synthesis and
processing of mycolic acids in Mycobacterium tuberculosis. Clin Microbiol 2005; 18:81-101.
8. Dubnau E, Chan J, Raynaud C, Mohan VP, Laneelle
MA, Yu K, Quemard A, Smith I, Daffe M. Oxygenated
mycolic acids are necessary for virulence of Mycobacterium
tuberculosis in mice. Mol Microbiol 2000; 36:630-637.
9. Glickman MS, Cox JS, Jacobs WR. A novel mycolic
acid cyclopropane synthase is required for cording, persistence, and virulence of Mycobacterium tuberculosis.
Molec Cell 2000; 5:717-727.
S 42
R e v i s t a
P o r t u g u e s a
10. Glickman MS, Jacobs WR. Microbial pathogenesis
of Mycobacterium tuberculosis: Dawn of a discipline. Cell
2001; 104:477-485.
11. Rao V, Gao F, Chen B, Jacobs WR Jr, Glickman,
MS. Trans-cyclopropanation of mycolic acids on trehalose dimycolate suppresses Mycobacterium tuberculosis
-induced inflammation and virulence. J Clin Invest
2006; 116:1660-1667.
12. Shimono N, Morici L, Casali N, Cantrell S, Sidders B,
Ehrt SLWR. Hypervirulent mutant of Mycobacterium tuberculosis resulting from the disruption of the mce1 operon.
PNAS 2003; 100:15918-15923.
13. Lima P, Sidders B, Morici L, Reader R, Senaratne R,
Casali N, Riley LW. Enhanced mortality despite control
of lung infection in mice aerogenically infected with a
Mycobacterium tuberculosis mce1 operon mutant. Microbes Infect 2007; 9:1285-1290.
14. Casali N, White AM, Riley LW. Regulation of the
Mycobacterium tuberculosis mce1 operon. J Bacteriol
2006; 188:441-449.
15. Uchida Y, Casali N, White A, Morici L, Kendall LV,
Riley LW. Accelerated immunopathological response of
mice infected with Mycobacterium tuberculosis disrupted
in the mce1 operon negative transcriptional regulator.
Cell Microbiol 2007; 9:1275-1283.
16. Casali N, Riley LW. A phylogenomic analysis of the Actinomycetales mce operons. BMC Genomics 2007; 8:60.
17. Trivedi OA, Arora P, Sridharan V, Tickoo R, Mohanty D, Gokhale RS. Enzymic activation and transfer
of fatty acids as acyl-adenylates in mycobacteria. Nature
2004; 428:441-445.
18. DiRusso CC, Black PN. Bacterial long chain fatty
acid transport: gateway to a fatty acid-responsive signaling system. J Biol Chem 2004; 279:49563-49566.
19. Dunphy KYSR, Masuzawa M, Kendall LV, Riley
LW. Journal of Infectious Disease (in press).
20. Pandey AK, Sassetti CM. Mycobacterial persistence
requires the utilization of host cholesterol. Proc Natl
Acad Sci USA 2008; 105:4376-4380.
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Uma nova vacina viva contra a tuberculose com base na
inativação do phoP
Jesus Gonzalo Asensio1
Ainhoa Arbues1
Dessi Marinova1
Carlos Martín1
A new live tuberculosis vaccine based on phoP inactivation
A BCG viva atenuada é a atual vacina contra a tuberculose (TB). Embora venha sendo usada há mais de 80 anos, esta vacina
não é eficaz na proteção contra a TB pulmonar no adulto, sendo necessárias novas vacinas e novas estratégias de vacinação que ofereçam melhor proteção contra esta doença
do que a vacina BCG existente. As vacinas
vivas atenuadas estão entre as mais eficazes
contra doenças infecciosas humanas devido
à resposta imune alargada e de longa duração por elas induzida.
Na última década verificou-se um ressurgimento global de tuberculose, e as estratégias
pioneiras para o desenvolvimento de vacinas mais eficazes levaram à descoberta de
subunidades de vacinas que provaram não
oferecer melhor proteção do que a BCG em
vários modelos animais, mas melhoravam a
eficácia da BCG quando usadas numa estratégia de vacinação prime-boost. Formas diferentes de BCG prime e boost com estratégias
de subunidades estão a ser utilizadas em ensaios clínicos, com o objetivo de melhorar a
1
The attenuated live BCG is the current vaccine against tuberculosis (TB). It has been
used for more than 80 years but is ineffective at providing protection against adult
pulmonary TB. New tuberculosis vaccine
candidates and TB vaccination strategies
conferring better protection against pulmonary tuberculosis than the current vaccine
BCG are needed. Live attenuated vaccines
are among the most effective vaccines
against human infectious disease due to the
broad and long-lived immune response they
induce.
In the recent decade, a global pipeline of
novel TB candidates has emerged. Pioneering strategies for the development of more
effective vaccines today have lead to the discovery of subunit vaccines, which have
proved ineffective at providing better protection than BCG in various animal mo
dels, but could improve the efficacy of BCG
used in a prime-boost strategy. Different
heterologous prime BCG and boost with
subunit strategies are in clinical trials with
Department of Microbiology, University of Zaragoza. Spain
http://genmico.unizar.es
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Uma nova vacina viva contra a tuberculose com base na inativação do phoP
Jesus Gonzalo Asensio, Ainhoa Arbues, Dessi Marinova, Carlos Martín
eficácia da BCG. Mais recentemente,
iniciaram-se ensaios clínicos com vacinas
BCG recombinantes, com o intuito de encontrar candidatas para serem usadas como
vacinas prime preventivas. Outra estratégia
inovadora, as vacinas vivas baseadas no Mycobacterium tuberculosis racionalmente atenuado, são candidatas promissoras (algumas
delas no fim da investigação pré-clínica),
um novo instrumento substituto da BCG,
com melhor eficácia protetora contra as formas pulmonares de TB.
Com base na observação de que a codificação pelo gene phoP do fator de transcrição
PhoP é essencial para a virulência do M. tuberculosis (Perez et al., 2001), atenuámos racionalmente o bacilo da TB por inativação
do phoP. O mutante demonstrou forte atenuação em modelos celular e animal e resultou mais atenuado do que a BCG em ratinhos SCID imunocomprometidos (Martin
et al., 2006). A vacina candidata protegeu
porquinhos-da-índia e primatas não humanos contra a infeção tuberculosa (Martin et
al., 2006, Verreck et al., 2009).
Estudos moleculares recentes (Gonzalo Asensio et al., 2008) demonstraram que o fator de
transcrição phoP controla a expressão de aproximadamente 80 genes (muitos dos quais implicados na virulência do M. tuberculosis),
responsável por cerca de 2% da open reading
frame (ORF) no genoma do M. tuberculosis.
Com base nestes estudos, pode pensar-se que
o fenótipo atenuado e a imunidade protetora
conferida pelo mutante phoP podem ser explicados pelo mecanismo de ação alterado do
fator de transcrição phoP, conforme descrito e
resumido na Fig. 1.
O mutante phoP diminui a regulação da
biossíntese dos complexos lípidos da parede
celular micobacteriana, incluindo a diacil-
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the aim to improve efficacy of BCG. More
recently, clinical trials with recombinant
BCG vaccines have started with the aim to
find candidates to be used as prime, preventive vaccines. Another innovative strategy,
live vaccines based on rationally attenuated
Mycobacterium tuberculosis are promising
candidates (some of which in late preclinical investigation), is a promising new BCGreplacement tool with better protective efficacy against pulmonary forms of TB.
Based upon the observation that the phoP
gene coding for the transcription factor
phoP is essential for M. tuberculosis virulence
(Perez et al., 2001), we rationally attenuated
the tubercle bacillus by inactivating phoP.
The mutant demonstrated strong attenuation in cellular and animal models and resulted more attenuated than BCG in immunocompromised SCID mice (Martin et
al., 2006). The vaccine candidate protected
guinea pigs and non-human primates
against tuberculosis infection (Martin et al.,
2006, Verreck et al., 2009).
Recent molecular studies (Gonzalo Asensio
et al., 2008) have demonstrated that phoP
transcription factor controls the expression
of approximately 80 genes (many of which
implicated in M. tuberculosis virulence), accounting for about 2% of the open reading
frame (ORF) in M. tuberculosis genome.
Based on these studies it could be deduced
that the attenuated phenotype and the protective immunity conferred by the phoP
mutant can be explained by the altered
mechanism of action of the transcription
factor phoP, as described below and summarized in Fig. 1.
The phoP mutant decreases byosinthesis
regulation of complex mycobacterial cellwall lipids, including diacyltrehalose (DAT)
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Uma nova vacina viva contra a tuberculose com base na inativação do phoP
Jesus Gonzalo Asensio, Ainhoa Arbues, Dessi Marinova, Carlos Martín
~2% ORF no genoma do M. tuberculosis sob controlo do phoP / ~2% ORFs in the M. tuberculosis genome under phoP control
RESPOSTAS AO STRESS
E AO CHOQUE TÉRMICO/STRESS
& HEAT-SHOCK RESPONSES
RESPIRAÇÃO AERÓBICA E ANAERÓBICA/
AEROBIC & ANAEROBIC RESPIRATION
METABOLISMO LIPÍDICO
SL, DAT, PAT.../
LIPID METABOLISM
SL, DAT, PAT...
REGIÃO RD1 E SECREÇÃO DE ESAT-6/
RD1 REGION & ESAT-6 SECRETION
PERSISTÊNCIA/
PERSISTENCE
H37Ra::rpsL
H37Ra
SO2
RESPOSTAS HIPÓXICAS PRECOCES
E DURADOURAS/EARLY & ENDURING
HYPOXIC RESPONSES
Gonzalo Asensio et al. JBC 2006
H37Rv
Membrana plasmática/
Plasma membrane
H37Ra::phoP
Mutante phoP/
PhoP mutant
Cell
lysate
Culture
filtrate
Frigui et al. PLoS Path 2008
Fig. 1 – Estudos moleculares de vacina baseada em phoP (Gonzalo-Asensio, et al., 2008)
Fig. 1 – Molecular studies of phoP-based vaccine (Gonzalo-Asensio et al., 2008)
trealose (DAT) e poliaciltrealose (PAT) implicadas na imunomodulação do sistema
imunológico hospedeiro (Saavedra R et al.
2005), não expressos pelo mutante phoP
(Gonzalo Asensio et al. 2006).
Também se observou desregulação dos genes
na região da diferença 1 (RD1) (Gonzalo
Asensio et al., 2008) necessária à virulência e à
secreção de ESAT-6 no mutante phoP, quando
comparado com a estirpe do tipo selvagem
(Friguie et al., 2008). A RD1 restringe-se às
estirpes virulentas do complexo M. tuberculosis
(CMTB) e perde-se por todas as subestirpes de
BCG (Behr et al., 1999, Pym et al., 2003).
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and polyacyltrehalose (PAT) implicated in
immunomodulation of the host immune
system (Saavedra R et al 2005), which are
not expressed by the phoP mutant (Gonzalo
Asensio et al 2006).
Down regulation of genes within the region
of difference 1 (RD1) (Gonzalo-Asensio et
al., 2008) required for virulence and ESAT6 secretion were also observed in the phoP
mutant when compared to wild-type strain
(Friguie et al 2008). RD1 is restricted to
virulent M. tuberculosis complex (MTBC)
strains and is lost by all BCG substrains
(Behr et al., 1999, Pym et al., 2003).
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Jesus Gonzalo Asensio, Ainhoa Arbues, Dessi Marinova, Carlos Martín
O mutante phoP evidenciou regulação alterada e controlo de outras funções-chave necessárias à sobrevivência dos microrganismos na
célula hospedeira. Como se mostra na Fig. 1,
estas funções incluem respostas hipóxicas
precoces e duradouras, respiração aeróbica e
anaeróbica, respostas ao stress e ao choque térmico, metabolismo lipídico (normalmente
desregulado na estirpe avirulenta H37Ra) e
expressão do fator de persistência ICL, importante para a persistência intracelular do
M. tuberculosis durante a infeção (Gonzalo
Asensio et al., 2008). O ICL é regulado positivamente no mutante phoP e esta característica poderia explicar o adequado estado de
persistência, importante para a apresentação
melhorada do antigénio pelo mutante durante a vacinação.
Estas observações foram usadas para construir
uma nova geração duma vacina viva baseada na
inativação do phoP com uma segunda mutação
adicional que afecta a síntese duma nova família de lípidos associada à virulência da M. tuberculosis depois do consenso de Genebra sobre os
passos essenciais para o desenvolvimento clínico de novas vacinas micobacterianas vivas atenuadas (Kamath et al., 2005). O elemento final é a primeira candidata a vacina viva
atenuada desenvolvida de acordo com o consenso e recomendações de Genebra para vacinas micobacterianas vivas, com o objectivo de
a apresentar ao Millennium Development Goal
no combate à TB. Ensaios pré-clínicos rigorosos feitos até à data com o protótipo da vacina
viva baseada no phoP demonstraram a sua adequada atenuação, segurança, imunogenicidade
e eficácia na proteção contra as formas respiratórias de TB em modelos animais (Perez et al.,
2001, Williams et al., 2005, Martin et al.,
2006, Aguilar et al., 2007, Cardona et al.,
2009, Verreck et al., 2009).
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The phoP mutant showed altered regulation and control of other key functions required for successful survival of the microorganism within the host cell. As illustrated
in Figure 1, these functions include early
and enduring hypoxic responses, aerobic
and anaerobic respiration, stress and heatshock responses, lipid metabolism (normally down regulated in the avirulent strain
H37Ra), and expression of the persistence
factor ICL, important for intracellular persistence of M. tuberculosis during infection
(Gonzalo-Asensio et al 2008). ICL is positively regulated in the phoP mutant and
this characteristic could explain the adequate persistence state important for improved antigen presentation by the mutant
during vaccination.
These observations were used to construct a
new generation of a live vaccine based on
phoP inactivation carrying a second additional mutation which affects the synthesis
of a new family of lipids associated with M.
tuberculosis virulence following the Geneva
consensus on essential steps towards clinical
development of new live attenuated
mycobacterial vaccines (Kamath et al.,
2005). The final construct is the first live
attenuated candidate vaccine developed
according with and fulfilling the Geneva
consensus
requirements
for
live
mycobacterial vaccines with the aim to
deliver on the Millennium Development
Goal to combat TB. Rigorous preclinical
studies to date with the live phoP-based
vaccine prototype have demonstrated proof
of principle for adequate attenuation, safety,
immunogenicity, and protective efficacy
against respiratory forms of TB in stringent
animal models (Perez et al., 2001, Williams
et al., 2005, Martin et al., 2006, Aguilar et
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Uma nova vacina viva contra a tuberculose com base na inativação do phoP
Jesus Gonzalo Asensio, Ainhoa Arbues, Dessi Marinova, Carlos Martín
Os estudos moleculares com o mutante
phoP ajudaram na compreensão do complexo puzzle por detrás do mecanismo de ação
do fator de transcrição do phoP necessário à
virulência, persistência e sobrevivência intracelular da estirpe do tipo selvagem durante a infeção, e cuja expressão alterada é
provavelmente responsável pela adequada
atenuação do fenótipo do protótipo da vacina. Além disso, a eliminação dos lípidos
imunomoduladores extraíveis da parede celular (DAT e PAT) resultante da inativação
do gene phoP e a deficiente síntese da complexa determinante da virulência e dos lípidos da parede celular PDIM (Camacho et
al., 1999, Cox et al., 1999), devido à segunda mutação geneticamente conseguida no
protótipo da vacina baseada no phoP, também contribuem para o fenótipo atenuado
desta nova candidata.
Investigação atual de qualidade proporciona
forte apoio científico a esta vacina de nova
geração em franco progresso e em vias de ser
clinicamente avaliada quanto à segurança e
eficácia em seres humanos. Tendo em conta
a falta de correlativos imunológicos de proteção, é indispensável realizar ensaios clínicos de fase 3 para conhecermos o valor real
de qualquer nova vacina usada profilaticamente contra a TB.
A parceria Stop TB estima que pelo menos
20 vacinas candidatas deveriam iniciar os
ensaios de segurança de fase I antes de 2015,
com o propósito de conseguir o licenciamento para uma vacina eficaz contra a TB
(Young, Dye, 2006). A descoberta e utilização de uma nova vacina contra a TB que
ofereça melhor proteção contra as formas
pulmonares da doença do que a atual BCG
é crucial para alcançar o objetivo de erradicar a tuberculose até 2050.
R e v i s t a
al., 2007, Cardona et al., 2009, Verreck et
al., 2009).
The molecular studies with the phoP mutant
have helped begin to understand the intricate
puzzle behind the mechanism of action of the
PhoP transcription factor required for
virulence, persistence and intracellular survival
of the wild-type strain during infection and
whose altered expression probably accounts
for the adequate attenuation phenotype of the
prototype vaccine. Moreover, elimination of
the extractable immunomodulatory cell-wall
lipids (DAT and PAT) as a result of the targeted
inactivation of the phoP gene, and the impaired
synthesis of the complex virulence determinant
and cell-wall lipid PDIM (Camacho et al.,
1999, Cox et al., 1999) due to the second
genetically engineered mutation in the
prototype phoP-based vaccine further
contribute to the attenuated phenotype of this
novel candidate.
Strong to date research provides robust
scientific support adducing this new
generation vaccine well–advanced to progress
from research to development and be
clinically evaluated for vaccine safety and
efficacy for the first time in human. Moreover,
considering the lack of immunological
correlates of protection, Phase 3 clinical trials
are indispensible to tell us the real value of
any new vaccine used as a prophylactic tool
against TB.
Stop TB partnership estimate that at least
20 vaccine candidates should enter phase I
safety trials before 2015 with the goal to
reach an effective licensed vaccine against
TB (Young and Dye, 2006). The discovery
and use of a new TB vaccine that confers
improved protection against pulmonary
forms of TB than the current BCG is key to
reach the 2050 objective of TB eradication.
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Bibliografia/Bibliography
Aguilar D, Infante E, Martin C, Gormley E, Gicquel B,
Hernandez Pando R. Immunological responses and protective immunity against tuberculosis conferred by vaccination of Balb/C mice with the attenuated Mycobacterium tuberculosis (phoP) SO2 strain. Clin Exp Immunol
2007; 147: 330-338.
Asensio JA, Arbues A, Perez E, Gicquel B, Martin C.
Live tuberculosis vaccines based on phoP mutants: a
step towards clinical trials. Expert Opin Biol Ther 2008;
8:201-211.
Camacho LR, Ensergueix D, Perez E, Gicquel B, Guilhot C. Identification of a virulence gene cluster of Mycobacterium tuberculosis by signature-tagged transposon
mutagenesis. Mol Microbiol 1999; 34: 257-267.
Cardona PJ, Asensio JG, Arbues A, Otal I, Lafoz C, Gil
O, Caceres N, Ausina V, Gicquel B, Martin C. Extended safety studies of the attenuated live tuberculosis vaccine SO2 based on phoP mutant. Vaccine 2009; 27:
2499-2505.
Cox JS, Chen B, McNeil M, Jacobs WR Jr. Complex
lipid determines tissue-specific replication of Mycobacterium tuberculosis in mice. Nature 1999; 402: 79-83.
Frigui W, Bottai D, Majlessi L, Monot M, Josselin E,
Brodin P, Garnier T, Gicquel B, Martin C, Leclerc C,
Cole ST, Brosch R. Control of M. tuberculosis ESAT-6
secretion and specific T cell recognition by PhoP. PLoS
Pathog 2008; 4: e33.
Gonzalo Asensio J, Maia C, Ferrer NL, Barilone N,
Laval F, Soto CY, Winter N, Daffe M, Gicquel B, Martin C, Jackson M. The virulence-associated two-component PhoP-PhoR system controls the biosynthesis of polyketide-derived lipids in Mycobacterium
tuberculosis. J Biol Chem 2006; 281: 1313-1316.
Gonzalo-Asensio J, Mostowy S, Harders-Westerveen J,
Huygen K, Hernandez-Pando R, Thole J, Behr M, Gicquel B, Martin C. PhoP: a missing piece in the intricate
puzzle of Mycobacterium tuberculosis virulence. PLoS
ONE 2008; 3: e3496.
S 48
R e v i s t a
P o r t u g u e s a
Kamath AT, Fruth U, Brennan MJ, Dobbelaer R, Hubrechts P, Ho MM, Mayner RE, Thole J, Walker KB,
Liu M, Lambert PH. New live mycobacterial vaccines:
the Geneva consensus on essential steps towards clinical
development. Vaccine 2005; 23: 3753-3761.
