UNIVERSIDADE DO ESTADO DO AMAZONAS
FUNDAÇÃO DE MEDICINA TROPICAL DR. HEITOR VIEIRA DOURADO
PROGRAMA DE PÓS-GRADUAÇÃO EM MEDICINATROPICAL
MESTRADO EM DOENÇAS TROPICAIS E INFECCIOSAS
AVALIAÇÃO IN VITRO DA FAGOCITOSE DE PLAQUETAS EM PACIENTES COM
MALÁRIA VIVAX
HELENA CRISTINA CARDOSO COELHO
MANAUS
2012
i
HELENA CRISTINA CARDOSO COELHO
AVALIAÇÃO IN VITRO DA FAGOCITOSE DE PLAQUETAS EM PACIENTES COM
MALÁRIA VIVAX
Dissertação apresentada ao Programa de
Pós-Graduação em Medicina Tropical da
Universidade do Estado do Amazonas em
Convênio com a Fundação de Medicina
Tropical Dr. Heitor Vieira Dourado, para
obtenção do grau de Mestre em Doenças
Tropicais e Infecciosas.
Orientador: Prof. Dr. Marcus Vinícius Guimarães de Lacerda
MANAUS
2012
Ficha Catalográfica
C672a
Coelho, Helena Cristina Cardoso.
Avaliação in vitro da fagocitose de plaquetas em pacientes
com malária vivax /. -- Manaus : Universidade do Estado do
Amazonas, Fundação de Medicina Tropical, 2012.
xiii. 141 f. : il.
Dissertação (Mestrado) apresentada ao Programa de PósGraduação em Medicina Tropical da Universidade do Estado do
Amazonas – UEA/FMT, 2012.
Orientador: Profº. Dr. Marcus Vinicius Guimarães de
Lacerda.
1.Malária 2.Plasmodium vivax 3.Plaquetopenia . Título
Ficha Catalográfica elaborada pela Bibliotecária Maria Eliana CDU:
N. Silva,
lotada na
616.928.5
Escola Superior de Ciências da Saúde - UEA
ii
FOLHA DE JULGAMENTO
AVALIAÇÃO IN VITRO DA FAGOCITOSE DE PLAQUETAS EM
PACIENTES COM MALÁRIA VIVAX
HELENA CRISTINA CARDOSO COELHO
“Esta Dissertação foi julgada adequada para obtenção do Título de Mestre em
Doenças Tropicais e Infecciosas, aprovada em sua forma final pelo Programa
de Pós-Graduação em Medicina Tropical da Universidade do Estado do
Amazonas em convênio com a Fundação de Medicina Tropical Dr. Heitor Vieira
Dourado”.
Banca Julgadora:
_____________________________________
Prof. Marcus Vinicius Guimarães de Lacerda, Dr.
Presidente
_______________________________
Prof. Fábio Trindade Maranhão, Dr.
Membro
______________________
Profª. Aya Sadahiro, Dra.
Membro
iii
À Deus, pela constante presença em
minha vida.
Aos meus pais, Gilberto e Cristina, por
todos os ensinamentos que me auxiliam na
jornada da vida.
Ao meu esposo, Genilson, pelo amor
e apoio incondicional em todas as horas.
Ao meu filho Pedro, pelas alegrias de
todos os dias.
Dedico-lhes
gratidão.
essa
conquista
com
iv
AGRADECIMENTOS
Ao meu orientador Dr. Marcus Vinícius G. Lacerda, pela oportunidade me
dada para desenvolver esse trabalho desafiador que me despertou grande interesse.
Agradeço também pelo conhecimento científico adquirido e pelo amadurecimento
que obtive durante todo esse período que me orientou.
Aos Professores Paulo Nogueira e Adriana Malheiro, pelo apoio, incentivo e
grandes contribuições no desenvolvimento do trabalho, e Profª Aya Sadahiro pelas
ótimas sugestões apresentadas no exame de qualificação.
À Stefanie Lopes, que me acompanhou durante grande parte dessa jornada e
que acabou tornando-se uma grande amiga. Obrigada Stef, por me ensinar com
tanta boa vontade, me aconselhar durante os longos momentos de reflexão, me
incentivar e me mostrar o valor da ciência, e é claro, obrigado pelos diversos
momentos de alegria, dentro e fora dos laboratórios.
Ao João Paulo Pimentel, que trouxe grande contribuição ao trabalho com sua
experiência, paciência e gentileza.
A minha querida amiga Gisely Melo, uma das primeiras pessoas que conheci
na FMT-HVD que me recebeu com muito carinho e que acabou tornando-se uma
grande companheira. E ao seu esposo e meu amigo Wuelton Monteiro pelo apoio,
grandes ensinamentos e bons momentos de descontração.