Martin C, Williams A, Hernandez-Pando R, Cardona PJ,
Gormley E, Bordat Y, Soto CY, Clark SO, Hatch GJ, Aguilar D, Ausina V, Gicquel B. The live Mycobacterium tuberculosis phoP mutant strain is more attenuated than BCG
and confers protective immunity against tuberculosis in
mice and guinea pigs. Vaccine 2006; 24: 3408-3419.
Perez E, Samper S, Bordas Y, Guilhot C, Gicquel B, Martin C. An essential role for phoP in Mycobacterium tuberculosis virulence. Mol Microbiol 2001; 41: 179-187.
Saavedra R, Segura E, Tenorio EP, López-Marín LM.
Mycobacterial trehalos-containing glycolipid with immunomodulatory activity on human CD4+ and CD8+
T-cells. Micr Inf 2006; 8:533-540.
Verreck FA, Vervenne RA, Kondova I, van Kralingen
KW, Remarque EJ, Braskamp G, van der Werff NM,
Kersbergen A, Ottenhoff TH, Heidt PJ, Gilbert SC,
Gicquel B, Hill AV, Martin C, McShane H, Thomas
AW. MVA.85A boosting of BCG and an attenuated,
phoP deficient M. tuberculosis vaccine both show protective efficacy against tuberculosis in rhesus macaques.
PLoS One 2009; 4: e5264.
Williams A, Hatch GJ, Clark SO, Gooch KE, Hatch
KA, Hall GA, Huygen K, Ottenhoff TH, Franken KL,
Andersen P, Doherty TM, Kaufmann SH, Grode L,
Seiler P, Martin C, Gicquel B, Cole ST, Brodin P, Pym
AS, Dalemans W, Cohen J, Lobet Y, Goonetilleke N,
McShane H, Hill A, Parish T, Smith D, Stoker NG,
Lowrie DB, Kallenius G, Svenson S, Pawlowski A, Blake
K, Marsh PD. Evaluation of vaccines in the EU TB vaccine cluster using a guinea pig aerosol infection model
of tuberculosis. Tuberculosis (Edinb) 2005; 85: 29-38.
Young D, Dye C. The development and impact of tuberculosis vaccines. Cell 2006; 124: 683-687.
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Simpósio: Testes rápidos para o rastreio preliminar da
tuberculose
Ruth McNerney1
Symposium: Point-of-care tests for tuberculosis
Pensa-se que há mais casos de tuberculose
no mundo hoje do que em qualquer outra
altura através da história. As estimativas da
Organização Mundial de Saúde (OMS) para
2007 sugeriam uma prevalência de 13,7 milhões, com 9,3 milhões de novos casos e 1,8
milhões de mortes. Esta é essencialmente
uma doença de pobreza, contando a Ásia e a
África juntas mais de noventa por cento dos
casos mundiais. Na ausência duma vacina
eficaz, o controlo depende da prontidão do
tratamento para reduzir a carga bacilar viável, diminuindo assim a contagiosidade de
indivíduos com formas pulmonares da doença. Através dos esforços de iniciativas de
saúde internacionais, como as do Global
Fund to fight HIV/AIDS, Tuberculosis and
Malaria, o tratamento da TB está agora disponível e de forma gratuita para a maioria
das pessoas. Porém, o acesso ao tratamento
está dependente do diagnóstico e os atrasos
na deteção podem resultar na deterioração
do doente e aumentar as oportunidades de
1
It is believed there are more cases of tuberculosis in the world today than at any
previous time in history. WHO estimates
for 2007 suggested a prevalence of 13.7
million, with 9.3 million incident cases
and 1.8 million deaths. It is primarily a
disease of poverty, with Asia and Africa
between them sharing over ninety percent
of the global TB burden. In the absence of
an effective vaccine control depends on
prompt treatment to reduce the viable
bacillary load, thus reducing the infectiousness of individuals with open pulmonary forms of the disease. Through the
efforts of international health initiatives
such as the Global Fund to fight HIV/
AIDS, Tuberculosis and Malaria treatment for TB is now widely available and
free for the majority patients. However,
access to treatment is dependent on diagnosis and delays in detection may result
in deterioration of the patient and increased opportunity for transmission.
Department of Infectious and Tropical Diseases,
London School of Hygiene & Tropical Medicine
Keppel Street, London, WC1E 7HT, UK
e-mail: [email protected]
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transmissão. Infelizmente, o diagnóstico
nem sempre é fácil, uma vez que os sintomas não são específicos, particularmente em
pessoas com problemas de saúde concomitantes, como a imunossupressão relacionada
com o VIH.
A maioria dos casos de TB residem em países com sistemas de saúde fracos e com poucos recursos, onde os laboratórios que realizam exames de diagnóstico são limitados
em número e distribuição geográfica, e cujo
acesso pode envolver longas viagens e despesa considerável. Esta situação é agravada
pela insensibilidade dos testes disponíveis e
pela necessidade de múltiplas consultas. Os
atrasos também se devem ao comportamento dos doentes, quando estes não procuram
uma consulta médica. A relutância em obter
um diagnóstico reflete a natureza crónica da
doença, a sua não perceção e, nalguns casos,
o receio de estigmatização e exclusão social.
As estimativas da OMS sugerem que, durante 2006, quatro milhões de casos de TB
não foram diagnosticados. Ao contrário do
que acontece com patologias como a malária ou o VIH, não há testes rápidos nem dispositivos (POC) suficientemente fiáveis para
uso de pessoal não especializado na comunidade.
Um painel de especialistas internacionais
foi convidado para um simpósio onde seria
abordado o tópico dos testes rápidos para o
diagnóstico da tuberculose. O evento foi
organizado pelo subgrupo POC do grupo
de trabalho STOP TB sobre novos diagnósticos, em colaboração com a Sociedade Europeia de Micobacteriologia. A intenção do
simpósio foi aumentar a perceção da necessidade de melhorar o acesso ao diagnóstico
da TB, atualizar os técnicos de diagnóstico
e os investigadores sobre o desenvolvimen-
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Unfortunately diagnosis is not straightforward as symptoms are not specific,
particularly in those with an underlying
health problem such as HIV related immunosupression. The majority of TB cases
reside in countries with weak and poorly
resourced health care systems where laboratory facilities for diagnosis are limited
in number and distribution. Access may
involve long journeys and considerable
expense, a situation compounded by the
insensitivity of the available tests and need
for multiple visits. Delays also arise from
health seeking behaviour, where individuals do not seek medical assistance. Reluctance to seek a diagnosis reflects the
chronic nature of the disease, a lack of
awareness and, in some settings a fear of
stigmatization and social exclusion. WHO
estimates suggest that during 2006 four
million cases of TB remained undiagnosed. Unlike conditions such as malaria
or HIV there are no sensitive rapid tests
and no reliable point-of-care (POC) devices that can be used by non specialist
personnel within the community. To address these issues a panel of international
experts were invited to speak in a symposium on the topic of point-of-care tests
for the diagnosis of tuberculosis disease.
The event was organized by the POC subgroup of the STOP TB working group on
New Diagnostics in collaboration with
the European Society of Mycobacteriology. The intention of the symposium was
to increase awareness of the need for improved access to TB diagnosis, to update
diagnostic practitioners and the research
community on the current status of POC
test development and to debate research
needs and priorities.
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to e o estado atual dos testes POC, e ainda
debater as necessidades e prioridades da investigação. O potencial dos testes POC na
ajuda ao controlo da TB foi discutido por
Catharina Boehme (Foundation for Innovative New Diagnostics). O teste ideal não deveria necessitar de técnicos especializados
nem de infraestruturas laboratoriais, facultaria o resultado no próprio dia da consulta, teria uma elevada especificidade e sensibilidade superior à da microscopia de
esfregaço. Os indivíduos com um resultado
positivo seriam imediatamente registados e
referenciados para tratamento. Os testes
com sensibilidade inferior à dos testes com
base no laboratório teriam um papel se pudessem ser usados para reduzir os atrasos no
diagnóstico. Alternativamente, um teste de
avaliação com elevada sensibilidade e especificidade razoável poderia ser usado no rastreio de populações de alto risco, em que,
com um resultado positivo, o doente seria
referenciado para confirmação do diagnóstico antes do início do tratamento.
As atuais dificuldades no acesso ao tratamento foram ilustradas pelo testemunho de
uma doente com TB natural da Zâmbia,
país severamente atingido por TB e VIH/
/SIDA. Em 2003, Carol Nyirenda (coordenadora nacional da Community Initiative for Tuberculosis, HIV/AIDS and Malaria)
dirigiu-se ao seu centro de saúde, queixando-se de tosse que durava havia mais de dois
meses. Como a doente não conseguia produzir expetoração para análise, foi enviada
para casa com suspeita de bronquite crónica. Durante os seis meses seguintes foi submetida a um total de quatro raios-X, mas
não lhe foi proposto um teste VIH nesse período. Foi-lhe, finalmente, proporcionado o
tratamento para a TB mais de seis meses de-
R e v i s t a
The potential of POC tests to aid TB
control was discussed by Catharina Boehme (Foundation for Innovative New Diagnostics). The ideal test would not require
specialist skills or a laboratory infrastructure, would yield a result within one visit,
would be highly specific and have a sensitivity superior to that of smear microscopy. Anyone found positive would be immediately registered and referred for
treatment. Tests that are not more sensitive than laboratory based tests would
have a role if they could be used to reduce
diagnostic delay. Alternatively, a screening test that had high sensitivity and a
reasonable specificity could be used to
screen high risk populations, where a positive result triggered referral for confirmation prior to initiation of treatment.
The current difficulties in accessing treatment were illustrated through the testament of a former TB patient from Zambia, a country hard hit by both TB and
HIV/AIDS. Carol Nyirenda (National
Co-ordinator, Community Initiative for
Tuberculosis, HIV/AIDS and Malaria) had
presented at her local health clinic in
2003 with a cough of over two months
duration. Unable to produce sputum for
testing she was sent home with suspected
chronic bronchitis. Over the following six
months she had a total of four chest xrays but was not offered an HIV test during that time. She was finally offered
treatment for her TB more than six
months after reporting symptoms. Successfully treated she now campaigns on
behalf of people living with HIV and TB.
She proposed that a test be developed that
could be used by community volunteers,
a test that could be used in rural settings
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pois de ter referido os sintomas. Tratada
com sucesso, Carol Nyirenda agora defende
ativamente os interesses de doentes com
VIH e TB, e propôs que seja desenvolvido
um teste que possa ser usado por voluntários comunitários, um teste que possa também ser usado em áreas rurais onde não há
eletricidade e que possa ser disponibilizado
a pessoas que residem em comunidades afetadas por TB e VIH.
Os países onde a TB é endémica frequentemente não dispõem da moldura legislativa
para regular dispositivos e testes de diagnóstico. Nessas regiões, os kits-teste podem ser
comercializados sem evidência de benefício
e, em algumas circunstâncias, foram acompanhadas de alegações enganadoras quanto
ao desempenho. Rosanna Peeling (London
School of Hygiene & Tropical Medicine) apresentou dados dum estudo em que o desempenho de 19 testes rápidos de diagnóstico
da tuberculose disponíveis comercialmente
foi avaliado usando um painel de 355 amostras séricas recolhidas em diversas regiões
geográficas. Infelizmente, nenhum dos testes demonstrou um desempenho suficientemente bom para substituir os atuais testes
de microscopia de esfregaço. Quando comparado com a combinação de referência-padrão (microscopia, cultura, radiografia e
follow-up clínico), o teste com a maior sensibilidade (59,71%) tinha uma especificidade de apenas 57,72%. Os dois testes com
maior especificidade (98,66%) tinham sensibilidades de 0,97 e 2,43%, respectivamente. É possível obter uma cópia deste relatório no website do Special Programme for
Research and Training in Tropical Diseases
(TDR): http://apps.who.int/tdr/svc/publi
cations/tdr-research-publicationsdiagnostics -evaluation-2.
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where there is no electricity and a test that
was affordable by people living in communities affected by TB and HIV.
Countries where TB is endemic frequently
lack the legistrative framework to regulate
diagnostic test devices. In such an environment test kits may be marketed without
evidence of their benefit and in some circumstances have been accompanied by
misleading claims regarding their performance. Rosanna Peeling (London School of
Hygiene & Tropical Medicine) presented
data from a study where the performance
of 19 commercially available rapid diagnostic tests for tuberculosis was assessed
using a panel of 355 sera collected from
eight geographically diverse sites. Unfortunately none of the tests were found to perform well enough to replace the current
test of smear microscopy. When compared
to a combined reference standard of microscopy, culture, radiography and clinical
follow-up the test with the highest sensitivity of 59.71% had a specificity of just
57.72%. The two tests with the highest
specificity (98.66%) had sensitivities of
0.97 and 2.43% respectively. A full copy of
the report may be downloaded from the
Special Programme for Research and Training in Tropical Diseases (TDR) website.
http://apps.who.int/tdr/svc/publications/
tdr-research-publications/diagnostics-evaluation-2. The failure of current knowledge to provide a rapid test for TB has led
to recognition of the need for novel approaches. Gerd Michel (Foundation for Innovative New Diagnostics) described the
application of genome, proteome and metabolomic based technologies to biomarker
discovery. The search for suitable detection
targets has expanded from traditional pro-
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O insucesso do conhecimento atual em fornecer um teste rápido para TB levou ao reconhecimento da necessidade de novas
abordagens. Gerd Michel (Foundation for
Innovative New Diagnostics) descreveu técnicas baseadas na aplicação de tecnologias
do conhecimento do genoma, proteoma e
metabolómica para a descoberta de biomarcadores. A busca de alvos adequados foi
além da química de proteínas tradicional
para um âmbito mais alargado de componentes estruturais e metabólitos. Os testes
baseados na deteção de lipoarabinomanano
(LAM), um componente lipossacarídeo da
parede celular, ainda não conseguiram sensibilidade suficiente, mas, curiosamente,
num estudo, pareceram funcionar melhor
em indivíduos positivos para VIH do que
nos que não estavam coinfectados.
Com o desenvolvimento de ensaios de ADN
transrenal fragmentado na urina, novas estratégias estão também a ser investigadas para
análises com base em ácidos nucleicos. Os
testes POC precisam de ser rápidos e fáceis de
fazer. Uma das maneiras mais simples e rápidas de identificar substâncias é pela análise
do seu aroma e a identificação de compostos
orgânicos voláteis (COV), e vem emergindo
como um campo de interesse. A prova de
conceito do diagnóstico da TB foi conseguida com o treino de ratos pouch africanos para
cheirarem TB em amostras de expectoração.
Embora não tão sensível como a microscopia
de esfregaço, os animais conseguem avaliar
uma amostra em apenas alguns segundos. Os
utensílios para detectar baixas concentrações
de COV estão a ser investigados, incluindo a
adaptação de novos instrumentos desenvolvidos para uso militar e para deteção de agentes
de bioterrorismo. No entanto, o optimismo
precoce no que respeita à aplicação da tecno-
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tein chemistry to a broader range of structural components and metabolites. Tests
based on detection of lipoarabinomannan
(LAM) a lipopolysaccharide component of
the cell wall have not so far achieved sufficient sensitivity but intriguingly in one
study appeared to function better in HIV
positive individuals than those that were
not co infected. New strategies are also being investigated for nucleic acid based
analysis with the development of assays for
fragmented transrenal DNA in urine. POC
tests need to be both rapid and easy to perform. One of the simplest and fastest ways
to identify substances is by their aroma and
volatile organic compound (VOC) analysis
is emerging as field of interest. Proof of
concept for TB diagnosis has been provided through the training of African pouch
rats to smell TB in sputum samples. Although not as sensitive as smear microscopy the animals are able to screen a sample
in just a few seconds. Tools for detecting
VOC at low concentrations are being investigated, including the adaptation of
novel instrumentation developed for military use and bioterror agent detection.
However, early optimism regarding the application of electronic nose technology has
dissipated following the realisation that
they lack the robustness required for a diagnostic test. A more analytical approach is
now being pursued and Ruth McNerney
(London School of Hygiene & Tropical Medicine) outlined some of the challenges facing those searching for VOC biomarkers
that are predictive of TB disease. Small
volatile molecules lack the distinctive character of macromolecules traditionally used
to predict infection and they are more often found in nature. To complicate matters
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logia do nariz electrónico dissipou-se depois
da constatação de que lhe falta a robustez necessária a um teste de diagnóstico. Uma abordagem mais analítica está agora em curso e
Ruth McNerney (London School of Hygiene
& Tropical Medicine) referiu alguns dos desafios que se apresentam a quem investiga biomarcadores em COV preditivos de TB. As
pequenas moléculas voláteis não têm o caráter distinto das macromoléculas tradicionalmente usadas para indicar a presença de infecção e encontram-se mais frequentemente
na natureza. Para complicar, os metabólitos
produzidos por Mycobacterium tuberculosis
variam, dependendo da disponibilidade de
nutrientes e outros fatores ambientais, pelo
que os COV emitidos durante uma infeção
podem diferir com o local da infecção e o estádio da doença. Do mesmo modo, os COV
emitidos pelo hospedeiro em resposta à infeção podem sofrer alterações com a progressão
da doença. A complexidade e a natureza dinâmica da emissão de COV sugere que para
atingir elevada sensibilidade e especificidade
será necessária uma abordagem analítica multivariada.
Há muitos passos ao longo do caminho do
desenvolvimento de testes e Amy P. Wong
(X PRIZE Foundation) discutiu a rota e
identificou as potenciais barreiras ao aparecimento de um novo diagnóstico de sucesso. Quando os biomarcadores tiverem sido
descobertos e testados com protótipos de
dispositivos, a capacidade de fabrico será
desenvolvida e estabelecer-se-á uma rota de
mercado. As novas tecnologias podem requerer métodos de fabrico inovadores que
assegurem a robustez dos dispositivos a preços competitivos. É necessária melhor
orientação das especificações de novos testes. A incerteza quanto ao mercado para
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metabolites produced by Mycobacterium
tuberculosis vary depending on the availability of nutrients and other environmental factors, thus VOC emitted during infection may differ with the site of infection
and stage of disease. Similarly, VOC emitted by the host in response to infection
may alter with disease progression. The
complexity and dynamic nature of VOC
emission suggest that to attain high sensitivity and specificity a multivariate analytical approach will be required.
There are many steps along the path of
test development and Amy P Wong (X
PRIZE Foundation) discussed the route
and identified potential barriers to the delivery of a successful new diagnostic. Once
biomarkers have been discovered and tested with prototype devices the capacity to
manufacture must be developed and a
route to market established. New technologies may require innovative manufacturing practices to ensure delivery of robust devices at a competitive price.
Improved guidance on optimum test
specification is badly needed. Uncertainty
regarding the market for POC TB tests
also acts as a disincentive to potential investors. Innovative mechanisms for rewarding test development are required to
stimulate participation and facilitate the
development of devices that can be used
to detect TB wherever in the world there
is a need. In the words of an ex TB patient
“TB infection and disease is not just about
numbers, for some of us it is a reality…
we sit and watch TB reversing all the efforts and gains of the HIV fight, as we die
one by one from TB. It is not about numbers, there are real people at the end of
the chain we have run out of time”.
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testes POC de TB também funciona como
desincentivo para potenciais investidores.
São necessários mecanismos inovadores que
compensem o desenvolvimento de testes,
de modo a estimular a participação e a facilitar o desenvolvimento de dispositivos que
possam ser usados para detectar a TB em
qualquer parte do mundo. Nas palavras de
um ex-doente de TB, “a infeção por TB e a
própria doença não são apenas números,
para alguns de nós é uma realidade… vamos assistindo à inversão, pela TB, de todos
os esforços e ganhos na luta do VIH, conforme vamos morrendo, um a um, de TB.
Não se trata de números, somos pessoas no
extremo da cadeia e o nosso tempo está a
terminar.”
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Criar capacidade laboratorial para a tuberculose:
Necessidades e estratégias
Thomas M Shinnick1
Building tuberculosis laboratory capacity: Needs and
strategies
Palavras-chave
Planos estratégicos nacionais, abordagem de
sistemas, rede de laboratórios de saúde pública.
Key-words
National strategic plans, systems approach,
public health laboratory networks.
Fundamentos
No relatório “TB Global 2009”1, a Organização Mundial de Saúde (OMS) utilizou
informação de modelos epidemiológicos e
dados de programas para estimar que, em
2007, havia 9,27 milhões de novos casos de
tuberculose (TB); 1,37 milhões (15%) de
casos entre pessoas infetadas com VIH;
511 000 novos casos de TB multirresistente
(MDR TB); e 50 000 novos casos de TB extensamente resistente a fármacos (XDR
TB). Estas são apenas estimativas, em parte
devido a falhas na disponibilidade de serviços laboratoriais de TB em muitas regiões
do mundo1.2. Apenas cerca de 60% dos novos casos de TB são confirmados pelo laboratório e só aproximadamente 5% dos casos
Background
In the 2009 Global TB report1, the World
Health Organization used information from
epidemiologic models and program data to
estimate that in 2007 there were 9.27 million new cases on TB; 1.37 million (15%)
cases among persons living with HIV;
511 000 new cases of multidrug-resistant
TB (MDR TB); and 50 000 new cases of
extensively drug-resistant TB (XDR TB).
These are only estimates, in part, because
there are large gaps in the availability of TB
laboratory services in many regions of the
world1,2. Only about 60% of new TB cases
are laboratory confirmed and only about
5% of MDR TB cases are actually identified
and reported.
1
PhD, Associate Director for Global Laboratory Activities, Division of TB Elimination,
Centers for Disease Control e Prevention
1600 Clifton Road, MS-G35, Atlanta Georgia 30333 USA
e-mail: [email protected]
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de MDR TB são realmente identificados e
reportados.
A inadequada capacidade laboratorial prejudica o diagnóstico, a gestão dos casos e a vigilância da doença. Isto é particularmente
importante para os doentes com TB resistente a fármacos porque, frequentemente, os
cuidados eficazes só se iniciam depois dos
resultados dos testes de suscetibilidade a fármacos. O aparecimento da MDR TB e da
XDR TB levou ao reconhecimento de que a
falta de capacidade laboratorial representa
uma crise global. Os fatores que têm contribuído para as falhas nos serviços laboratoriais de TB incluem: 1) falta de reconhecimento da importância do laboratório no
tratamento e no controlo da doença; 2) má
comunicação entre os programas nacionais
de TB e as entidades que fornecem os serviços laboratoriais de TB; 3) recursos humanos
e financeiros inadequados nesses laboratórios; 4) falta de infraestruturas e de instalações; e 5) preocupações com biossegurança4.
Ao reconhecer o importante papel do laboratório, a 2007 World Health Assembly5 endossou o apelo do Global Plan to Stop TB2 para
acesso universal a testes de cultura e de suscetibilidade a fármacos. O acesso universal representa capacidade para fazer anualmente
120 milhões de exames microscópicos, 60
milhões de testes de cultura e 6 milhões de
testes de suscetibilidade a fármacos3,6. Cumprir com este objetivo representará criar 5000
novos centros de microscopia, treinar 9000
novos técnicos de microscopia e criar ainda
2000 novos laboratório para testes de cultura
e testes de suscetibilidade a fármacos e treinar
23 000 novos técnicos. Serão necessários
anualmente pelo menos mil milhões de dólares americanos para construir e manter infraestruturas laboratoriais de TB. No entanto,
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The inadequate laboratory capacity hinders
diagnosis, case management, and disease
surveillance. This is particularly important
for patients with drug-resistant TB because
effective care often does not begin until results of drug-susceptibility tests are available. Indeed, the emergence of MDR TB
and XDR TB has led to the recognition that
the lack of TB laboratory capacity is a global
crisis. Factors that have contributed to the
gaps in TB laboratory services include: 1) a
lack of recognition of the importance of the
laboratory in TB treatment and control; 2)
poor communication between National TB
Programs and those providing TB laboratory services; 3) inadequate human and financial resources for TB laboratories; 4)
lack of infrastructure and physical facilities;
and 5) biosafety concerns4.
Recognizing the important role of the laboratory, the 2007 World Health Assembly5
endorsed the call of the Global Plan to Stop
TB2 for universal access to culture and drugsusceptibility testing. Universal access will
require the capacity perform 120 million
microscopy investigations, 60 million culture investigations, and 6 million drug-susceptibility investigations per year3,6. Meeting this goal will require establishing 5000
new microscopy centers and training 9000
new microscopy technicians as well as establishing 2000 new culture and drug-susceptibility testing laboratories and training
23 000 new technicians. At least US$ 1 billion will be needed annually for building
TB laboratory infrastructure and recurring
costs. However, the benefit to cost ratio of
such investments is estimated to be 9:1 in
populations with a high prevalence of HIV
infection7. Meeting the universal access
goals could save countries in sub-Saharan
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estima-se que o rácio custo-benefício de tais
investimentos seja de 9:1 em populações com
elevada prevalência de infeção por VIH7. Se
se conseguir o objetivo de acesso universal, os
países na África subsariana poderão poupar
cerca de 52 mil milhões de dólares americanos anualmente.
Planeamento estratégico para
laboratórios nacionais
Para criar e manter os serviços laboratoriais
em falta serão necessários recursos consideráveis, dedicação e vontade política. A Declaração de Maputo de 2008 sobre Fortalecimento dos Sistemas de Laboratórios8
apelou para os governos tomarem posse das
redes de serviços laboratoriais e implementarem estratégias nacionais de reforço e
apoio a esses serviços e, consequentemente,
às principais doenças que ameaçam a saúde
pública. Os governos deveriam criar um departamento de sistemas laboratoriais, no
âmbito do Ministério da Saúde, capaz de
gerir essa rede de sistemas e assumir como
prioridade elaborar uma política laboratorial inserida no plano nacional de saúde.
Os esforços de planeamento estratégico deveriam incluir programas para todas as doenças, serviços clínicos e outras estruturas e a
padronização dos serviços laboratoriais assente na qualidade. Para implementar e
manter o planeamento estratégico, os governos deveriam: 1) reconhecer a importância
dos serviços laboratoriais no controlo das
doenças; 2) assegurar que o fortalecimento
dos planos do sector da saúde inclui componentes adequadamente orçamentados para
o desenvolvimento da capacidade laboratorial; 3) coordenar os esforços de todos os
departamentos, programas específicos para
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Africa alone as much as US$ 52 billion annually.
National laboratory strategic
plans
creating and sustaining the needed laboratory services will require considerable resources, efforts, and political will. The 2008
Maputo Declaration on Strengthening Laboratory Systems8 calls upon national governments to take ownership of their laboratory systems and develop national strategic
laboratory plans that integrate laboratory
support for the major diseases of public
health importance. National governments
should establish a department of laboratory
systems within the Ministry of Health to
provide leadership and should set as a priority developing a laboratory policy within
the health development plan that will guide
the implementation of a strategic laboratory
plan.
Strategic planning efforts should include all
disease programs, clinical services, and other
stakeholders and strive to create a laboratory
system based on quality laboratory management principles. To implement and sustain
the strategic plans, national governments
should: 1) acknowledge the importance of
laboratory services in disease control; 2) ensure that health sector strengthening plans
include adequately budgeted components
for laboratory capacity development; 3) coordinate efforts of all departments, diseasespecific programs, donors, and technical
partners responsible for laboratory services;
4) commit adequate human and financial
resources for laboratory services; 5) develop
and implement a human resource policy to
create a qualified laboratory workforce and
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cada doença, dadores e parcerias técnicas
responsáveis pelos serviços de laboratório;
4) consignar recursos humanos e financeiros
adequados para esses serviços; 5) desenvolver e implementar uma política de recursos
humanos, a fim de se obterem profissionais
qualificados para os laboratórios, e identificar e afastar barreiras ao desenvolvimento
das suas carreiras e remunerações, bem como
a sustentação da sua competência técnica; e
6) contratar fornecedores de serviços de laboratório (e.g., privados, académicos e laboratórios de saúde pública) para melhorar o
acesso aos testes de qualidade controlada
para o diagnósticos da TB.
Construção de redes de
laboratórios
Uma componente essencial no esforço de
combate a doenças infeciosas é uma rede de
laboratórios que forneça testes de diagnóstico
fiáveis, tratamento e monitorização da terapêutica. Tal rede deveria ser posta ao serviço
de patologias com particular importância na
saúde pública, especialmente o VIH e a TB.
Uma abordagem de sistemas na criação dessa
rede é fundamental para otimizar os testes de
laboratório e a troca de informações necessária para assegurar que os serviços apropriados
estarão disponíveis em cada programa. A
abrangência desse esforço envolve avaliação e
compreensão da estrutura, do desempenho e
do custo da rede de laboratórios; desenvolvimento duma rede de referência e informação
que assegure o rápido fluxo de amostras e de
resultados; e adoção de princípios de melhoramento da qualidade para avaliar e melhorar
o desempenho da rede de laboratórios9.
Um desafio na abordagem integrada é o facto
do nível global de programas e de subsídios
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identify and remove barriers to laboratory
staff career development, remuneration, retention, and sustainability of technical competency, and 6) engage all laboratory service
providers (e.g., private, academic, and public health laboratories) to improve access to
quality-assured TB diagnostic testing.
Building laboratory networks
An essential component of efforts to combat infectious diseases is a network of laboratories that can provide reliable laboratory
testing for diagnosis, treatment, and monitoring of therapy. Such a network should
strive to be integrated across the diseases of
public health importance, especially HIV
and TB. A systems approach to creating
such a network is necessary to optimize laboratory testing and information exchange
and to ensure that appropriate services are
available in every program. Comprehensive
laboratory strengthening efforts involve assessment and understanding of the structure, performance, and cost of the laboratory network; development of a referral and
information network to ensure prompt flow
of specimens and information; and use of
quality-improvement principles to evaluate
and improve the performance of the laboratory network9.
A challenge to an integrated approach is
that at the global level programs and funding are directed to specific diseases. Such
vertical global programs are often mirrored
by vertical national programs and laboratories. For a variety of reasons including funding sources, technical requirements, and
biosafety concerns, the national reference
laboratory for a disease in many countries is
a specialized, free-standing institution sepa-
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serem dirigidos a doenças específicas. Esses
programas verticais globais refletem frequentemente programas e laboratórios verticais
nacionais. Por uma variedade de razões, incluindo origens de recursos, necessidades técnicas e preocupações com a biossegurança, o
laboratório de referência nacional para uma
doença em muitos países é uma instituição
individual especializada, separada do sistema
geral de laboratórios de saúde pública e de
outros laboratórios nacionais de referência.
Além disso, os laboratórios nacionais de referência concentram-se frequentemente no
apoio a actividades epidemiológicas, de vigilância ou de investigação, e podem ter um
papel direto limitado no cuidado de doentes,
o que pode ser uma limitação no que respeita
ao valor da integração de serviços de laboratórios nacionais de referência nos programas
dirigidos a doenças específicas.
Por outro lado, os laboratórios nacionais de
referência têm frequentemente a responsabilidade de desenvolver, dirigir e monitorizar as
redes de laboratórios de saúde pública que
fornecem serviços de diagnóstico e de cuidados a doentes. Essas redes podem fornecer
serviços para mais de um programa de doenças específicas, representando oportunidades
de associação de programas e harmonização
ou integração de serviços. Na verdade, ao nível mais periférico do sistema de saúde, os
serviços de laboratório estão muitas vezes totalmente integrados, com um técnico que
executa os testes para todas as doenças.
Conceber a capacidade do laboratório é, por
si só, uma oportunidade para construir ligações entre programas e para integrar serviços, isto é, embora o treinamento de técnicos
assente na experiência específica de determinadas doenças, a capacidade de construir laboratórios tem mais que ver com perícia
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rate from the general public health laboratory system and other national reference
laboratories. Also, national reference laboratories often concentrate on support for epidemiologic, surveillance, or research activities and may have a limited, direct role in
patient care. Such an emphasis may limit
the perceived value of integrating the services of national reference laboratories across
disease programs.
On the other hand, national reference laboratories often have responsibility for developing, leading, and monitoring a network of public health laboratories that
provide services for diagnosis and patient
care. Such network laboratories may provide services for more than one diseasespecific program and represent opportunities to build linkages between programs
and harmonize or integrate services. Indeed, at the most peripheral level of the
health system, laboratory services are often
fully integrated with one technician doing
testing for all diseases.
Building laboratory capacity is by itself
an opportunity to build linkages between
programs and integrate services. That is,
although training of bench-level technicians relies on disease-specific expertise,
laboratory capacity building relies more
on cross-cutting expertise in infrastructure, biosafety, facilities, human resource
development, specimen referral, supply
chain management, logistics, equipment,
maintenance, quality assurance programs, management principles, information systems, data management, and accreditation processes. A potential role
for a department of laboratory systems
within the Ministry of Health of a country would be to oversee programs that
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transversal em infraestruturas, biossegurança, instalações, recursos humanos, referência
de espécimes, gestão da cadeia logística, logística, equipamento, manutenção, programas de garantia de qualidade, princípios de
boa gestão, sistemas de informação, gestão
de dados e acreditação de processos. Um papel potencial para um departamento de sistemas de laboratórios, sob a alçada do ministério da saúde de um país, seria o de
inspecionar os programas que tratam destes
temas de modo coordenado e integrado.
Geralmente, deveria ser responsabilidade do
laboratório de TB nacional de referência, em
colaboração e coordenação com o programa
nacional de TB, programas de outras doenças
e departamentos do ministério da saúde, para
desenvolver, implementar e monitorizar uma
rede de laboratórios que assegure o acesso a
testes de TB com qualidade. Em muitos países, existem redes de laboratórios de microscopia de esfregaços para bacilos ácido-álcool
resistentes (BAAR) inspecionados pelo laboratório de TB de referência nacional que poderiam servir de ponto de partida para o desenvolvimento da capacidade laboratorial
necessária ao processamento de testes de TB
resistente a fármacos e TB associada a VIH.
Porém, há que ter o cuidado de evitar criar
uma rede de laboratórios de TB isolada e vertical. Sempre que possível, os serviços dos laboratórios TB devem ser uma parte integral
dos cuidados de saúde primários e duma rede
nacional de laboratórios de saúde pública.
Recursos e assistência
Começam a surgir recursos de apoio a países
com capacidade para construir laboratórios
para testar TB. A Global Laboratory Initiative
(GLI) da Stop TB Partnership (http://www.
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address these issues in coordinated, integrated manner.
Generally, it should be the responsibility of
the national TB reference laboratory, in collaboration and coordination with the national TB program, other disease programs,
and MOH departments, to develop, implement, and monitor a laboratory network
that assures access to quality TB testing and
complete, timely reporting. In many countries, there exists a network of AFB-smear
microscopy laboratories overseen by the national TB reference laboratory, which could
serve as a starting point for developing the
laboratory capacity needed to address drugresistant TB and HIV-associated TB. However, care must be taken to avoid creating an
isolated, vertical TB laboratory network.
Where possible, TB laboratory services
should be an integral part of primary health
care and a national public health laboratory
network.
Resources and assistance
Resources are becoming available to assist
countries with TB laboratory capacity building. The Global Laboratory Initiative (GLI)
of the Stop TB Partnership (http://www.
stoptb.org/wg/gli/; 6) is working to develop
1) a roadmap for ensuring quality TB diagnostics services within national laboratory
strategic plans, 2) guidance on norms and
standards for TB laboratory testing, equipment, and biosafety, 3) global policy guidance, 4) training materials, and 5) human
resource strategies.
Laboratory consultants are needed to assist
countries with assessment, strategic planning, implementation, and evaluation. Currently expert laboratory diagnostic advice
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stoptb.org/wg/gli/6 está a trabalhar para desenvolver: 1) um roteiro para assegurar serviços de diagnóstico de TB com qualidade,
integrados no planeamento estratégico de
laboratórios; 2) orientação nas normas e padrões para laboratórios de testes de TB, equipamento e biossegurança; 3) orientação na
política global; 4) materiais de formação; e
5) estratégias de recursos humanos.
São necessários consultores de serviços de laboratório para ajudar os países na avaliação,
planeamento estratégico e implementação.
Presentemente, aconselhamento e experiência têm sido fornecidos por peritos de laboratórios de países industrializados, frequentemente numa base ad hoc, com pouca ou
nenhuma coordenação com parceiros globais. O Plano Global 2006-2015 da OMS2
indicava que o actual sistema de curtas
workshops e visitas de aconselhamento são
insuficientes para suprir a falta de pessoal de
laboratório em países com estas carências e
para criar sistemas laboratoriais sustentáveis.
A GLI está a trabalhar no desenvolvimento
duma estratégia de consenso para criar capacidade laboratorial, cursos para consultores
de implementação da estratégia de consenso
e um mecanismo que assegure a coordenação de esforços em cada país e entre parceiros. Os aspetos críticos no processo de consultadoria são: desenvolver uma abordagem
consistente para criar a capacidade laboratorial; promover o uso de sistemas e testes de
laboratório apropriados para países com recursos limitados; e assegurar que a experiência necessária para desenvolver, implementar
e avaliar o planeamento estratégico referido
seja disponibilizada. De notar que há uma
maior necessidade de experiência para criar
capacidade laboratorial do que para as técnicas específicas de laboratório. Um dos obje-
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and expertise has been provided by industrialized-world laboratory experts, often on an
ad hoc basis with little or no coordination
with global partners. The WHO Global
Plan 2006-20152 indicated that the current
system of short workshops and consultancy
visits are insufficient to bridge the training
gap of laboratory personnel in high burden
countries and to build sustainable laboratory systems. The GLI is working to develop a
consensus strategy for laboratory capacity
building, courses to train consultants in the
implementation of the consensus strategy,
and a mechanism to ensure coordination of
capacity building efforts within a country
and among partners. The critical aspects in
the consulting process are to develop a consistent approach to laboratory capacity
building; to promote the use of laboratory
systems and tests appropriate for resourcelimited, high-burden countries; and to ensure that the expertise needed to develop,
implement, and evaluate a strategic plan for
laboratory capacity building will be available to countries. Note that there is greater
need for expertise in laboratory capacity
building than there is for expertise in disease-specific laboratory techniques. A goal
is to have cadre of consultants who can be
stationed in country for extended periods of
time that would be able to assist countries
in building an integrated laboratory network capable of providing the laboratory
services needed to combat TB, HIV, and
malaria.
Conclusion
An effective response to the call for universal access to TB diagnostics requires a massive scale-up of TB laboratory services and
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tivos é conseguir quadros de consultores que
possam ser colocados nos países durante longos períodos de tempo, a fim de ajudarem na
criação duma rede integrada de laboratórios
capaz de fornecer os serviços necessários ao
combate de tuberculose, VIH e malária.
Conclusão
Uma resposta eficaz ao apelo para acesso
universal ao diagnóstico da TB exige um aumento maciço dos serviços laboratoriais e
correspondente atribuição de recursos. Para
maximizar o impacto e a sustentabilidade
destes investimentos é imprescindível uma
abordagem alargada dos sistemas de saúde.
A harmonização de esforços na criação
duma sólida rede de sistemas laboratoriais
capaz de responder às necessidades de todas
as patologias de importância para a saúde
pública é um benefício para todos os programas de saúde e contribui para o fortalecimento do setor da saúde em general e para a
qualidade dos cuidados aos doentes.
correspondingly large commitment of resources. To maximize the impact and sustainability of the investments in TB laboratory capacity building, a broad health
systems strengthening approach is needed.
Harmonized efforts using a systems approach to build to a strong laboratory network that addresses the needs of all diseases
of public health importance is a benefit to
all disease programs and contributes to general health sector strengthening and quality patient care.
Bibliografia/Bibliography
1. World Health Organization. Global tuberculosis
control: epidemiology, strategy, financing: WHO report 2009 (WHO/HTM/TB/2009.411). Geneva:
World Health Organization; 2009. Available online at
http://www.who.int/tb/publications/global_report/
en. Accessed Nov. 1, 2009.
2. Stop TB Partnership e World Health Organization. Global Plan to Stop TB 2006–2015 (WHO/
HTM/STB/2006.35). Geneva: World Health Organization; 2006. Available online at http://www.
stoptb.org/globalplan/assets/documents/GlobalPlanFinal.pdf. Accessed Nov. 1, 2009.
3. Report of a Ministerial Meeting of High M/XDR-TB Burden Countries, Beijing China, April 1-3,
2009. Key bottlenecks in M/XDR-TB control and patient care. 5. Responding to the laboratory bottleneck.
S 64
R e v i s t a
P o r t u g u e s a
Geneva: World Health Organization; 2009. Available
online at http://www.who.int/tb/challenges/mdr/bottlenecks/bottlenecks_chapter5.pdf. Accessed Nov. 1,
2009.
4. Abdel Aziz M, Ryszewska K, Laszlo A, Blanc L. Strategic approach for the strengthening of laboratory
services for tuberculosis control, 2006-2009 (WHO/
HTM/TB/2006.364). Geneva: World Health Organization; 2006. Available online at: http://whqlibdoc.
who.int/hq/2006/WHO_HTM_TB_2006.364_eng.
pdf. Accessed on Nov. 1, 2009.