Aos queridos amigos Belisa Magalhães e Kleber Alexandre pelo carinho,
incentivo e momentos especiais vividos ao lado deles.
Ao Dr. André M. Siqueira, que desde o inicio do meu ingresso sempre esteve
disposto a me auxiliar. Obrigada André pelos ensinamentos, incentivos e momentos
divertidos.
Aos queridos Raimunda, Wellington, Allyne, Marcela, Sra. Ericilda pela
gentileza e empenho no processo de inclusão e coleta de amostra dos pacientes do
estudo.
Aos colegas do laboratório de virologia da FMT-HVD, especialmente ao
Bosco pelo profissionalismo, dedicação e grande auxilio que nos prestou.
As pessoas do laboratório do Centro de Pesquisa Leônidas e Maria Deane
(FIOCRUZ), pela colaboração e pelos momentos agradáveis e descontraídos vividos
nesses últimos anos.
Aos amigos do HEMOAM, Allyson Guimarães e Walter Luiz, pela importante
contribuição na realização do estudo.
A todos os professores que, com seus ensinamentos, participaram de minha
formação.
Ao grande companheiro de “gincanas” pelos laboratórios, Alejandro com o
seu eterno bom humor, tornava o nosso dia muito melhor.
A toda equipe da malária (alunos, pesquisadores e funcionários) que de
alguma forma contribuíram no desenvolvimento desse trabalho e aqueles que
passaram um período conosco e que deixaram saudades como Mireia e Letusa.
A querida Teresa, pessoa de boa índole, com muita força de vontade.
Obrigada por sempre estar disposta a me ajudar.
Aos colegas de turma de pós-graduação que foram importantes no início
dessa caminhada. Em especial, à Patrícia e a Luciana pela amizade, espírito de
cooperação e pelos momentos de descontração e reflexão.
Aos pacientes que contribuíram no desenvolvimento dessa pesquisa.
v
Ao professor Ronei Mamoni (FCM-UNICAMP) que gentilmente doou as
células THP-1 para execução dos ensaios de fagocitose.
Ao excelente pesquisador Bernardo, pela oportunidade me dada de fazer
parte de uma de suas pesquisas.
À Fundação de Medicina Tropical do Amazonas e à Universidade do Estado
do Amazonas UEA, pelo programa de pós-graduação.
À Superintendência da Zona Franca de Manaus - SUFRAMA, financiadora do
programa de pós-graduação da UEA.
À Fundação de Hematologia e Hemoterapia do Estado do Amazonas HEMOAM e Centro de Pesquisa Leônidas e Maria Deane, por me autorizarem a
utilizar parte das instalações na execução das minhas atividades.
Ao meu marido Gê, por me fazer acreditar na felicidade que alcançaríamos
após passarmos um período turbulento com diversas mudanças em nossas vidas.
Por não me deixar desistir dos meus sonhos, e por estar sempre ao meu lado nos
momentos de tristeza e alegria, oferecendo apoio, incentivo, carinho e muita
paciência.
À minha linda família, Gilberto (pai), Cristina (mãe), Monize (irmã) Júnior e
Viviane (irmão e cunhada) por serem pessoas maravilhosas, por compreenderem a
minha ausência em muitos momentos de suas vidas e por muitas vezes através de
conversas ao vivo ou ao telefone renovar minhas forças para o cumprimento do que
me propus a fazer. E aos pequenos anjos da minha vida, Katarina, Walentina e
Breno que me tiraram muitos sorrisos, mesmos nos momentos de grande aflição.
Aos meus queridos amigos Aline e Moacyr que foram os grandes
incentivadores ao meu ingresso no programa de pós–graduação e responsáveis por
muitos momentos felizes da minha vida. Obrigada Aline por ter entrado em minha
vida e me acrescentar tantas coisas boas. Algo me diz que por algum motivo isso
aconteceria mais cedo ou mais tarde, em Manaus, São Paulo ou em qualquer outro
lugar.
À minha grande amiga Fabiana, pela longa amizade sincera, pelo carinho,
incentivo e pelas longas conversas sobre nossa simultânea trajetória científica.
E como uma veterinária e apaixonada por animais, agradeço as minhas filhas
de focinho que me fazem esquecer por alguns instantes os problemas do mundo a
fora.
vi
RESUMO
A plaquetopenia é uma alteração hematológica comumente relatada em
pacientes com malária, entretanto, pouco se conhece sobre os mecanismos exatos
que causam essa alteração hematológica nesses indivíduos. O objetivo desse
trabalho foi estudar o papel da fagocitose na plaquetopenia da malária vivax.