5. World Health Organization. 60th World Health Assembly. Resolutions and Decisions. World Health Assembly Resolution WHA 60.19: Tuberculosis control:
progress and long-term planning. Geneva: World Health
Organization; 2007. Available online at http://apps.
d e
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Thomas M Shinnick
who.int/gb/ebwha/pdf_files/WHA60/A60_R19 -en.
pdf. Accessed Nov. 1, 2009.
6. Global Laboratory Initiative. Moving tuberculosis (TB)
laboratory capacity strengthening forward: A Global Laboratory Initiative. Supporting document for 15th Annual
Meeting of the Stop TB Partnership Coordinating Board,
Bagamoyo Tanzania, November 28-29, 2008. Geneva:
Stop Tb Partnership; 2009. Available online at http://
www.stoptb.org/cb/meetings/20081028_Bagamoyo_
Tanzania/assets/documents/2.08-11.1%20GLI%20Syn
opsis%20FINAL.pdf. Accessed Nov. 1, 2009.
7. Laxminarayan R, Klein E, Dye C, Floyd K, Darley S,
Adeyi O. Economic Benefit of Tuberculosis Control
(August 1, 2007). World Bank Policy Research Working
Paper Series, No. 4295, 2007. Available at http://www.
wds.worldbank.org/servlet/WDSContentServer/
R e v i s t a
WDSP/IB/2007/08/01/000158349_20070801103922
/Rendered/PDF/wps4295.pdf. Accessed Nov. 1, 2009.
8. World Health Organization. Maputo Declaration on
Strengthening Laboratory Sistems. Presented at: Consensus Meeting on Clinical Laboratory Testing Harmonization and Standardization; January 22-24, 2008;
Maputo, Mozambique. Available at: http://www.who.
int/diagnostics_laboratory/Maputo-Declaration_2008.
pdf. Accessed Nov. 1, 2009.
9. Centers for Disease Control and Prevention (Shinnick TM, Iademarco MF, Ridderhof JC). National plan
for reliable tuberculosis laboratory services using a sistems approach: recommendations from CDC and the
Association of Public Health Laboratories Task Force on
Tuberculosis Laboratory Services. MMWR (RR-6)
2005; 54:1-12.
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Experiência da Rede Brasileira de Pesquisa em
Tuberculose no desenvolvimento e avaliação de novos
métodos de diagnóstico em tuberculose
Afranio Kritski1
The experience of the Brazilian Tuberculosis Research
Network in the development and evaluation of new
methods of diagnosing tuberculosis
Introdução
De acordo com estimativa da Organização
Mundial de Saúde (OMS) de 2009, o Brasil
está em 18.º lugar entre os 22 países que constituem 80% da carga global da tuberculose
(TB). A TB continua a ser a principal causa de
morte em indivíduos infetados com VIH,
mesmo nos submetidos a HAART (terapia
antirretroviral potente) oferecida gratuitamente a todas as pessoas infetadas com VIH, desde
1997. Tal como acontece noutros países em
desenvolvimento, há alguma dificuldade em
harmonizar a comunicação e o entendimento
entre especialistas programáticos em TB, académicos e comunidade, e as organizações não
governamentais. Em 2001, a investigação da
TB não estava sujeita a quaisquer linhas de
orientação quanto a: a) desenvolvimento de
novos produtos; b) inovação tecnológica; e c)
incorporação de novos instrumentos nos setores público e privado. A Rede Nacional de Investigação da Tuberculose (REDE-TB) foi
criada, em 2002, para aproximar as diferentes
1
Introduction
Brazil ranks 18th out of the 22 countries
that bear 80% of the worldwide tuberculosis (TB) burden, according to the World
Health Organization (WHO) in 2009. TB
is the leading cause of mortality in HIV infected persons, despite all HIV infected persons having received highly active antiretroviral therapy (HAART) free of charge since
1997.
As in other developing nations, there is a
significant gap in communication and understanding between TB programme experts, academics, the community, and nongovernmental organisations. In 2001, there
was no TB research policy in place regarding: a) the development of new products, b)
technological innovation and c) the incorporation of new tools in public and private
sectors.
In recognition of this gap, a National TB
Research Network was established in 2002
to bring together different constituencies to
Coordinator, Diagnostic Area, Brazilian TB Research Network (Rede TB). Academic Tuberculosis Programme, Medical School of the Federal University
of Rio de Janeiro, Brazil
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sensibilidades e promover uma estratégia integrada, multidisciplinar e multi-institucional
para controlo e investigação da TB no Brasil.
(Rede-TB – www.redetb.org). Nesta apresentação, serão comentadas as atividades da área
diagnóstica da Rede TB.
promote an integrated, multi-disciplinary
and multi-institutional strategy to TB control and research in Brazil (www.redetb.org).
This article describes the Network’s work in
the area of diagnosis.
Desenvolvimento, avaliação
e incorporação de testes
diagnósticos no Brasil
A avaliação da incorporação de novas tecnologias diagnósticas em TB tem sido também
priorizada pela Organização Mundial de
Saúde após o lançamento do Plano Global
de TB da OMS/STOP, em 2006 (http://
www.who.int/tb/strategy/stop_tb_strategy/
en/). A partir de 2002, apercebemo-nos de
que o desenvolvimento diagnóstico e o processo de avaliação proposto pela OMS receberam das partes interessadas atenção e
apoios diferentes. Verificou-se um enorme
interesse e disponibilidade de fundos relativamente às seguintes fases: a) pré-clínica:
descoberta e investigação, desenvolvimento;
b) fase 1: avaliação – prova de princípio; c)
fase 2: avaliações laboratoriais; e d) fase 3:
avaliações de ensaios de campo para análise
de desempenho. Mas a fase 4 despertou menos interesse e obteve menos fundos: custo,
estudos de impacto e, para a transferência
de políticas, pré-qualificação, aprovisionamento, preços negociados, análise de acesso.
Em 2007, procedeu-se à avaliação das tendências dos artigos científicos sobre tuberculose no Brasil publicados entre 1986 e
2006. Entre os 1054 trabalhos avaliados,
que continham a palavra tuberculose e cujos
autores estavam ligados a instituições brasileiras, 486 (46,1%) artigos estavam relacionados com descrição e revisão de casos e séries. Apenas 70 (6,7%) artigos eram estudos
The development, evaluation
and incorporation of diagnostic
tests in Brazil
Evaluating the incorporation of new TB
diagnostic techniques was prioritised by the
WHO following the 2006 launch of the
Global Plan Against TB – WHO/STOP
(http://www.who.int/tb/strategy/stop_tb_
strategy/en/). Since 2002, we have learned
that the diagnostics development and
evaluation process proposed by WHO has
received different attention and funding by
the stakeholders. A lot of interest and available funding has been allocated to the following stages: a) pre-clinical: discovery and
research, development, b) phase 1: evaluation – proof of principle; c) phase 2: laboratory evaluations, and d) phase 3: evaluation
of field trials for performance analysis. There
has been a scarcity of interest and available
funding allocated, however, to phase 4: cost,
impact studies and policy transfer, prequalification, bulk procurement, negotiated pricing and access analysis.
In 2007 there was an evaluation of the trends
in scientific articles on TB in Brazil published between 1986 and 2006. Of 1054 TB
publications assessed containing the word
‘tuberculosis’ with the authors affiliated to
Brazilian Institutions, 486 (46.1%) articles
were related to descriptive, review and case
series reports. Only 70 (6.7%) articles described operational/effectiveness studies or
clinical trials (Kritski AL et al., 2007).
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operacionais/de eficácia ou ensaios clínicos.
(Kritski AL, et al., 2007).
Em 2002, fomos abordados por uma empresa que manifestou interesse em ter os seus
testes serológicos registados numa agência
reguladora brasileira (ANVISA) para futura
comercialização. A avaliação desses testes serológicos demonstrou uma elevada especificidade (95%) entre os controlos saudáveis,
mas baixa especificidade (46%) entre os indivíduos suspeitos de serem portadores de
TB (Gounder C, et al., 2002). Felizmente, a
empresa deixou o mercado brasileiro. Esses
resultados são semelhantes aos descritos na
revisão sistemática publicada em 2007
(Steingart et al., 2007). Os autores concluíram que no momento não há indicação de
kits sorológicos comercializados ou testes in
house no diagnóstico da TB no mundo. Os
profissionais de saúde deveriam ser alertados
para não utilizarem tais tecnologias na sua
prática clínica. Lamentavelmente, esses testes ainda estão à venda em países em desenvolvimento, não tendo sido publicitadas
quaisquer recomendações sobre este facto
pelas organizações internacionais. Em 2005,
iniciou-se uma interação com a “Gerência
de produtos para diagnóstico de uso in vitro”
da Agência Reguladora Nacional (ANVISA)
e efetuou-se uma avaliação dos testes diagnósticos de tuberculose registados na ANVISA
no período de 2000 a 2004: 48 foram registados desde 2000, 33 não tinham registo válido mas continuavam no mercado e 14
(30%) eram testes imunosserológicos. Estes
testes foram registados em diferentes cenários clínicos no Brasil, sem validação prévia.
Estes resultados acentuam a dificuldade de
harmonização entre a agência reguladora, os
académicos e os decisores que tratam da incorporação de novas tecnologias no Brasil.
R e v i s t a
In 2002, we were approached by a company
interested in having its serological tests registered at the Brazilian regulatory agency (ANVISA) for future commercial use. The evaluation of these serological tests showed a high
specificity (95%) in healthy control subjects,
but a low specificity in TB suspects (46%)
(Gounder C et al., 2002). Fortunately, they
withdrew from the Brazilian market. These
results are similar to those described in the
systematic review published in 2007 (Steingart et al., 2007). The authors concluded
that there is currently no indication for commercial serologic testing kits or in-house
tests in the diagnosis of TB in the world.
Healthcare professionals should be warned
not to use these techniques in clinical practice. Unfortunately, these tests are still sold
in developing nations and no strict recommendations on them has been issued by international organisations. In 2005 contact
was made with the management section of
the in vitro diagnostic products section of
the National Regulatory Agency (ANVISA)
and an evaluation of TB diagnostic products
tested registered at ANVISA in the period
2000 to 2004 was carried out: 48 had been
registered since 2000; 33 had no valid registration, but still remained on the market and
14 (30%) were immunoserological tests.
These tests had been registered without prior
validation in different clinical settings in
Brazil. These results highlighted the gap between the regulatory agency, academics and
the policy makers who deal with the incorporation of new technologies in Brazil.
Over the last few years, Brazil, along with
other developing countries, has seen new
molecular technique kits for diagnosing TB
or resistant TB enter the market, both polymerase reaction (PCR) and phenotype
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Nos últimos anos, como ocorre noutros países em desenvolvimento, no Brasil foram
comercializadas novas tecnologias moleculares (kits de PCR) ou fenotípicas (MGIT960)
no diagnóstico de TB e TB resistente. Entretanto, tais tecnologias têm sido disponibilizadas praticamente na rede privada de laboratórios. Eventualmente foram incluídos em
unidades de saúde do sistema público de
saúde, mas sem continuidade, por não terem
sido incorporados na lista de prioridades do
ministério.
Testes fenotípicos para o
diagnóstico de TB resistente
Por meio de estudo realizado em três laboratórios de micobacteriologia participantes
da Rede-TB, observou-se elevada concordância entre a performance do MGIT960 e
os três métodos considerados padrão-ouro
para o diagnóstico de TB resistente: a) Método de proporções; b) Bactec 460; e c) Razão da resistência (Giampaglia et al., 2007).
Em outro estudo, em colaboração com pesquisadores nas Honduras e na Universidade
John Hokpins, avaliamos a performance do
MODS em 854 doentes suspeitos de TB
pulmonar. A sensibilidade e a especificidade do MODS foi respectivamente de 96,5%
e de 92,6%, o tempo do diagnóstico inferior para MODS (6 dias; interquartil de 5 a
7), em comparação com LJ (21 dias; interquartil 17 a 25 dias) (Arias M et al., 2007)
Os estudos sobre a técnica MODS sugerem
que ela pode ser útil no diagnóstico da TB
sensível às drogas, pois, além de apresentar
sensibilidade e especificidade similares aos
meios de cultura padronizados, o diagnóstico é bem mais rápido. Entretanto, requer
técnicos de laboratório com elevado grau
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(MGIT960) kits. These technologies were
made available almost exclusively in privately owned laboratories. They were eventually incorporated into healthcare units of
the National Health System, but were discontinued as they were not considered
Ministry of Health priorities.
Phenotype techniques for
diagnosing resistant TB
A study performed in three TB Research
Network mycobacteriology laboratories
showed a strong agreement between
MGIT960 performance and the three gold
standard methods for diagnosing resistant
TB; the proportion method, Bactec 460
and the resistance ratio method (Giampaglia et al., 2007). Another study, performed
by researchers in the Honduras and at the
John Hopkins University, evaluated the performance of the microscopic-observation
drug-susceptibility (MODS) assay in 854
patients with suspected pulmonary TB.
MODS sensitivity and specificity was
96.5% and 92.6%, respectively, and time to
diagnosis was quicker for MODS (6 days; 5
– 7 interquartile), than Lowenstein Jensen
(LJ) medium (21 days; 17 – 25 interquartile), (Arias M et al., 2007).
Studies on the MODS assay suggest that
MODS may be of use in diagnosing drugsusceptible TB as in addition to its sensitivity and specificity – similar to that of standardised culture media – it offers a far
quicker diagnosis. It does, however, require
laboratory techniciens with a high degree of
proficiency and biosafety as it uses a liquid
medium in Petri dishes. Furthermore, we
evaluated the MODS performance for drug
resistant TB (DR-TB). In 351 DR-TB suspects, MODS had sensitivity and specificity
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de proficiência e de biossegurança, em razão do uso de meio líquido em placas de
Petri. Avaliámos, ainda, o desempenho do
MODS com a TB resistente (DR-TB). Entre 351 indivíduos suspeitos de DR-TB, o
MODS teve sensibilidade e especificidade
para isoniazida e rifampicina de 97,2%,
79%, 96,4% e 86,5%, respetivamente
(Mello FCQ, et al., 2007). Estes resultados
sugerem que o MODS poderá ser usado
para triagem no diagnóstico da DR-TB.
Tecnologias de biologia molecular
para diagnóstico de TB e TB
resistente
Há grande variabilidade da precisão dos
testes moleculares comercializados no diagnóstico da TB ativa, principalmente em
doentes imunossuprimidos, com menores
valores de sensibilidade em relação a especificidade (Palomino, 2009, Ling 2008,
Barnard, 2008). Além disso, em razão dos
resultados obtidos em estudos de performance, alguns testes moleculares foram
aprovados nos órgãos regulatórios de países
industrializados e comercializados para uso
em amostras respiratórias, ou seja, para a
investigação de TB pulmonar, em doentes
adultos, sem história prévia de tratamento
antiTB. Portanto, os testes moleculares comercializados no momento não deveriam
ser utilizados para o diagnóstico de outras
formas de TB, monitoramento do tratamento, e não substituem o exame de cultura para micobactérias.
Nos países desenvolvidos, os resultados positivos da microscopia direta têm um elevado valor indicativo positivo no diagnóstico
da tuberculose. Nestas regiões, os testes
moleculares podem ter maior impacto no
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for isoniazid and rifampicin respectively of
97.2%, 79%, and of 96.4%, 86.5% (Mello
FCQ, et al., 2007). These results suggest
that MODS might be used as screening
technique for DR-TB diagnosis.
Molecular biology techniques for
diagnosing TB and resistant TB
There is a great variation in the accuracy of
commercial molecular tests for the diagnosis of active TB, mainly in immunosuppressed patients, with lesser sensitivity than
specificity (Palomino, 2009, Ling 2008,
Barnard, 2008). Further, performance results from studies have led to some molecular techniques being approved by the regulatory bodies of industrialised countries and
sold for use in respiratory samples, that is to
investigate pulmonary TB in adult patients
with no prior history of TB treatment. The
molecular tests currently sold should not be
used to diagnose other forms of TB or monitor treatment and do not replace a mycobacterial culture exam.
In developing nations, positive direct microscopy results have a high positive predictive value for tuberculosis diagnosis. In
those regions, molecular tests may provide a
higher impact for diagnosis of smear negative TB cases (SNTB), especially among
HIV seropositive cases. However, little data
are available on the cost effectiveness and
clinical utility of PCR in SNTB, in a setting
with a high burden of TB/HIV co-infection
(van Cleef, 2005). We evaluated the performance of the PCR dot-blot (developed by
Brazilian scientists) in parallel with pretest
probability (clinical suspicion) in patients
suspected of having SNTB, in a prospective
study of 213 individuals with clinical and
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diagnóstico de casos de TB com esfregaço
negativo TB (ENTB), especialmente entre
seropositivos para VIH. Porém, há poucos
estudos sobre a relação custo-benefício e
utilidade clínica da PCR na ENTB, em regiões com elevada incidência de coinfeção
TB/VIH (van Cleef, 2005). Avaliámos o
desempenho da PCR dot-blot (desenvolvida
por cientistas brasileiros) paralelamente à
probabilidade pré-teste (suspeita clínica)
em doentes suspeitos de ENTB, num estudo prospectivo de 213 indivíduos com suspeita clínica e radiológica de ENTB num
hospital de referência de TB/VIH, no Sul
do Brasil. Não se observou diferença na
sensibilidade da PCR relativamente ao estatuto de VIH(Scherer LC, et al., 2007). No
mesmo grupo de estudo, comparámos duas
estratégias: o uso de microscopia de esfregaço com bacilos acid fast por coloração Ziehl-Neelsen (esfregaço AFB) e cultura; e esfregaço AFB e teste colorimétrico (PCR
dot-blot), e efectuámos uma análise de custos que incluiu serviços de saúde e custos
com os doentes. Os custos totais de screening foram 3,7 vezes para o esfregaço AFB
e cultura versus os custos para esfregaço
AFB e PCR dot-blot (US$ 5 651 560 versus
US$ 1 513 ,760). O esfregaço AFB e PCR
dot-blot mostrou melhor relação custo-benefício do que o esfregaço AFB e cultura
quando se considerou o custo de tratar todos os casos corretamente diagnosticados
(Scherer LC et al., 2009), Estes resultados
mostraram que os testes moleculares, mesmo nos países desenvolvidos, são bem aceites e desempenham um papel importante
no diagnóstico da ENTB, diminuindo não
só o tempo necessário para o diagnóstico,
mas ainda a morbilidade/mortalidade e a
transmissão do M. tuberculosis à comunida-
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radiological suspicion of SNTB in a TB/
HIV referral hospital in southern Brazil.
There was no difference in the sensitivity of
PCR in relation to HIV status (Scherer LC
et al., 2007).
In the same study group we compared two
strategies: use of acid fast bacilli smear microscopy by Ziehl-Neelsen staining (AFB
smear) plus culture and AFB smear plus
colorimetric test (PCR dot-blot) and performed a cost analysis that included health
services and patient costs. The total screening costs were 3.7 times higher for AFB
smear plus culture than for AFB smear plus
PCR dot-blot costs (USD 5,651.560 versus
USD 1,513. 760). AFB smear plus PCR
dot-blot was more cost-effective than AFB
smear plus culture, when the cost of treating
all correctly diagnosed cases was considered
(Scherer LC et al., 2009). These results show
that molecular tests, even in developing nations, are welcome and play an important
role in SNTB diagnosis, cutting diagnostic
delay, morbid-mortality and M. tuberculosis
transmission to the community, even in regions with a high rate of HIV infection.
Molecular tests for diagnosing
multi-drug resistant TB (MDR-TB)
Of the molecular tests sold worldwide for a
swift diagnosis of resistant TB, the following
are accurate: INNO-LIPA Rif.TB kit (Innogenetics, Zwijndrecht, Belgium), GenoType®
MDRTB and GenoType®MDRTB plus assays (Hain Lifescience, GMBH, Germany),
TB Research Network researchers undertook a study into the accuracy of selected
samples using the INNO-Lipa Rif.TB kit
and found a high level of agreement with
the reference tests in selected samples (Oli-
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de, mesmo em regiões com grande incidência de infecção por VIH.
Testes moleculares para o
diagnóstico de TB multirresistente
Entre os testes moleculares comercializados, a
nível mundial, para o diagnóstico rápido de
TB resistente que mostraram boa acurácia: o
kit INNO-LIPA Rif.TB (Innogenetics, Zwijndrecht, Bélgica), o ensaio de GenoType®
MDRTB e GenoType® MDRTBplus (Hain
Lifescience, GMBH, Alemanha), pesquisadores da Rede TB realizaram estudo de acurácia em amostras selecionadas ao utilizarem o
kit INNO-Lipa Rif.TB e observaram elevada
concordância com testes de referência em
amostras selecionadas. [Oliveira M, 2005).