Plaquetas de 35 pacientes com malária vivax atendidos na Fundação de Medicina
Tropical – Dr. Heitor Vieira Dourado, foram marcadas com diacetato de 5clorometilfluoresceína e incubadas com células THP-1 por 1 hora. A fagocitose de
plaquetas foi verificada por citometria de fluxo. Como controle negativo, foram
utilizadas plaquetas de 8 pessoas saudáveis. A expressão da P-selectina foi
avaliada utilizando anticorpo anti CD62-P humano conjugado com ficoeritrina (PE).
Concentrações séricas de citocinas do tipo IL-2, IL-4, IL-6, IL-10, fator de necrose
tumoral alfa (TNF-α), interferon gama (IFN-γ) e IL-17 foram mensurados também por
citometria de fluxo. A fagocitose de plaquetas foi maior nos pacientes
plaquetopênicos do que nos não-plaquetopênicos (p=0,042) e pessoas saudáveis
(p=0,048). Além disso, foi encontrada uma correlação negativa entre a fagocitose e
contagem de plaquetas (p=0,016; r=-0,402). Não houve diferença significativa da
expressão de P-selectina nas plaquetas entre os pacientes plaquetopênicos e
pessoas saudáveis (p=0,092). Concentrações de IL-6, IL-10 e IFN-γ foram mais altas
em pacientes com malária comparadas com pessoas saudáveis. Valores de IL-6 e
IL-10 foram mais elevadas em pacientes plaquetopênicos quando comparados a
pacientes não-plaquetopênicos (p=0,016; p=0,045). Concentração de TNF-α foi mais
elevada em pacientes plaquetopênicos do que em pessoas saudáveis (0,007) e uma
correlação positiva foi encontrada entre TNF-α e fagocitose de plaquetas (p=0,010;
r=0,425). Os resultados sugerem que a fagocitose de plaquetas por monócitos pode
ser considerada, em parte, um mecanismo que contribui na plaquetopenia da
malária vivax. Esse fenômeno mostrou-se independente da ativação de plaquetas.
Contudo, mais estudos são necessários para determinar os mecanismos
moleculares que envolvem a fagocitose de plaquetas nessa doença.
Palavras-chaves: Malária. Plasmodium vivax. Plaquetopenia. Fagocitose.
vii
ABSTRACT
Thrombocytopenia is a hematological change commonly reported in patients with
malaria, however, the exact mechanism has not been elucidated. The objective of the
present work was to study the role of phagocytosis in malaria thrombocytopenia.
Platelets from of thirty and five patients with vivax malaria were collected in
Fundação de Medicina Tropical – Dr. Heitor Vieira Dourado, labeled with 5-diacetate
clorometilfluoresceína (CMFDA) and incubated with THP-1 cells for 1 hour. Platelet
phagocytosis was evaluated by flow cytometry. As a negative control, we used
platelets from eithg healthy volunteers. The expression of P-selectin was evaluated
using PE Mouse Anti-Human CD62P. Serum IL-2, IL-4, IL-6, IL-10, TNF-α (Tumor
Necrosis Factor alpha), IFN-γ (Interferon gamma) and IL-17 were also measured by
flow cytometry. The platelet phagocytosis was greater in thrombocytopenic patients
than in those non-thrombocytopenic patients (p=0.042) and healthy volunteers
(p=0.048). Furthermore, we found a negative correlation between phagocytosis and
platelet counts (p=0.016, r=-0.402). No significant difference was found in the
expression of P-selectin between thrombocytopenic patients and healthy volunteers
(p=0.092). IL-6, IL-10 and IFN-γ were elevated in malaria patients compared to HV.
Even more, IL-6 and IL-10 values were higher in thrombocytopenic patients that nonthrombocytopenic one (p=0.016; p=0.045). TNF-α was only elevated in
thrombocytopenic patients compared to HV (p=0.007) and a positive correlation was
found between TNF-α and platelet phagocytosis (p=0.010; r=0.425). This data
suggest that phagocytosis platelet might be part of the pathogenic process involved
in thrombocytopenia in vivax malaria. This phenomenon seems to be independent of
platelet activation. However, further studies are needed to determine the molecular
mechanisms involving platelet phagocytosis in this disease.
Keywords: Malaria. Plasmodium vivax. Thrombocytopenia. Phagocytosis.
viii
LISTA DE FIGURAS
Figura 1:
Figura 2:
Figura 3:
Figura 4-A:
Figura 4-B:
Figura 4-C:
Figura 5:
Áreas de transmissão da malária ..........................................
Mapa do Brasil com distribuição de casos de malária por
1.000 habitantes no Brasil .....................................................
Imagem da formação das plaquetas......................................
Imagem de uma plaqueta quiescente, apresentando forma
discoide, com superfície rugosa (aumento 30.000 X)............
Aberturas na superfície da plaqueta conectadas ao sistema
canicular aberto (aumento 26.000 X).....................................