Entretanto, até ao momento, não há estudos
publicados na literatura sobre a performance
de tais testes moleculares em condições de rotina, em países em desenvolvimento. Recentemente, avaliamos o desempenho da sequenciação de ADN em 38 (26%) doentes (99
amostras clínicas) com resultados discordantes entre MODS e métodos proporcionais.
Surpreendentemente, verificámos um elevado
índice de heterorresistência a RIF e/ou INH
(21,7%) e de superinfeção (28,9%), usando a
técnica spoligotyping, sendo a maioria Lam,
Haarlem, e T1 [Andrade et al., 2009]. Resultados semelhantes foram observados na África
do Sul e no Usbequistão. Van Rie et al. (2005)
avaliaram 186 doentes com TB, encontrando
superinfeção em 23% (14/62) de casos relatados e em 17% (21/1254) dos casos. Hofmann-Thiel et al. (Hofmann-Thiel S, et al., 2009)
avaliaram 35 casos de TB, encontrando superinfeção em 8,6%. Em ambos estudos, a maioria das superinfeções esteve relacionada com a
família M.tb Beijing. Mais recentemente,
R e v i s t a
veira M, 2005). That said, there are as yet
no published studies in the literature into
the performance of these molecular tests
under routine conditions in developing
countries.
We recently evaluated the performance of
DNA sequencing in 38 (26%) patients (99
clinical samples) with discordant results between MODS and the proportion method.
Surprisingly, we found high rate of heteroresistance to RIF and/or INH (21.7%) and
of superinfection (28.9%) using the spoligotyping technique, with the majority Lam,
Haarlem, and T1 (Andrade et al., 2009).
Similar results were seen in South Africa
and in Uzbekistan. Van Rie et al. (Van Rie
2005) evaluated 186 TB patients, finding
superinfection in 23% (14/62) retreatment
cases and in 17% (21/1254) of cases. Hofmann-Thiel et al. (Hofmann-Thiel S et al.
2009), evaluating 35 TB cases, found superinfection in 8.6%.
In both studies the majority of superinfection was related to the M.tb Beijing family.
More recently, Hillemann et al. (Hillemann
D, 2009), compared the accuracy of the
new MTBDRsl assay for extensively drugresistant TB (XDR-TB) which included detection of fluoroquinolone, amikacin-capreomycin and ethambutol resistance testing.
Among 106 selected clinical isolates, resistance to rifampicin and isoniazid was found
in 63 (59.4%) cases. Ofloxacin resistance
was found in 32 (30.2%) cases and heteroresistance was observed in 21.9% (7/32).
The high rates of heteroresistance and superinfection identified in those studies in
clinical samples collected from DR-TB suspects highlight the need to evaluate the impact of the use of line probe assays in DRTB suspects before its incorporation into
P o r t u g u e s a
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Hillemann et al. (Hillemann D, 2009), compararam a precisão do novo ensaio MTBDRsl para XDR-TB, que incluiu a deteção da
resistência aos testes com fluoroquinolona,
amikacin-capreomicina e etambutol. Entre
106 isolados clínicos selecionados, observaram resistência a rifampicina e a isoniazida em
63 (59,4%) dos casos. Observaram resistência
à ofloxacina em 32 (30,2%) e heterorresistência em 21,9% (7/32). Os elevados índices de heterorresistência e de superinfeção
identificados nesses estudos em amostras clínicas colhidas de indivíduos suspeitos de DR-TB acentuam a necessidade de avaliar o impacto do uso de ensaios line probe nestes
suspeitos, antes da sua incorporação na prática clínica, especialmente nos países em desenvolvimento.
Mais recentemente, a Rede TB, juntamente
com pesquisadores da Union International
Contra Tuberculosis, do Management Science
for Health, do Centro de Referência Prof. Hélio Fraga, da Fiocruz, e técnicos do Programa
Nacional de TB e do Departamento de Ciência e Tecnologia do Ministério da Saúde, elaboraram uma plataforma de protocolos de
pesquisa de viabilidade e impacto econômico
a serem realizados em diferentes regiões do
país, que inclui o uso do teste Xpert™ MTB/
Rif (Cepheid, Sunnyvale, CA, EUA) para o
diagnóstico de TB e TB resistente e o uso do
teste GenoType® MDRTBplus e MTBDRsl
assay (Hain Lifescience, GMBH, Alemanha)
para o manuseio do doentes suspeitos de TB-MDR e TB-XDR, respetivamente
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clinical practice, especially in developing
nations.
More recently, the TB Research Network
along with researchers from the International Union Against Tuberculosis of the
Management Science for Health of the
Centro de Referencia Prof Helio Fraga da
Fiocruz and technicians from the National
TB programme and the Ministry of Health’s
Department of Science Technology drew
up feasibility and economic impact research
protocols agreements. These are to be carried out in different regions of Brazil and
include use of the Xpert™ MTB / Rif (Cepheid, Sunnyvale, CA, USA) test for diagnosing TB and resistant TB and use of the
GenoType® MDRTB plus test and MTBDRsl assay (Hain Lifescience, GMBH,
Germany) for the management of patients
with suspected MDR-TB and XDR-TB,
respectively.
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Afranio Kritski
Bibliografia/Bibliography
Andrade MK, Dalcolmo M, Marsico AG, Mello FCQ,
Dorman S, Rossetti ML, Fonseca LS, de Oliveira MM,
Kritski A. 40th World Conference on Lung Health.
Cancun, México 3-7 December, 2009 (www.worldlunghealth.org).
Arias M, Mello FC, Pavon A, Marsico AG, Alvarado-Galvez C, Rosales S, Pessoa CL, Perez, Andrade MK,
Kritski AL, Fonseca LS, Chaisson RE, Kimerling ME,
Dorman SE. The microscopic observation drug susceptibility (MODS) assay for detection of tuberculosis and
tuberculosis drug resistance: results from a multi-center
study. Clin Infect Dis 2007;44(5):674-80. Epub 2007
Jan 22.
Barnard M, Albert H, Coetzee G, O’Brien R, Bosman
ME. Rapid molecular screening for multidrug-resistant
tuberculosis in a high-volume public health laboratory
in South Africa. Am J Resp Crit Care 2008, 177: 787-792.
de Oliveira MM, da Silva Rocha A, Cardoso Oelemann M, Gomes HM, Fonseca L, Werneck-Barreto
AM, Valim AM, Rossetti ML, Rossau R, Mijs W, Vanderborght B, Suffys P. Rapid detection of resistance
against rifampicin in isolates of Mycobacterium tuberculosis from Brazilian patients using a reverse-phase
hybridization assay. J Microbiol Methods 2003;
53(3):335-342.
Flores LL, PaiM, Colford JM, Riley LW. In-house nucleic
acid amplification tests for the detection of Mycobacterium
tuberculosis in sputum specimens: meta-analysis and metaregression. BMC Microbiol 2005; 5: 55.
Giampaglia MS, Martins MC, Vieira GBO, Vinhas SA,
da Silva Telles MA, Palaci M, Marsico AG, Hadad DJ,
Mello FCQ, Kritski A, Siddiqi S, Fonseca LS. Multi-center Evaluation of Automated Bactec MGIT 960
System for testing susceptibility of M. tuberculosis as
compared with BACTEC 460TB, proportion and resistance ratio methods in Southeast of Brazil. Int J Tuberc Lung Dis 2007; 11(9):986-991.
Global Plan against TB – WHO/STOP em 2006
(http://www.who.int/tb/strategy/stop_tb_strategy/en/).
Gounder C, Mello FCQ, Conde MB; Kritski A, Chaisson RE, Dorman S. Field evaluation of a rapid immunochromatographic test for tuberculosis. J Clin Microbiol
2002; 40(6):1477-1451.
Hofmann-Thiel S, van Ingen J, Feldmann K, Turaev L,
Uzakova GT, Murmusaeva G, van Soolingen D, Hoff-
R e v i s t a
mann H. Mechanisms of heteroresistance to isoniazid
and rifampin of Mycobacterium tuberculosis in Tashkent,
Uzbekistan. Eur Respir J 2009;33(2):368-374. Epub
2008 Oct 1.
Hillemann D, Rüsch-Gerdes S, Richter E. Feasibility of
the GenoType MTBDRsl assay for fluoroquinolone,
amikacin-capreomycin, and ethambutol resistance testing of Mycobacterium tuberculosis strains and clinical
specimens. J Clin Microbiol 2009; 47(6):1767-1772.
Epub 2009 Apr 22.
Kritski AL, Villa TS, Trajman A, Lapa e Silva, JR Medronho RA, Ruffino-Netto A. Two decades of research
on tuberculosis in Brazil: state of the art of scientific
publications. Rev Saude Publica 2007;41(Supl):9-14.
Ling DI, Flores LL, Riley LW, Pai M. Commercial
nucleic-acid amplification tests for diagnosis of pulmonary tuberculosis in respiratory specimens: meta-analysis
and meta-regression. PLoS One 2008; 2:e1536.
Mello FCQ, Arias M, Pavón A, Marsico AG, Alvarado-Gálvez C, Rosales |S, Pessoa CEC, Pérez M, Andrade
MK, Kritski AL, Fonseca LS, Chaisson RE, Kimerling
M, Dorman, SE. Clinical evaluation of the microscopic observation drug susceptibility (MODS) assay
for detection of Mycobacterium tuberculosis resistance to isoniazid or rifampin. J Clin Microbiol 2007;
45(10):3387-3389.
Palomino JC. Molecular detection, identification and
drug resistance detection in Mycobacterium tuberculosis.
Minireview. FEMS Immunol Med Microbiol 2009; 1-9.
Rede Brasileira de Pesquisa em Tuberculose – Rede Tb
(www.redetb.org)
Scherer LC, Sperhacke RD, Mello FCQ, C Jarczewski,
Cafrune P, Minghelli S, Osorio M, Rossetti ML, Kritski
AL. PCR colorimetric dot-blot assay and clinical pretest
probability for diagnosis of pulmonary tuberculosis in
smear-negative patients BMC Public Health 2007;
7(1):356.
Scherer LC, Sperhacke RD, Ruffino-Netto A, Rossetti,
MLR, Kritski AL. Cost-effectiveness analysis of the
PCR associated with Ziehl Neelsen smear examination
(ZN) for the rapid diagnosis of pulmonary tuberculosis
in subjects with and without HIV in a hospital setting.
BMC Public Health 2009 (in press).
Steingart KR, Henry M, Laal S, Hopewell PC, Ramsay
A, Menzies D, Cunningham J, Weldingh K, Pai M.
Commercial serological antibody detection tests for the
P o r t u g u e s a
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diagnosis of pulmonary tuberculosis: a systematic review. PLoS Med 2007 Jun; 4(6):e202. Erratum in: PLoS
Med 2007; 4(8):e254.
Van Cleeff M, Kivihya-Ndugga L, Githui W, Ng’ang’a
L, Kibuga D, Odhiambo J, Klatser P. Cost-effectiveness
of polymerase chain reaction versus Ziehl-Neelsen smear
microscopy for diagnosis of tuberculosis in Kenya. Int J
Tuberc Lung Dis 2005; 9(8):877-883.
Van Rie A, Victor TC, Richardson M, Johnson R, van
der Spuy GD, Murray EJ, Beyers N, Gey van Pittius
NC, van Helden PD, Warren RM. Reinfection and
mixed infection cause changing Mycobacterium tubercu-
S 76
R e v i s t a
P o r t u g u e s a
losis drug-resistance patterns. Am J Respir Crit Care
Med 2005; 172(5):636-642. Epub 2005 Jun 9.
WHO – 2007 recomendations – use of liquid médium
for TB and drug resistant TB diagnosis http://www.
who.int/tb/dots/laboratory/en/index.html.
WHO – 2008 recommendations – use of line molecular
probe assays for drug resistant TB diagnosis (http://
www.who.int/tb/features_archive/mdrtb_rapid_tests/
en/index.html\).
[WHO] World Health Organization 2009. Global Tuberculosis Control. WHO Report. Geneva, Switzerland.
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Ensaios clínicos de novas drogas e testes diagnósticos
em tuberculose: Desafios micobacteriológicos
Moisés Palaci1
Clinical trials of new tuberculosis drugs and diagnostic
tests: Mycobacteriological challenges
Desde que a Organização Mundial de Saúde
declarou a da tuberculose (TB) como emergência global em 1993, esta doença, historicamente importante e igualmente negligenciada, vem recebendo mais atenção por parte
das agências dedicadas ao seu controlo e ao
financiamento de pesquisas. Como consequência deste facto e do desenvolvimento
científico e tecnológico alcançado nos últimos anos, novos testes diagnósticos e compostos com potencial terapêuticos têm surgido e obrigado os fabricantes e sites de
pesquisa clínica a avaliá-los para serem registados em agências regulatórias. Em ensaios
clínicos para avaliação de novas drogas, a
principal metodologia utilizada é a atividade
bactericida precoce (early bactericidal activity
– EBA) descrita por Mitchson1, que consiste
em quantificar a carga bacilar (CFU) presente em amostras de escarro recolhidas por um
período de 12 horas durante os primeiros 7
dias de tratamento. Tal metodologia é baseada no facto de a redução do número de CFU
Since the World Health Organization’s 1993
declaration that tuberculosis (TB) was a
worldwide emergency, this historically important and equally neglected disease has
received more attention from agencies dedicated to its controlling and financing research. As a consequence of this and of recent scientific and technological advances,
new diagnostic tests and compounds with
therapeutic potential have emerged. These
have obliged manufacturers and clinical research sites to evaluate and register them in
regulatory bodies. The main methodology
used in clinical trials to evaluate new drugs
is early bactericidal activity (EBA), described
by Mitchison1, which consists of quantifying the colony forming unit (CFU) in sputum samples collected over a 12-h period
for the first 7 days of treatment.
This methodology is based on the reduction
in the number of CFU over the first two days
being statistically related to the efficacy of the
treatment regimens instituted, making it a
Núcleo de Doenças Infecciosas da Universidade Federal do Espírito Santo, Brasil/Infectious Diseases Unit, Universidade Federal do Espírito Santo, Brazil
e-mail: [email protected]
1
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desafios micobacteriológicos
Moisés Palaci
durante os primeiros dois dias estar estatisticamente relacionada com a eficácia dos esquemas terapêuticos empregues e representa
um parâmetro clínico de redução da infecciosidade2,3. Os estudos de EBA são realizados para comparar a atividade de várias doses
de uma droga, diferentes drogas dentro da
mesma classe e diferentes classes de drogas4.
A realização deste tipo de ensaio clínico requer elevado investimento financeiro, profissionais qualificados em boas práticas clínicas
e laboratoriais e um sério comprometimento
e compreensão dos doentes. Diante destes
factos, o laboratório de micobacteriologia assume grande responsabilidade e enfrenta
muitos desafios para cumprir com êxito o seu
papel nos ensaios clínicos. A seguir são descritos resumidamente alguns dos obstáculos e
estudos realizados para superá-los.
Recolha de amostras 12 a 16
horas
Um pool de escarro recolhido nestas condições requer internação do doente, o seu distanciamento da família, custo financeiro de
internação, maior risco de contaminação da
cultura e longo período de tempo para monitoramento. Com o objetivo de verificar se
o pool de escarro recolhido durante 5 horas
pela manhã poderia conter uma carga bacilar
semelhante ao pool recolhido num período
de 12 horas, Nascimento et al (estudo em
fase final), ao comparar a carga bacilar em
amostras de escarro de doentes com tuberculose pulmonar, recolhidas por períodos de 5
e 12 horas, não observaram diferenças estatísticas significativas entre os dois grupos
avaliados (6,88 log10 CFU/ml, e 6,95 log10
CFU/ml, respectivamente) e demonstraram
assim que uma recolha monitorizada de es-
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clinical parameter of reduced infectiousness2,3.
EBA studies are performed to compare the
activity of various doses of a drug, different
drugs of the same class and different classes of
drugs4. This type of clinical trial implies heavy
financial investment, professionals qualified
in good clinical and laboratory practices and a
serious commitment to and understanding of
patients. Accordingly, the mycobacteriology
laboratory faces heavy responsibility and a series of challenges to be able to play a successful role in clinical trials. Some of the obstacles
encountered and studies undertaken to overcome them are summarised below.
Sample collection over 12 and 16 hrs
Collecting a pool of sputum under these conditions requires the patient being admitted to the
hospital, which involves separation of the patient from his/her family, the costs of hospitalization stay, an increased risk of culture contamination and increased length of patient
monitoring. To see if a pool of sputum collected
over 5-h in the morning contained similar CFU
to a pool collected over a 12-h period, Nascimento et al. (study currently in final stage) when
comparing CFU in the sputum of patients with
pulmonary TB collected over 5-h and 12-h periods, did not find any statistically significant
differences in the two groups evaluated (6.88
log10 CFU/ml vs. 6.95 log10 CFU/ml, respectively) and, thus, showed that sputum collected
over 5-h could be used in future clinical trials.
New markers of bacteriological
clearance in response to antituberculosis therapy
Microbiological parameters can easily be assessed in spontaneously expectorated spu-
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carro durante 5 horas poderá ser utilizada
em futuros ensaios clínicos.
Novos marcadores de depuração
bacteriológica em resposta à
terapia antituberculose
Os parâmetros microbiológicos podem ser
facilmente avaliados na expectoração espontânea, mas a cultura quantitativa é demorada
e trabalhosa. O marcador ideal mediria eventos no decurso do início do tratamento e seria rigoroso, independentemente da ação ou
do regime a ser testado. Recentemente,
Liwen et al.5 avaliaram e reportaram níveis
de mRNA por RT-PCR quantitativa em
amostras recolhidas de doentes com TB sob
monoterapia, num anterior estudo de atividade bactericida em fluoroquinolonas, e nos
que estavam sob um regime-padrão com
base em rifampina num ensaio de IL-2. Os
autores demonstraram que o mensageiro
RNA para a isocitrato liase da enzima do ciclo glioxilato teve taxas de declínio semelhantes em doentes a receberem monoterapia
com isoniazida, gatifloxicina, levofloxacina e
moxifloxacina. A isocitrato liase (icl) mRNA
esteve altamente relacionada com as unidades que formavam colónias na expectoração
antes da terapia e durante 7 dias de monoterapia em todos os grupos de tratamento.
Detectou-se icl mRNA na expectoração de
doentes com cultura positiva de TB em regime de tratamento com base em rifampina
durante 2 meses. Além disso, também demonstraram que a proteína de ligação à fibronectina (fbpB) mRNA diminuiu 0,47
log10 moléculas/ml/d desde o início até ao
segundo dia, o único mRNA que se relacionou com a atividade bactericida precoce da
monoterapia com isoniazida. Em conclusão,
R e v i s t a
tum, but quantitative culture is time consuming and labour intensive. The ideal
marker would measure events early during
treatment and be accurate regardless of the
drug action or regimen being tested. Recently, Liwen et al.5 assessed and reported
mRNA levels by quantitative RT-PCR in
sputum specimens from TB patients receiving monotherapy in an early bactericidal
activity study of fluoroquinolones and in
those receiving a standard rifampin-based
regimen in an IL-2 trial. These authors
demonstrated that messenger RNA for the
glyoxylate cycle enzyme isocitrate lyase declined at similar rates in patients receiving
isoniazid, gatifloxacin, levofloxacin, and
moxifloxacin monotherapy. Isocitrate lyase
(icl) mRNA correlated highly with CFU in
sputum prior to therapy and during 7 days
of monotherapy in all treatment arms. icl
mRNA was detectable in sputum of culturepositive TB patients receiving a rifampinbased regimen for 2 months. In addition,
they also demonstrated that fibronectinbinding protein (fbpB) mRNA decreased
0.47 log10 molecules/ml/d from baseline to
day 2, the only mRNA correlating with the
early bactericidal activity of isoniazid monotherapy. In conclusion, icl and fbpB mRNAs
are reliable markers of M. tuberculosis viability, however, both of these uses will require
larger longitudinal studies to validate the reliability of icl mRNA as a surrogate marker
of response to drug therapy for TB.
Lower rates of contamination
of mycobacterial culture
In clinical trials to evaluate treatment regimes that require patient follow-up for up
to 2 years after cure, and in studies to eva-
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Ensaios clínicos de novas drogas e testes diagnósticos em tuberculose:
desafios micobacteriológicos
Moisés Palaci
icl e fbpB mRNAs são marcadores fiáveis de
viabilidade do M. tuberculosis; no entanto,
estas duas utilizações carecem de estudos
longitudinais mais extensos que validem a
fiabilidade de icl mRNA como marcador
substituto de resposta à terapia farmacológica para TB.
Diminuição das taxas de
contaminação de culturas
de micobactérias
Em ensaios clínicos para avaliação de regimes
terapêuticos que exigem o monitoramento do
doente em até dois anos após a sua cura, e em
estudos para avaliação de novos testes diagnósticos, culturas de escarro contaminadas podem
causar prejuízos económicos, eliminação de
dados da análise estatística (time points onde
ocorreram contaminação) ou até a exclusão do
doente do estudo. Diante destas situações problemáticas, Peres et al. (artigo submetido a publicação) realizaram um ensaio clínico pragmático para avaliar a eficácia de métodos de
antissepsia intrabucal na redução da taxa de
contaminação de culturas de doentes suspeitos
de tuberculose. Os autores constataram que,
dentre os três procedimentos de higienização
bucais utilizados isoladamente (somente água,
digluconato de clorexidina e cloreto de cetilpiridínio), apenas o realizado como digluconato
de clorexidina permitiu uma redução significativa da taxa de contaminação das culturas,
sobretudo no meio líquido MGIT (Becton
Dickinson). Verificaram ainda que o uso de
uma concentração maior do antimicrobiano
PANTA (PANTA 2x) nas amostras-controlo
dos doentes reduziu significativamente a população de organismos contaminantes no
meio MGIT, sem, contudo, reduzir a taxa de
deteção do M. tuberculosis.
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luate new diagnostic tests, contaminated
sputum cultures could result in high costs,
elimination of data from the statistical analysis (time points where contamination occurred) or even exclusion of the patient
from the study. In the face of these difficulties, Peres et al. (article submitted for publication) performed a pragmatic clinical trial
to evaluate the efficacy of methods of intraoral antisepsis in reducing the contamination rate of cultures from patients suspected
of TB. The authors found that of the three
intra-oral hygiene procedures employed
separately (water only; chlorhexidine digluconate and cetylpyridinium chloride), only
the one using chlorhexidine digluconate resulted in a significant reduction of the culture contamination rate, particularly in
MGIT liquid medium (Becton Dickinson).
They also found that using a higher concentration of PANTA antimicrobial solution
(PANTA 2x) in patients’ control samples,
significantly reduced the population of contaminating organisms in the MGIT liquid
medium, without, however, lowering the
rate of M. tuberculosis detection.
Sputum sample division
Performing clinical trials to evaluate new diagnostic methods and treatments often requires sputum sample division for comparative analysis. In this case, the samples must
be divided equally, maintaining a similar
CFU in each part. The use of a mucolitic
agent is recommended for this, but there are
no studies proving its efficacy or that of
other procedures for sample division. Morais et al. (unpublished data) used a quantitative culture technique to verify if CFU
was similar in divided samples after the di-
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Moisés Palaci
Divisão em amostras de escarro
A realização de ensaios clínicos para avaliação
de novos métodos diagnósticos e terapêuticos
frequentemente requer a divisão da amostra de
escarro para análise comparativa. Neste caso é
necessário que a divisão da amostra ocorra de
maneira equitativa, mantendo a carga bacilar
semelhante entre as partes. Recomenda-se o
uso de um agente mucolítico para esta finalidade; contudo, por não se dispor de estudos que
comprovem a eficiência deste ou de outros
procedimentos de divisão de amostras, Morais
et al (dados ainda não publicados), utilizando
técnica de cultura quantitativa, propuseram-se
verificar se após a digestão de amostras escarro
por procedimento químico (N-acetil-L-cisteina
50 mg/ml- 10% do volume da amostra durante 15 minutos), ou mecânico (agitação com
pérolas de vidro), a carga bacilar seria semelhante nas amostras divididas. Ao comparar os
resultados dos grupos entre si não observaram
diferenças estatísticas significativas. Para o primeiro grupo que fez uso imediato do NALC
foi observada na aliquota I uma média de 5,66
(±0,92) log10, enquanto na aliquota II 5,65
(±0,94) log10 UFC/ml. Para o segundo grupo,
processado com pérolas de vidro, foi observado
uma média de 5,53 (±0,91) e 5,49 (±0,94)
log10 UFC/ml em cada aliquota, respetivamente, demonstrando assim que tanto o procedimento químico quanto o físico podem ser utilizados com igual eficiência para divisão de
amostras de escarro.
Como destacado anteriormente, os ensaios
clínicos terapêuticos em tuberculose são
complexos e onerosos. Poucos sites no mundo possuem os requisitos e equipas necessárias à sua realização. Se considerarmos o modesto investimento feito nesta área,
importantes avanços ocorreram nos últimos
anos. Entretanto, muitos outros desafios
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gestion of sputum samples by chemical procedure (N-Acetyl-L-Cysteine 50mg/ml10% of the sample volume for 15 minutes),
or mechanically (agitation with glass beads).
No statistically significant differences were
seen when the results of the groups were
compared. In the first group (NALC), aliquot I had mean 5.66 (±0.92) log10 and aliquot II mean 5.65 (±0.94) log10 CFU/ml. In
the second group (glass beads) the means
were 5.53 (±0.91) and 5.49 (±0.94) log10CFU/ml for each aliquot, respectively, thus
showing that both chemical and physical
procedures can be used with equal efficacy
for division of sputum samples.
As mentioned above, clinical treatment trials
for TB are complex and expensive. Few sites
worldwide have the necessary requirements
and teams to perform them. If we consider
the modest investment made in this area,
important advances have been seen in recent years. There are, however, many challenges which have yet to be met. One such
challenge is the validation of the results described above and the development of simple tests able to determine sensitivity to
drugs for latent bacteria and bacterial viability over the long course of TB treatment.
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Moisés Palaci
ainda necessitam de ser superados. Dentre os
quais a validação dos resultados descritos e o
desenvolvimento de testes simples e capazes
de determinar a sensibilidade a drogas de
bactérias em fase de latência e a viabilidade
bacteriana durante o longo período de tratamento da tuberculose.
Bibliografia/Bibliography
1. Mitchison DA. Assessment of new sterilizing drugs
for treating pulmonary tuberculosis by culture at 2
months. Am Rev Respir Dis 1993; 147: 1062-1063.
2. Jindani A, Aber VR, Edwards EA, Mitchison DA.
The early bactericidal activity of drugs in patients with
pulmonary tuberculosis. Am Rev Respir Dis 1980;
121:939-949.
3. Kennedy N, Fox R, Kisyombe GM, Saruni AOS,
Uiso LO, Ramsay ARC, Ngowi FI, Gillespie S. Early
bactericidal and sterilizing activities of ciprofloxacin in
pulmonary tuberculosis. Am Rev Respir Dis 1993;
148:1547-1551.
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4. Johnson JL, Hadad DJ, Boom WH, Daley CL, Peloquin CA, Eisenach KD, Jankus DD, Debanne SM,
Charlebois ED, Maciel E, Palaci M, Dietze R. Early and
extended early bactericidal activity of levofloxacin, gatifloxacin and moxifloxacin in pulmonary tuberculosis.
Int J Tuberc Lung Dis 2006; 10:605-612.
5. Li L, Mahan CS, Palaci M, Horter L, Loeffelholz L,
Johnson JL, Dietze R, Debanne SM, Joloba ML, Okwera A, Boom WH, Eisenach KD. Sputum Mycobacterium tuberculosis mRNA as a marker of bacteriologic
clearance in response to anti-tuberculosis therapy. J Clin
Microbiol 2009; 18 [Epub ahead of print].
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Avaliação das moléculas com atividade antiTB das
plantas do cerrado brasileiro
Karina de Prince1
Fernando R Pavan1
Daisy N Sato2
Wagner Villegas5
Sergio RA Leite5
Clarice QF Leite1*
Screening of molecules with anti-TB activity from the
brazilian cerrado plants
Introdução
A tuberculose (TB) ainda é, em todo o mundo, a doença infeciosa mais frequente e importante, causando morbilidade e morte.
Um terço da população mundial está infetada com Mycobacterium tuberculosis (MTB) e
aproximadamente dois milhões de mortes
são atribuídas à TB anualmente1. Entre todos os países do continente americano, o
Brasil tem o segundo maior índice de morbilidade e de morte por TB, com uma prevalência de 62/100 0002. O ressurgimento global da TB e o rápido aparecimento da
tuberculose multirresistente (MDR-TB) sublinha a importância do desenvolvimento de
novos fármacos antituberculose3.
Na busca de novos compostos a partir de
plantas, as árvores forneceram muitos fármacos no passado e permanecem uma importante fonte de novos compostos. Recentemente
os produtos naturais têm sido alvo de muita
atenção como potenciais agentes antiTB4-6.
Introduction
Across the world, tuberculosis (TB) remains
the most frequent and important infectious
disease causing morbidity and death. A
third of the world’s population is infected
with Mycobacterium tuberculosis (MTB),
and approximately two million deaths are
attributable to TB annually1. Among all the
countries in the Americas, Brazil reports the
second-highest TB mortality and morbidity,
with a prevalence of 62 in /100 0002. The
global resurgence of TB and the rapid emergence of MDR-TB, underscore the importance of the development of new antituberculous drugs3.
Concerning the search for new compounds
from Plants, trees have provided many drugs
in the past, and remain a rich source of novel compounds. Natural products have recently received a lot of attention as potential
anti-TB agents4-6. In Brazil, there is a traditional knowledge of how to use native plants
Faculdade de Ciências Farmacêuticas, UNESP, CEP 14801-902, Araraquara (SP), Brasil
Instituto Adolfo Lutz, Ribeirão Preto, CEP 14085-410, Ribeirão Preto (SP), Brasil
3 Departamento de Química, Universidade Federal de São Carlos, CP 676, CEP 13565-905, São Carlos (SP), Brasil
4 Instituto de Química de São Carlos, Universidade de São Paulo, CP 780, 13560-970, São Carlos – SP, Brasil
5 Instituto de Química de Araraquara, UNESP, CEP 14801-902, Araraquara – SP, Brasil.
e-mail: [email protected]
1
2
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No Brasil, existe um conhecimento tradicional de como utilizar plantas nativas no tratamento de várias doenças, porque muitas comunidades não têm acesso a medicamentos e
usam essas plantas para se tratarem7.
Embora tenham sido encontradas várias
centenas de produtos naturais de plantas
com atividade antimicobacteriana, nenhuma delas atingiu a fase de desenvolvimento
para fármaco devido a dificuldades, como a
escassez de compostos, a elevada complexidade estrutural e a falta de estudos de acompanhamento de indícios promissores.
Objetivo
Neste contexto, iniciámos o projeto integrando análises químicas e testes de actividade antiTB de plantas, especialmente de
espécies que compõem o cerrado brasileiro,
bioma do tipo savana que predomina no
planalto central brasileiro e que inclui milhares de espécies vasculares nativas agrupadas em centenas de famílias. Muitas destas
plantas são habitualmente usadas como remédios naturais pela população local para
tratar doenças várias.
Material e métodos
Os especímenes de plantas do cerrado foram
colhidas nos estados de Tocantins (aproximadamente 11° S, 48° O) e Mato Grosso do Sul
(aproximadamente 21° S, 56° O). Para a separação fitoquímica, usámos técnicas cromatográficas, essencialmente adequadas para separação de substâncias polares (GPC, XAD2,
DCCC, HSCC, HPLC, etc.). Para determinar a estrutura dos compostos isolados usámos
principalmente métodos espectrofotométricos, como NMR, IR, UV e MS. Avaliámos a
atividade dos extratos, frações enriquecidas e
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to treat several diseases because many communities don’t have access to synthetic medicines and use those plants in treatments7.
Although several hundred natural plant
products with antimycobacterial activity
have been found, none of them have
moved towards drug development, because of difficulties, such as very low compound availability, high structural complexity and lack of follow-up studies of
promising leads
Objective
In this context, we started the project integrating chemical analysis and anti-TB
activity tests of plants, especially species
that compose the “Brazilian Cerrado”, a
savannah like biome that predominates in
the center-west region of the country. It
includes more than several thousands native vascular plant species, grouped in
hundreds of families. Many of these plants
are commonly used as natural remedies
by people living in this area to treat various illnesses.
Material and methods
The cerrado plant specimens were collected
in the states of Tocantins (at nearly 11° S by
48° W) and Mato Grosso do Sul (approximately 21° S by 56° W). To perform the
phytochemical separation, we used chromatographic techniques, mainly suitable for
polar substances (GPC, XAD2, DCCC,
HSCC, HPLC, etc). To determine the
structure of the isolated compounds we
used mainly spectrophotometric methods
such as NMR, IR, UV and MS. To evaluate
the activity of the extracts, enriched frac-
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substâncias puras contra o M. tuberculosis por
resazurin microtiter assay (REMA), de acordo
com Palomino et al. (2002) e M. tuberculosis
H37Rv ATCC 272948.
Resultados e discussão
Sessenta e cinco extratos de 37 plantas distribuídas por 18 famílias foram testadas contra
M. tuberculosis. Vinte e seis por cento dos extratos avaliados demonstraram actividade
promissora, nomeadamente concentração
inibitória mínima (CIM) ≤ 125 μg/m, 13
(76%) deles extratos de clorofórmio e 4
(24%) de metanol. Esses extratos foram selecionados para fracionamento guiado por
atividade e avaliação detalhada das propriedades antituberculosas, sendo a sua composição química analisada (Quadro I).
Do extrato de clorofórmio de B. fagifolia, a
mistura de lupeol, α-amirina e β-amirina revelaram CIM mais baixa (31,25 μg/mL) do
que lupeol, α-amirina ou β-amirina isolados, cujos valores CIM foram ≥ 62,5 μg/mL.
A mistura de lupeol e acetatos de α-amirina
e β-amirina mostraram o mesmo valor CIM
(31,25 μg/mL), sugerindo que a acetilação
de α-amirina e β-amirina não influencia a
sua atividade9. Observou-se a mesma situação nas frações enriquecidas de extrato de
clorofórmio de B. crassa. As CIM de 31,25
para a mistura de α-e β-amirina e de 62,5
μg/mL, o dobro do valor para o acetato puro
de α-amirina, confirma o efeito sinergístico
entre os componentes destas misturas contra
M. tuberculosi10.
Para C. adamantium, a 5,7-dihidroxi-6,8-di
-C-metilflavanona (A) isolada mostrou uma
CIM superior a 250 e 2’, 4’-dihidroxi-3’,5’dimetil-6’-metoxicalcona (B) uma CIM de
62,5 μg/mL, enquanto as suas misturas
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tions and pure substances against M. tuberculosis we used the Resazurin Microtiter Assay (REMA) according Palomino et al.
(2002) and the M. tuberculosis H37Rv ATCC
27294 strain8.
Results and discussion
sixty five extracts from 37 plants, distributed in 18 families have been tested against
M. tuberculosis. Out of all the extracts assayed, 26% showed promising activity,
namely MICs below or equal to 125 μg/mL,
13 (76%) of these coming from chloroform
extract and 4 (24%) from methanol extract.
These extracts were selected for activity
guided-fractionation and detailed evaluation of the anti-tuberculosis properties, and
their chemical composition was analysed
and showed in Table I.
From the B. fagifolia chloroform extract, the
mixture of lupeol, α-amyrin and β-amyrin
displayed a lower MIC (31.25 μg/mL) than
isolated lupeol, α-amyrin or β-amyrin
whose MIC value were higher than or equal
to 62.5 μg/mL. The mixture containing lupeol and α-amyrin and β-amyrin acetates
showed the same MIC value of 31.25 μg/
mL, suggesting that the acetylation of
α-amyrin and β-amyrin does not influence
their activity9. The same situation was found
in the enriched fractions from the B. crassa
chloroform extract. MICs of 31.25 for the
mixture of α-and β-amyrin and 62.5 μg/
mL, double the value for the pure acetate of
α-amyrin, confirms the synergistic effect
among the components of these mixture
against M. tuberculosis10.
For C. adamantium, the isolated 5,7-dihydroxy-6,8-di-C-methylflavanone (A) showed
higher MIC of 250 and 2’,4’-dihydroxy-3’,5’-
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Quadro I – Valores CIM das frações enriquecidas e compostos testados contra M. tuberculosis
Compostos
Fração enriquecida / compostos de clorofórmio extrato de B. fagifolia
Mistura de lupeol, α-e β-amirina
Mistura de lupeol, acetatos de α- e β-amirina
α-amirina
α-amirina acetato
Dotriacontano
Ácido básico
Fração enriquecida / compostos de extrato de clorofórmio de B. crassa
Mistura de α-e β- amirina
Mistura de acetato de α-e β- amirina
Mistura de ácidos ursólico e oleanólico
Compostos de extrato de clorofórmio de C. adamantium
5,7-dihidroxi-6,8-di-C-metilflavanona (A)
2’,4’-dihidroxi-3’,5’-dimetil-6’-metoxicalcona (B)
Mistura A + B (2:8)
Mistura A + B (3:7)
Mistura A + B (7:3)
Mistura A + B (8:2)
Compostos de extrato de clorofórmio de Qualea parviflora
Lupeol
Lupenona
Ácido betulínico
Ácido 3 epibetulínico
Friedelina
β sitosterol
CIM (μg/mL)
31,25
31,25
62,5
62,5
62,5
2,5
31,25
31,25
62,5
250
62,5
7,8
15,6
31,25
62,5
62,5
125
31,25
62,5
125
125
Table I – MIC values of enriched fractions and compounds tested against M. tuberculosis
Compounds
Enriched fraction/compounds from chloroform extract of B. fagifolia
Mixture of lupeol, α-and β-amyrin
Mixture of lupeol, acetates of α- and β-amyrin
α-amyrin
α-amyrin acetate
Dotriacontane
Bassic acid
Enriched fraction/compounds from chloroform extract of B. crassa
Mixture of α-and β-amyrin
Mixture of α-and β-amyrin acetate
Mixture of ursolic and oleanolic acid
Compounds from chloroform extract of C. adamantium
5,7-dihydroxy-6,8-di-C-methylflavanone (A)
2’,4’-dihydroxy-3’,5’-dimethyl-6’-methoxychalcone (B)
Mixture A + B (2:8)
Mixture A + B (3:7)
Mixture A + B (7:3)
Mixture A + B (8:2)
Compounds from chloroform extract of Qualea parviflora
Lupeol
Lupenona
Betulinic acid
3 epi betulinic acid
Friedelin
β sitosterol
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MIC (μg/mL)
31.25
31.25
62.5
62.5
62.5
2.5
31.25
31.25
62.5
250
62.5
7.8
15.6
31.25
62.5
62.5
125
31.25
62.5
125
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(A+B), em vários rácios, mostrou vários
CIM, entre 62,5 μg/mL para o rácio 8:2
(A+B) e a atividade muito melhor de 7,8
μg/mL para o rácio 2:8. Há aqui claro sinergismo entre os compostos A e B quando
misturados, e este sinergismo depende fortemente do seu rácio de concentração5. Nos
compostos isolados de Qualea parviflora,
apesar da grande semelhança estrutural de
lupeol e lupenona, observou-se uma redução duas vezes maior da atividade antiTB de
lupeol, que se deveu à substituição de
β-hidroxilato C3 no lupeol para cetona na
lupenona. Do mesmo modo, também se
observou uma redução duas vezes maior da
atividade antiTB entre os ácidos betulínico
(CIM 31,2 μg/mL) e epibetulínico (62,5μg/
mL), devido à epimerização no ácido epibetulínico de β-hidroxil C3. Esses resultados
corroboram os do trabalho de Cantrells et
al. (2001), em que nos triterpenos, β-hidroxil
em C3 é importante para a atividade antiTB4. A melhor MIC observada foi no triterpeno do ácido básico de B. fagifolia, que
mostrou forte atividade antitubercular, com
valores CIM de 2,5 μg/mL9. Este valor de
concentração inibitória é comparável aos
dos fármacos antiTB de primeira linha,
como etambutol (1-5 μg/mL) e estreptomicina (2-8 μg/mL), e melhor do que a pirazinamida (20-100μg/mL)11.
dimethyl-6’-methoxychalcone (B) a MIC of
62.5 μg/mL, while their mixtures (A+B), in
several ratios, showed various MICs, ranging
from 62.5 μg/mL for the ratio 8:2 (A+B)
down to the much higher activity of 7.8 μg/
mL, for the ratio 2:8. Here there is a clear synergism between compounds A and B when
mixed and this synergism depends strongly
on their concentration ratio5. For compounds
isolated from Qualea parviflora, despite the
great structural similarity of lupeol and lupenone a two-fold reduction of anti-TB activity
was observed with lupeol. This was due to the
substitution of β hydroxylat C3 in lupeol to
the ketone on lupenone. Similarly a two fold
reduction of anti-TB activity was observed
between betulinic (MIC 31,2 μg/mL) and
epi-betulinic acid (62,5μg/mL), due to the
epimerization in epi-betulinic acid of the β
hydroxyl C3. Those results corroborate the
report of Cantrells et al. (2001) in that within
triterpenes, the β hydroxyl on C3 is important for anti-TB activity4. The best MIC
found was for the triterpene bassic acid from
B. fagifolia, this showed strong antitubercular
activity, with MIC values of 2.5 μg/mL9. This
inhibitory concentration value is comparable
to those of first-line tuberculosis drugs, such
as ethambutol (1-5 μg/mL) and streptomycin
(2-8 μg/mL), and better than pyrazinamide
(20-100μg/mL)11.
Conclusão
Os resultados indicam que as plantas do
cerrado podem proporcionar frações e compostos com atividade antituberculose promissora.
Conclusion
The results indicated that plants of the “cerrado” can provide fractions and compounds
with promising anti – tuberculosis activity.
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Karina de Prince, Fernando R Pavan, Daisy N Sato, Wagner Villegas, Sergio RA Leite, Clarice QF Leite
Bibliografia/Bibliography
1. Global Alliance for TB Drug Development, www.
tballiance.org, accessed June 1, 2009.
2. Malaspina AC, Cavalcanti HR, Leite CQF, Machado
SM, Viana BH, Silva RM, Hage EF, Figueiredo WM,
Marques E, Ferrazoli L, Arbex M, Lessi M, Fonseca LS,
Rigouts L, Saad MH. Jpn J Infect Dis (2008); 231-233.
3. Lourenço MCS, Ferreira ML, Souza MV, Peralta MA,
Vasconcelos TR, Henriques MGO. Eur J Med Chem
2008; 43:1344-1347.
4. Cantrell CL, Franzblau SG, Fischer NH. Planta Med
2001; 67:1-10.
5. Pavan FR, Leite CQF, Coelho RG, Coutinho ID,
Honda NK, Cardoso CAL, Vilegas W, Leite SRA, Sato
DN. Quim Nova 2009; 32:1222-1226.
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6. Honda NK, Pavan FR, Coelho RG, Leite SRA,
Micheletti AC, Lopes TIB, Misutsi MY, Beatriz A, Brum
RL, Leite CQF. Phytomedicine (2009) in press.
7. Almeida SP, Proença CEB, Sano SM, Ribeiro Cerrado
JF. Espécies vegetais úteis. In: Sano SM, Almeida SP
(Eds.). Planaltina, Distrito Federal, Brazil, 38-39.
8. Palomino JC, Martin A, Camacho M, Guerra H,
Swings J, Portaels F. Antimicrob Agents Chemoter
2002; 2720-2722.
9. Higuchi CT, Sannomiya M, Pavan FR, Leite SRA,
Sato DN, Franzblau SG, Sacramento LVS, Vilegas W,
Leite CQF. eCAM (2008) doi:10.1093/ecam/nen077.
10. Higuchi CT, Pavan FR, Leite CQF, Sannomiya M,
Vilegas W, Leite SRA, Sacramento LVS, Sato DN.
Quim Nova 2008; 31:1719-1721.
11. Collins L, Franzblau SG. Antimicrob Agent Chemother 1997; 1004-1009.
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Novas ferramentas de fácil utilização para genotipagem
padronizada e com qualidade controlada de estirpes do
complexo Mycobacterium tuberculosis
Caroline Allix-Béguec3
Christine Hubans3
Stéphanie Ferreira3
Philip Supply1,2,3
New, easy-to-use tools for standardised and qualitycontrolled genotyping of Mycobacterium tuberculosis
complex strains
A tipagem harmonizada e fiável de bactérias
patogénicas permite a fácil identificação de
clones em circulação local ou internacionalmente, o que é essencial para bons serviços de
vigilância epidemiológica e de controlo de
doenças. Isto aplica-se particularmente a
doenças como a tuberculose (TB), com incidência mundial e emergência global de estirpes resistentes a fármacos. Além do mais, a
tipagem pode ajudar nas decisões terapêuticas, por exemplo, em casos de suspeição de
erros ou contaminações laboratoriais. A tipagem pela metodologia MIRU-VNTR (mycobacterial interspersed repetitive unit-variable
number of tandem repeat) tornou-se importante para a genotipagem rápida e de alta resolução dos isolados do complexo Mycobacterium tuberculosis. Um consórcio internacional
que inclui 10 laboratórios europeus e americanos1 propôs um sistema baseado em 24 loci
para padronização internacional. Estudos de
população mostraram que a tipagem MIRU-VNTR padrão tem um valor de previsão
Harmonized and reliable typing of pathogenic bacteria permits easy identification
of locally or internationally circulating
clones, which is essential for optimal epidemiological surveillance and disease control. This is especially true for diseases such
as tuberculosis (TB), with worldwide distribution and global emergence of drugresistant strains. In addition, typing can
guide therapeutic decisions, for instance in
case of suspected laboratory errors or contaminations. Mycobacterial interspersed
repetitive unit-variable number of tandem
repeat (MIRU-VNTR) typing has become
a major method for fast and high-resolution genotyping of Mycobacterium tuberculosis complex isolates. A system based on
24 loci has been proposed for international
standardization by an international consortium including 10 European and American laboratories1. In population-based
studies, standard MIRU-VNTR typing
was shown to have an equal to slightly bet-
INSERM U629
Institut Pasteur de Lille, Lille
3 Genoscreen, Lille, France
e-mail: [email protected]
1
2
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Caroline Allix-Béguec, Christine Hubans, Stéphanie Ferreira, Philip Supply
igual a ligeiramente melhor do que o anterior
padrão-ouro, IS6110 RFLP, no estudo da
transmissão de TB, em ambientes com características epidemiológicas representativas das
de muitos países desenvolvidos. A interrogação baseada na PCR de até 24 marcadores
independentes e bem calibrados facilita a rápida e fiável elucidação dos mecanismos moleculares de situações complexas que envolvam potenciais surtos, infeções mistas ou
reinfeções2-6. Como resultado, este método
vem sendo adoptado internacionalmente,
frequentemente em combinação com spoligotyping, como o novo método de referência
para epidemiologia molecular da TB, por
exemplo, pelo Center for Disease Control
(CDC) nos Estados Unidos, grandes consórcios europeus de investigação e de vigilância
epidemiológica e por centros de referência
nacionais ou regionais.
Novos equipamentos e serviços de uso fácil
que ficaram recentemente disponíveis vieram facilitar o controlo de qualidade da utilização desta técnica, bem como a interpretação dos resultados obtidos. Os serviços de
tipagem MIRU-VNTR são propostos e usados já por centros de referência e laboratórios internacionais para realização de genotipagem (incluindo de estirpes de M. bovis) e/
/ou para avaliação do sistema de garantia de
qualidade QA/QC. Os kits de calibração,
validação e tipagem MIRU-VNTR de qualidade controlada, bem como o software para
gestão de dados e formação on-site, facilitam
enormemente a padronização e a utilização
eficiente da tipagem MIRU-VNTR no laboratório do utilizador.
Com o kit de tipagem MIRU-VNTR, 24
marcadores são ampliados a partir de ADN
purificado ou de extrato bruto de ADN de
colónias micobacterianas ou de grânulos de
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ter predictive value than the previous gold
standard IS6110 RFLP for the study of TB
transmission, in settings with epidemiological characteristics representative of
those of many developed countries. PCRbased interrogation of up to 24 independent and well-calibrated markers facilitates
prompt and reliable molecular-guided elucidation of complex situations involving
potential outbreak cases, mixed infections
or re-infections2-6. As a result, this method
is being internationally adopted, often in
combination with spoligotyping, as the
new reference method for TB molecular
epidemiology, e.g. by the US CDC, large
European research and epidemiological
surveillance consortiums and National or
Regional reference Centers.
New, easy-to-use tools and services have recently become available, which facilitate
quality-controlled use of this technique, as
well as interpretation of the results obtained.
MIRU-VNTR typing services are proposed
and already used by international Reference
Centers and laboratories, for outsourcing
their genotyping (including of M. bovis
strains) and/or for QA/QC evaluation.
Quality-controlled MIRU-VNTR Calibration, Validation and Typing kits, as well as
dedicated data management software and
on-site trainings greatly facilitate standardized set up and efficient use of MIRUVNTR typing in user’s laboratory.
With the MIRU-VNTR Typing Kit, 24
markers are amplified from purified DNA
or crude DNA extract from mycobacterial
colonies or cell pellets from liquid cultures, using 8 triplex PCR and fluorescent
primers specific for the flanking regions of
the targeted loci. Amplified fragment are
separated by capillary electrophoresis on
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células de culturas líquidas, usando 8 triplex
PCR e primers fluorescentes específicos para
os flancos dos loci alvo. Os fragmentos ampliados são separados por eletroforese capilar em plataformas ABI para determinação
das dimensões dos produtos da PCR. Conforme se vão sabendo os tamanhos das unidades repetidas, estes refletem os números
das sequências dos loci ampliados. A análise
é feita usando o software ABI GeneMapper®,
equipado com módulos optimizados fornecida no GENOSCREEN MIRU-VNTR typing calibration kit. O resultado final é um
genótipo numérico portátil, que corresponde ao número repetido em cada locus. As
estirpes dos genótipos podem então ser analisadas e comparadas com as estirpes de genótipos de referência, recorrendo a bancos
de dados locais ou a www.miru-vntrplus.
org, um banco de dados de identificação
multifuncional de livre acesso na Internet.
O kit de calibragem MIRU-VNTR foi desenhado para implementação e validação
padronizada da técnica MIRU-VNTR no
laboratório do utilizador. A migração relativa entre o padrão de tamanho e os produtos
do PCR depende dos locus e alelos do
MIRU-VNTR e difere segundo o polímero
usado para eletroforese capilar e entre instrumentos4. Assim, os bin sets GeneMapper
específicos dos instrumentos são criados
com o fim de calibrar para estes efeitos.
Cada bin define o âmbito de tamanhos observados para um dado número repetido
(alelo) determinado a partir de 4 sequências
diferentes. Estes bin sets são criados usando
o software Bin Set Creator e 24 marcadores allelic ladders, de acordo com os tamanhos dos allelic ladder obtidos no analisador de ADN do utilizador. Para validar o
processo, é fornecido um painel de 12
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ABI platforms to determine the PCR
product sizes. As the length of the repeat
units is known, these sizes reflect the numbers of repeated sequences in the amplified loci. Analysis is done using ABI GeneMapper® software customized with
optimized modules provided in GENOSCREEN MIRU-VNTR Typing Calibration Kit. The final result is a portable numerical genotype, corresponding to the
repeat number in each locus. Strain genotypes can then be analyzed and compared
to reference strain genotypes using local
databases or www.miru-vntrplus.org, a
multi-functional identification database
freely accessible via the Internet.
The MIRU-VNTR Calibration Kit is designed for standardised implementation and
validation of MIRU-VNTR technique in
user’s laboratory. Relative migration between the size standard and the PCR products depends on the MIRU-VNTR locus
and alleles, and differs upon polymer used
for capillary electrophoresis and between
instruments4. Therefore, instrument-specific GeneMapper bin sets are created in order
to calibrate for these effects. Each bin defines the observed size range for a given repeat number (allele) determined from 4 different runs. These bin sets are specifically
created using the Bin Set Creator software
and 24 marker-specific Allelic Ladders, in
accordance with the Allelic Ladder sizes
obtained on user’s DNA Analyzer. To validate the process, a panel of 12 reference
samples (including one negative control)
with known allelic profiles is provided. After PCR amplification and electrophoretic
separation, allelic profiles are determined
using the created bin sets and checked for
validation.
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Caroline Allix-Béguec, Christine Hubans, Stéphanie Ferreira, Philip Supply
Fig. 1 – Tipagem MIRU-VNTR. A curva, as caixas e as setas à direita representam um cromossoma da estirpe do complexo
M. tuberculosis, unidades repetidas dos loci MIRU-VNTR e os primers usados para ampliação PCR, respectivamente.
Vinte e quatro loci MIRU-VNTR são analisados via 8 PCR multiplex. O resultado final é um genótipo numérico, que corresponde ao número de unidades repetidas de cada um dos 24 marcadores
Fig. 1 – MIRU-VNTR typing. Close curve, orange boxes and arrows on the right represent a M. tuberculosis complex strain
chromosome, repeat units of MIRU-VNTR loci, and labeled primers used for PCR amplification, respectively. Twenty-four
MIRU-VNTR loci are analysed via 8 multiplex PCRs. The final result is a numerical genotype, corresponding to the repeat
unit number of each of the 24 markers
amostras de referência (incluindo um controlo negativo) com perfis alélicos conhecidos. Após ampliação por PCR e separação
eletroforética, determinam-se os perfis alélicos usando os bin sets criados e validados.
O software do gestor de dados MIRU-VNTR foi desenhado para facilitar a gestão
de dados após análise do GeneMapper. As
características incluem operações de controlo de qualidade (verificação dos controlos,
etc.), gestão dos dados (dados em falta e alelos duplos, criação de novas folhas de cálculo, intercalação e controlo cruzado dos resultados do projecto GeneMapper®) e
formatação final para compatibilidade com
o banco de dados MIRU-VNTRPlus. A for-
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The MIRU-VNTR Data Manager software
is designed to facilitate data management
after GeneMapper analysis. Features include
quality control operations (verification of
controls, etc), data management (missing
data and double alleles, creation of new run
spreadsheets, merging and cross-control of
GeneMapper® project results), and final formatting for compatibility with MIRU-VNTRPlus database. MIRU-VNTR training is
proposed in user’s laboratory, and includes
calibration and validation of user’s platform,
as well as training on technical and scientific interpretation. Dedicated MIRU-VNTR Support is provided to kit users via specific e-mailbox and telephone.
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mação MIRU-VNTR, que se propõe tenha
lugar no laboratório do utilizador, inclui calibragem e validação da plataforma do utilizador, bem como formação quanto à interpretação técnica e científica. Aos utilizadores
dos kits MIRU-VNTR é oferecido apoio
específico via e-mail e telefone.
Espera-se que a disponibilidade destes aparelhos para genotipagem em tempo real
mais fácil e eficiente contribua para melhorar o controlo e a vigilância molecular da
tuberculose.
It is hoped that the availability of these tools
for easier and more efficient real-time genotyping will contribute to improved molecular-guided TB control and surveillance.
Bibliografia/Bibliography
1. Supply P, et al. Proposal for standardization of optimized mycobacterial interspersed repetitive unit-variable-number tandem repeat typing of Mycobacterium tuberculosis. J Clin Microbiol 2006 44(12):4498-510.
2. Alonso-Rodriguez N, et al. Evaluation of the new advanced 15-loci MIRU-VNTR genotyping tool in Mycobacterium tuberculosis molecular epidemiology studies.
BMC Microbiol 2008; 8:34.
3. Oelemann MC, et al. Assessment of an optimized
mycobacterial interspersed repetitive-unit-variable-number tandem-repeat typing system combined with
spoligotyping for population-based molecular epidemiology studies of tuberculosis. J Clin Microbiol 2007;
45(3):691-697.
R e v i s t a
4. Allix C, Supply P, Fauville-Dufaux M. Utility of fast
mycobacterial interspersed repetitive unit-variable
number tandem repeat genotyping in clinical mycobacteriological analysis. Clin Infect Dis 2004; 39(6):783-789.
5. Allix-Béguec C, Fauville-Dufaux M, Supply P. Three-year population-based evaluation of standardized mycobacterial interspersed repetitive-unit-variable-number
tandem-repeat typing of Mycobacterium tuberculosis. J
Clin Microbiol 2008; 46(4):1398-1406.
6. Shamputa IC, et al. Mixed infection and clonal representativeness of a single sputum sample in tuberculosis
patients from a penitentiary hospital in Georgia. Respir
Res 2006; 7:99.
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Simpósio: Avaliação externa da qualidade
Elvira Richter1
Symposium: External quality assurance
Os objetivos deste simpósio foram:
• avaliar a situação actual
• explorar os progressos na AEQ
• desenvolver redes
• promover melhor colaboração
The aims of this symposium were:
• to estimate the current situation
• to explore advances in EQA
• to develop networks
• to promote better collaboration
Três oradores informaram quanto aos diferentes aspectos destes objetivos.
Three speakers provided information to different aspects of these aims.
A AEQ num país com baixa
incidência e elevados
rendimentos (Alemanha)
Há anos que a situação da tuberculose (TB)
na Alemanha se caracteriza por uma diminuição do número de doentes, com uma
incidência de 5,5 por 100 000 habitantes
em 2008. Quarenta e três por cento dos
doentes com TB pulmonar confirmada por
cultura tiveram esfregaços negativos. Vinte
por cento de todos os doentes apresentavam exclusivamente TB extrapulmonar. O
índice de estirpes resistentes atinge 11%
para qualquer resistência e 2% para a TB
EQA in a low incidence, high
income country (Germany)
The TB situation in Germany is characterized since years by a decrease of the number
of patients with an incidence of 5.5 per
100,000 inhabitants in the year 2008. 43%
of patients with culture confirmed pulmonary TB were smear negative. 20% of all
patients present with exclusively extrapulmonary TB. The rate of resistant strains
reaches 11% for any resistance and 2% for
MDR. XDR strains are reported. Infections
with non tuberculous mycobacteria (NTM)
comprise lymphadenitis in children, adults
1
NRL, Borstel, Germany
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multirresistente (MDR). Estão descritas estirpes extensivamente resistentes (XDR).
As infecções com micobactérias não tuberculosas (MNT) incluem a linfadenite em
crianças, adultos com doenças subjacentes
[e.g., doença pulmonar obstrutiva crónica
(DPOC), bronquiectasia), ou outras (como
o granuloma de piscina).
Esta situação reflete-se no tipo específico de
AEQ com o objetivo de avaliar:
1) A especificidade e sensibilidade da microscopia (lâminas positivas e negativas
preparadas para coloração com a técnica
de rotina do laboratório);
2) A sensibilidade das técnicas de cultura
[amostras com ou sem micobactérias
(TB ou MNT), preparados para isolamento primário e identificação de TB ou
MNT];
3) A sensibilidade e especificidade das técnicas de ampliação de ácidos nucleicos
(as amostras de expetoração são preparados para deteção de ácidos nucleicos específicos de TB);
4) A fiabilidade e rapidez dos testes de suscetibilidade (INH, RMP, EMB, SM, e
PZA);
5) O rigor da identificação das bactérias da
TB e das MNT (diferentes espécies micobacterianas são preparadas para identificação a nível de espécie).
Antes do envio das amostras para AEQ nos
laboratórios, é necessário esclarecer os regulamentos (e.g., transporte, ou autorização
para o manuseamento de bactérias de TB).
Algumas causas importantes para os falsos
resultados podem ser detectadas através da
análise dos dados e reanálise adicional desses
dados com a colaboração do laboratório que
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with underlying diseases (e.g. COPD, bronchiectasis), or others (like swimming pool
granuloma).
This situation is reflected by the specific
kind of EQA aiming to the evaluation of:
1) specificity and sensitivity of microscopy
(positive and negative slides prepared for
staining with the routine technique of
the laboratory)
2) sensitivity of culture techniques (specimens with or without mycobacteria [TB
or NTM] are prepared for primary isolation and identification of TB or NTM)
3) sensitivity and specificity of nucleic acid
amplification techniques (sputum specimens are prepared for detection of specific nucleic acids of TB)
4) reliability and rapidity of susceptibility
testing (INH, RMP, EMB, SM, and
PZA)
5) the accuracy of identification of TB bacteria and NTM (different mycobacterial
species are prepared for identification to
species level)
Before sending EQA specimens to the laboratories, regulations (e.g transportation, or
the permission for handling with TB bacteria) have to be clarified.
Some important causes for false results can
be found by analysis of the data and additional reanalysis in cooperation with the
laboratory that reported false results. Reasons for false positive microscopic results
were found to follow from staining in cuvettes, not as single slides, or misidentification of staining artefacts. False positive culture results derive from laboratory cross
contamination. Incorrect susceptibility results are most often found when testing SM,
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forneceu os falsos resultados. Observaram-se falsos positivos de microscopia após coloração em cuvettes, e não em lâminas individuais, ou identificação errada dos produtos
de coloração. Os resultados falsos positivos
de culturas resultam de contaminação cruzada no laboratório. Encontram-se com alguma frequência resultados com suscetibilidade incorreta ao testar SM e, em parte,
PZA. Ao fazer a identificação de especis,
ocorrem por vezes erros significativos, sendo
a principal razão a insuficiência da utilização exclusiva de análises bioquímicas.
Na preparação de amostras para AEQ há
que abordar alguns desafios.
– A questão da verdadeira avaliação da sensibilidade (no que respeita a microscopia,
ampliação dos ácidos nucleicos e isolamento primário). A preparação de amostras com pequenas quantidades de bactérias é difícil, porque pode resultar em
amostras individuais sem micobactérias.
– Para evitar o uso de estirpes MDR, são
selecionados isolados com padrões de resistência pouco comuns. Isto não reflete
uma “situação da vida real”.
– Há um contraste entre as necessidades
científicas e as do diagnóstico, como se
observa no espantoso número de espécies de MNT com sequências que constam das bases de dados de nucleótidos,
com apenas algumas espécies diferentes
isoladas em laboratório.
Organização e AEQ na Rede de
Laboratórios de TB na Letónia
A incidência de TB na Letónia foi caracterizada por uma contínua diminuição até
aproximadamente 1990, quando se verifi-
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and in part PZA. Performing species identification, sometimes significant errors occur.
The main reason for this is the insufficiency
of biochemical analyses alone.
Some challenges have to be addressed in the
preparation of samples for EQA.
– The question of a real estimation of sensitivity (with regard to microscopy, nucleic acid amplification, and primary isolation). The preparation of samples with
low amount of bacteria is difficult since it
can result in single samples without mycobacteria.
– To avoid the use of MDR strains, isolates with uncommon resistance patterns
are selected. This does not reflect a ‚real
life situation‘.
– There is a contrast between scientific vs.
diagnostic needs as we see in the overwhelming number of NTM-species with
sequences delivered in the Nucleotide
Databases, but only few different species
isolated in the routine laboratory.
Organization and EQA in the
Latvian TB laboratory network
The TB incidence in Latvia was characterized by a continuous decrease until approx.
1990, when a sharp increase was noted,
which peaked around 2000. Since then, the
incidence again decreased as to 40.3 per
100,000 inhabitants for the year 2008.
The Latvian TB-Laboratory Network is
composed of the National Reference Laboratory in Riga, of 5 regional culture and microscopy laboratories, and of several laboratories (at district hospitals, policlinics,
private labs) facilitating AFB microscopy at
PHC level in each of 26 districts.
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cou um brusco aumento, com pico cerca do
ano 2000. Desde então, a incidência voltou
a descer, atingindo 40,3 por 100 000 habitantes em 2008.
A Rede de Laboratórios de TB na Letónia é
composta pelo Laboratório Nacional de Referência (LNR), em Riga, 5 laboratórios regionais para trabalho de cultura e microscopia e
ainda vários outros laboratórios (em hospitais
distritais, policlínicas, laboratórios privados)
que proporcionam microscopia de esfregaço
para pesquisa de bacilos ácido-álcool resistentes (BAAR) a nível PHC em cada um dos 26
distritos.
Todos os centros de microscopia são auditados e certificados com base em padrões internacionais (ISO 17025, ISO 15189). O controlo de qualidade interno é feito por coloração
e microscopia de esfregaços positivos e negativos conhecidos. Os laboratórios de cultura
fazem descontaminação de amostras e de culturas. As culturas são enviadas para o LNR
para identificação e testes de suscetibilidade a
fármacos. O laboratório nacional de referência realiza microscopia de esfregaços, cultura
de amostras, testes de susceptibilidade para
fármacos de 1.ª linha (+ Kanamicina) e TSF
de 2ª linha para estirpes de MDR-TB.
Além disso, o LNR efectua o controlo da
qualidade externo de todos os laboratórios,
duas vezes por ano, utilizando um painel de
5 esfregaços fixados. Com este fim, 5 lâminas preparadas a partir de expetoração de
doentes, contendo amostras negativas e positivas, são fornecidas aos laboratórios, que
processam e interpretam as lâminas, registam os resultados e enviam-nos ao LNR
juntamente com as respectivas lâminas. Finalmente, o LNR reexamina as lâminas,
avalia a coloração e os resultados e emite relatórios sobre o desempenho. Este LNR
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All microscopy centers are audited and certified based on international standards for
testing and medical laboratories (ISO
17025, ISO 15189). Internal Quality Control is performed by staining and microscopy of known positive and negative smears.
The culture laboratories perform decontamination of specimens and culture. Cultures will be sent to NRL for identification
and drug susceptibility tests (DST). The
National Reference Laboratory performs
smear microscopy, culture of specimens,
DST for 1st line drugs (+ Kanamycin), and
2nd line DST for MDR-TB strains.
Furthermore, the NRL provides the external
quality assurance for all laboratories by a
panel of 5 fixed smears twice a year. For this,
5 slides prepared from patient sputum, comprising negative, scanty, and positive specimens, are provided to the laboratories. These
stain and read the slides, record the results
and send back results and slides to NRL. Finally, the NRL rereads the slides, assesses
staining and results, and issues reports on
performance. Furthermore, the NRL monitors the quality of sputum smear microscopy
procedure during supervisory visits.
For EQA of the performance of the culture,
2 specimens are sent to each lab twice a year.
In detail, each specimen is divided into two
parts, one part is decontaminated and incubated at NRL, and the other is sent to the
culture laboratory. The culture laboratory
performs decontamination, inoculation, incubation, record of results and sends the results back to the NRL. Beside this, the NRL
monitors the quality of culture procedures
during supervisory visits.
The NRL itself yearly participated in an external proficiency programme for 1st line
DST provided by in Swedish Institute for
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Elvira Richter
também controla a qualidade do procedimento da microscopia dos esfregaços de expetoração durante visitas de inspecção.
Para a AEQ do desempenho da cultura, 2
amostras são enviadas a cada laboratório
duas vezes por ano. Cada amostra é dividida
em duas partes, sendo uma parte descontaminada e incubada no LNR e a outra enviada para o laboratório de cultura. Este procede à descontaminação, inoculação, incubação
e registo dos resultados, que envia ao LNR.
Além disto, o LNR também controla a qualidade dos procedimentos de cultura durante
visitas de inspecção.
O próprio LNR participou anualmente
num programa externo de proficiência para
TSF de 1.ª linha disponibilizado pelo Instituto Sueco para o Controlo de Doenças Infecciosas. A partir de 2008, o LNR passou a
ser o Laboratório Supranacional de Referência, tomando, assim, parte nas atividades
AEQ anuais para TSF de 1.ª e 2.ª linha.
A implementação do controlo da
qualidade como requisito para a
introdução de um novo teste de
diagnóstico
A integração de laboratórios de saúde pública
é um dos principais alvos na luta contra as doenças infeciosas. O controlo da qualidade do
procedimento dos testes é um requisito essencial para a obtenção de resultados úteis e fiáveis. O controlo da qualidade compreende o
controlo interno da qualidade e a avaliação externa da qualidade. Para a integração de novas
técnicas moleculares num laboratório de TB,
o controlo interno da qualidade compreende
vários aspectos, como uma infraestrutura elaborada, utilização e manutenção adequadas
do equipamento e orientação nos procedi-
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Infectious Diseases Control. Since 2008 the
NRL itself became Supranational Reference
Laboratory and thus taking part in the yearly EQA rounds for 1st and 2nd line DST.
The implementation a of quality
assurance as prerequisite for the
introduction of a new diagnostic
test.
The integration of public health laboratories is one of the main targets in fighting
emerging infectious diseases. Quality assurance of the test procedures is a prerequisite
to obtain reliable and useful results. Quality
assurance is composed of internal quality
control and external quality assessment. For
the integration of new molecular techniques
into a TB laboratory, internal quality control comprises several aspects, like elaborate
infrastructure, equipment that is well maintained and operated and procedures guided
by efficient SOPs. Test reagents need to be
controlled with every LOT, its integrity
needs to be checked for every shipment and
proper transport and storage has to be controlled. The test procedures itself require
several quality control steps, like contamination controls, negative controls at different levels of the test process, the correct interpretation of adequate and inadequate
runs, and the implementation of certain
quality indicators. Furthermore, personnel
need to be trained and re-trained.
External proficiency testing can be conducted by analysis of duplicate specimens from
one patient. As example, proficiency testing
for line probe assays was performed in four
laboratories in India as prerequisite for the
implementation of the new test. The advantage of panel testing for external quality
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Elvira Richter
mentos por SOP eficientes. Os reagentes têm
de ser controlados com cada LOT, a sua integridade verificada em cada remessa e o transporte e armazenagem devidamente fiscalizados. O processamento dos testes envolve vários
passos de controlo da qualidade, como controlo de contaminação, controlos negativos
em diferentes níveis do processo, interpretação
correta das sequências adequadas e inadequadas e implementação de certos indicadores de
qualidade. Há ainda que treinar o pessoal e
proporcionar a sua atualização contínua.
Os testes externos de proficiência podem ser
efetuados pela análise de duplicados de
amostras de um doente. Como exemplo, os
testes de proficiência para ensaios line probe
foram realizados em quatro laboratórios, na
Índia, um requisito essencial para a implementação dos novos testes. A vantagem dos
testes de painel para controlo externo da
qualidade consiste na revisão de todo o procedimento, no uso direcionado de estirpes
selecionadas com determinadas resistências,
com mutações comuns e incomuns, bem
como na utilização de espécies de MNT.
Há, no entanto, que ter em conta o custo
dos testes de painel (incluindo preparação e
expedição). O envio de resultados digitalizados para interpretação, bem como para
reverificação, pode constituir uma alternativa menos dispendiosa para AEQ dos ensaios
line probe, devendo a melhor solução ser
apreciada para cada situação.
Foram oradores: Elvira Richter, NRL, Borstel, Alemanha; Girts Skenders, State Agency
of TB and Lung Disease, Letónia; Akos Somoskövy, FIND, Geneva, Suissa.
O simpósio foi apoiado por INSTAND e.V.
Society for Research Promotion of Quality Assurance in Medical Laboratories, WHO Collaborating Centre, Düsseldorf, Alemanha.
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control is the revision of the entire procedure, the directed use of selected strains
with certain resistances, with common and
uncommon mutations, as well as the use of
NTM species. However, costs of panel tests
(including preparation and shipment) have
to be taken into account. Cheaper alternatives for EQA of line probe assays may be
achieved by sending of digitized results for
interpretation as well as for re-checking.
The best solution has to be found for different situations.
The speakers were: Elvira Richter, NRL,
Borstel, Germany; Girts Skenders, State
Agency of TB and Lung Disease, Latvia;
Akos Somoskövy, FIND, Geneva, Switzerland.
The symposium was sponsored by INSTAND e.V., Society for Research Promotion of Quality Assurance in Medical Laboratories, WHO Collaborating Centre,
Düsseldorf, Germany.
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Normas de Publicação
Instructions for Authors
Normas de publicação na
Revista Portuguesa de Pneumologia
Portuguese Journal of Pulmonology
Instructions for authors
A Revista Portuguesa de Pneumologia considera para
publicação trabalhos (artigos originais, de revisão, de
actualização, casos clínicos, cartas ao editor, resumos
críticos a livros, etc.) relacionados directa ou indirectamente com o Aparelho Respiratório.
As opiniões expressas são da exclusiva responsabilidade dos autores.
Revista Portuguesa de Pneumologia / The Portuguese
Journal of Pulmonology publishes papers (original articles, revised articles, updated articles, case reports,
letters to the editor, book reviews, etc) which are directly or indirectly related to the respiratory system.
The opinions expressed are the exclusive responsibility of the authors.
Os artigos publicados ficarão propriedade da Revista Portuguesa de Pneumologia, não podendo ser
reproduzidos, no todo ou em parte, sem autorização do editor.
The articles published are the intellectual property
of Revista Portuguesa de Pneumologia/ The Portuguese Journal of Pulmonology and may not be reproduced, in part or in whole, without permission
from the editor.
A aceitação dos originais enviados para publicação é
condicionada à avaliação pelo Conselho Científico
da Revista. Nesta avaliação os artigos poderão ser:
All submissions are subject to a screening process by
the Journal’s Scientific Board. There are three assessments possible:
a) aceites sem alterações;
b) aceites após as modificações propostas e aceites pelos autores;
c) recusados.
a) accepted for publication as is;
b) accepted for publication after the proposed alterations, accepted by the authors;
c) rejected.
Apresentação dos trabalhos – Os textos devem ser
escritos em português, dactilografados, com margens
largas (25 mm), a dois espaços, numa só face do papel
e em três exemplares com as páginas numeradas no
canto superior direito.
Manuscript instructions – The manuscripts submitted should be in Portuguese, typed, doubled spaced
with ample (25mm) margins, and on one side of A4
sized paper. Three copies should be submitted, with
the pages numbered in the upper right corner.
Solicita-se a todos os autores que enviem artigos para
publicação que o façam acompanhados do respecti-
All articles submitted for publication must be sent
in addition in a computerised format. The compu-
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NORMAS DE PUBLICAÇÃO/INSTRUCTIONS FOR AUTHORS
vo suporte magnético, que indiquem o programa
de computador em que foram executados e que tenham em atenção à reprodução das imagens (que
deverá ser feita, idealmente, em suporte JPG ou
TIFF) de modo a que fiquem nítidas na sua impressão tipográfica.
Chama-se a atenção que a transcrição de imagens, quadros ou gráficos de outras publicações deverá ter a prévia autorização dos respectivos editores para dar cumprimento às normas que regem os direitos de autor.
Poder-se-ão considerar para publicação artigos redigidos em inglês. Neste caso, deve incluir-se o resumo, o
título e as palavras-chave, também em português.
Deverão ser referenciados, pelos próprios autores,
como artigos originais, de revisão, cartas ao editor, ou
outros.
Todos os artigos originais serão também publicados
em inglês, após retroversão para esta língua, pela(s)
tradutora(s) da Revista Portuguesa de Pneumologia.
Caso os autores assim o entendam, poderão enviar os
artigos já traduzidos.
Estrutura – Sempre que possível será adoptado o esquema convencional em que se iniciará cada parte do
trabalho numa nova página pela seguinte ordem:
a) Na primeira página:
– título do trabalho em português e inglês
b) Na segunda página:
– o nome dos autores com os respectivos títulos
académicos e/ou profissionais;
– os serviços onde foi realizado, nome dos seus directores e os respectivos endereços.
c) Na(s) página(s) seguinte(s):
– o resumo em português que não deverá ultrapassar 250 palavras para os trabalhos originais e de
150 para os casos clínicos;
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ter program used must be clearly stated, and particular attention must be paid to the reproduction of
images, which should ideally be in JPG or TIFF format, to give a clear printed quality.
If an image, figure or graph has been previously published, written permission from the editor in question must be submitted to safeguard the author’s intellectual property rights.
Articles may also be submitted in English. In this
case, the summary, the title and key words should
also be submitted in Portuguese.
The authores should categorise their submissions as
original articles, revised articles, case studies, letters
to the editor, technical notes, etc.
All original articles shall be also printed in English,
after being translated by the Revista Portuguesa de
Pneumologia / The Portuguese Journal of Pulmonology´s
translators. Authors may submit their articles already
translated, if they so wish.
Form – As far as possible, the convention will be observed of beginning each new part of the paper on a
new page and in the following order:
a) Title page:
– the full title of the paper in Portuguese and
English;
b) Second page:
– the full names of the authors and their academic
and /or professional titles;
– the full name and address of the institution(s) at
which the work was carried out and the full
name of the institution(s) director(s).
c) Following page(s):
– a summary in Portuguese, not exceeding 250
words for original articles and 150 for case reports;
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NORMAS DE PUBLICAÇÃO/INSTRUCTIONS FOR AUTHORS
d)
e)
f)
g)
h)
– os resumos em inglês com características idênticas ao do inicial em português;
– as palavras-chave, em português e inglês (3 a
10), que servirão de base à indexação do artigo,
de acordo com a terminologia do Index Medicus “Medical Subject Headings”.
O texto que, no caso dos artigos originais, terá em
geral: Introdução, Material e Métodos, Resultados, Discussão e Conclusões
O texto, também em inglês, tratando-se de um artigo
original, e caso o(s) autor(es) assim o entendam fazer
Agradecimentos
Bibliografia
Quadros e Figuras.
d)
e)
f)
g)
h)
– a summary in English, with identical characteristics to that in Portuguese;
– between three and 10 key words in Portuguese
and English, which will be used to index the article, using terms from the Medical Subject Headings list of the Index Medicus.
Original articles shall include, as a general rule,
the following: Introduction, Subjects and Methods, Results, Discussion and Conclusions.
Original articles may be written in English if the
authors so wish.
Acknowledgments.
References.
Tables and Figures.
Bibliografia – As referências bibliográficas devem ser
numeradas por ordem consecutiva da sua primeira
citação no texto. Devem ser identificadas no texto
com números árabes. As referências devem conter, no
caso das revistas, o nome do primeiro autor (apelido
e nome), seguido dos restantes, do título do artigo,
do nome da publicação e da sua identificação (ano,
volume e páginas).
References – The bibliographical references should be
numbered consecutively, in the order in which they are
cited in the text and should be identified in the text with
Arabic numerals. Each reference to a journal article
should contain the surname and initial of the first author
followed by the rest. These should be followed by the title of the article, the title of the publication and its identifying years, volume number and the page numbers.
Quadros e figuras – Os quadros e figuras devem ser
apresentados em páginas separadas, em condições de
reprodução. Devem ser acompanhados da respectiva legenda em página à parte, mencionando no verso a lápis
o número de ordem. Todos os gráficos deverão ser apresentados através de fotografia do respectivo original.
Tables and figures – Each table and figure should be
on a separate page, and in such conditions as to be reproduced. Each table and figure should have its own
brief description on a separate page and bear its sequence number on its back, written in pencil. Each table and
figure should be represented via a copy of the original.
Modificações e revisões – No caso da aceitação do artigo ser condicionada a modificações, estas devem ser
realizadas pelos autores no prazo máximo de vinte dias.
Alterations and changes – Should an article be accepted for publication subject to alteration, these must
be made by the authors within a twenty day period.
As provas tipográficas serão da responsabilidade da
Redacção, se os autores não indicarem o contrário.
Neste caso elas deverão ser feitas no prazo determinado pela Redacção, em função das necessidades editoriais da Revista.
Publishing proofs – These are the editorial board’s
responsibility, unless the authors state otherwise.
Should this latter be the case, the proofs should be
concluded within a deadline set by the editorial board, in line with the editorial norms of the Journal.
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NORMAS DE PUBLICAÇÃO/INSTRUCTIONS FOR AUTHORS
Cartas ao editor – Devem constituir um comentário
crítico a um artigo da Revista ou uma pequena nota
sobre um tema ou caso clínico. Não devem exceder as
500 palavras, nem conter mais de um quadro ou figura e um máximo de 6 referências bibliográficas. As
respostas do(s) autor(es) devem obedecer às mesmas
características.
Pedido de publicação – Os trabalhos deverão ser enviados à Redacção, em nome do editor, para a sede da
SPP, Rua Ivone Silva, n.º 6 – 6.º Esq., Edifício Arcis,
1069-130 Lisboa, Portugal, acompanhados de uma
carta com pedido de publicação, subscrito por todos
os autores, indicação da cedência do copyright e que
não foram publicados ou enviados para publicação
em outra revista nacional ou estrangeira. Não serão
aceites trabalhos já publicados ou enviados simultaneamente a outras revistas.
Deverão ser acompanhados pelo endereço electrónico do autor principal para eventuais pedidos de
esclarecimentos por parte da Redacção.
Os trabalhos também poderão ser enviados por via
electrónica (e-mail: [email protected]).
Nota final – Para um mais completo esclarecimento
sobre este assunto aconselha-se a leitura dos requisitos do International Commitee of Medical Journal Editors, publicados na integra no N Engl J Med 1991;
324:424-428.
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Letters to the editor – These should contain a critical appraisal of a Journal article or a small comment
on a theme or a case study. They should not exceed
500 words, contain more than one table or figure and
have a maximum of 6 bibliographical references. Authors’ replies should observe these same norms.
Submissions for publication – Papers should be sent to
the editorial board, addressed to the editor: Sociedade
Portuguesa Pneumologia office: SPP – Rua Ivone Silva,
n.º 6 – 6.º Esq., Edifício Arcis, 1069-130 Lisboa, Portugal. Submissions should be accompanied by a letter requesting that the work be submitted for publication,
signed by all of the authors, stating that they waive their
intellectual property rights and that they work has not be
published or submitted for publication in any other Portuguese of international journal. Work already published or already sent to other journals will be rejected.
Submissions must bear the e-mail address of the
corresponding author in order to facilitate contact
with the editorial team should any clarification be
necessary.
Papers may should be sent via electronic mail to:
[email protected]
Final note – For a fuller clarification of this matter, a
reading of the requirements of the International Committee of Medical Journal Editors, published in full in
the N Engl J Med 1991; 324:424-428, is advised.
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