Grânulos α (G) e grânulos densos (D) no citoplasma de
uma plaqueta (aumento 32.000 X)........................................
Etapas do experimento de fagocitose de plaquetas..............
1
3
5
6
6
6
27
ix
LISTA DE TABELAS
Tabela 1:
Tabela 2:
Grânulos plaquetários e seus constituintes principais............ 7
Classificação dos pacientes de acordo com a contagem de
plaquetas ................................................................................ 25
x
LISTA DE QUADROS
Quadro 1:
Quadro 2:
Classificação fisiopatológica da plaquetopenia.........................
Medicamentos que podem causar plaquetopenia.....................
9
23
xi
LISTA DE ABREVIATURAS, SÍMBOLOS E UNIDADES DE MEDIDA
ADP – Adenosina difosfato
ATP – Adenosina trifosfato
BSGC – Solução salina tamponada com glicose e citrato (Buffered Saline
Glucose-Citrate)
CD36 – Receptor de membrana celular
CD40L – Ligante do receptor de membrana celular CD40
CID – Coagulação Intravascular Disseminada
CMFDA – Diacetato de 5-clorometilfluoresceína (5-ChloroMethylFluorescein
DiAcetate)
CPqLMD – Centro de Pesquisa Leônidas Maria Deane
FITC – Isotiocianato de fluorosceína (Fluorescein IsoThioCyanate)
FL1 – Canal 1 de Fluorescência à citometria de fluxo
FMT-HVD – Fundação de Medicina Tropical Dr. Heitor Vieira Dourado
FFg – Frequência de fagocitose
GM-CSF – Fator Estimulador de Colônia de Granulócitos e Monócitos
(Granulocyte macrophage colony-stimulating factor).
GP – Glicoproteina
HBV – Vírus da Hepatite B
HCV – Vírus da Hepatite C
HDL – Lipoproteina de alta densidade (High Density Lipoprotein)
HEMOAM – Fundação de Hematologia e Hemoterapia do Amazonas
HIV – Vírus da Imunodeficiência Humana (Human Immunodeficiency Virus)
IC – Intervalo de Confiança
ICAM – Molécula de adesão intercelular (Intercellular adhesion molecule)
LDL – Lipoproteína de baixa densidade (Low Density Lipoprotein)
IFg- Índice de fagocitose
IFN-y – Interferon gama
IgG – Imunoglobulina G
IL – Interleucina
µL – Microlitro
µM – Micromolar
MAC-1 – Antígeno de macrófago 1 (Macrophage-1 antigen)
xii
Mg – Miligrama
mL – Mililitro
M-CSF – Fator estimulador de Colônias de Macrófagos (Macrophage colonystimulating factor)
MDA – Malondialdeído
MHC – Complexo principal de histocompatibilidade (Major histocompatibility
complex)
MIF – Mediana de intensidade de fluorescência
OMS – Organização Mundial de Saúde
PAIgG – Anticorpos da classe IgG associados a plaquetas
PBMC – Células Mononucleares de Sangue Periférico (Peripheral Blood
Mononuclear Cell)
PCR – Reação em cadeia pela polimerase (Polymerase Chain Reaction)
PDF – Produtos de degradação da fibrina
PE – Ficoeritrina (Phycoerythrin)
PESCLIN – Enfermaria de Pesquisa Clínica da FMT-HVD
Ph – Potencial Hidrogeniônico
PMA – Forbol 12-miristato 13-acetato (Phorbol Miristate Acetate)
PNCM – Plano Nacional de Controle da Malária
PRP – Plasma Rico em Plaquetas
P-selectina – Molécula de adesão celular
PSGL-1- Ligante glicoproteico de P-selectina (P-selectin glycoprotein ligand-1)
RNA – Ácido Ribonucleico (RiboNucleic Acid)
SSC – Granulações das células à citometria de fluxo (Side Scatter)
TCLE – Termo de Consentimento Livre e Esclarecido
TGF-β – Fator de crescimento de transformação - beta (Transforming growth
factor beta)
THP-1 – Linhagem comercial de monócitos
TNF – Fator de Necrose Tumoral (Tumor Necrosis Factor)
TPO – Trombopoietina
TLRs_ Receptores Toll-like (Toll-like receptors)
VPM – Volume Plaquetário Médio
˚C – Graus Celsius
xiii
SUMÁRIO
1 INTRODUÇÃO.......................................................................................
1.1
Aspectos epidemiológicos da malária..........................................
1.2
Aspectos clínicos da malária........................................................
1.3
Aspectos imunológicos da malária: produção de citocinas..........
1.4
Plaquetas......................................................................................
1.4.1 Formação da plaquetas................................................................
1.4.2 Aspectos estruturais e funcionais das plaquetas.........................
1.4.3 Fagocitose de plaquetas por monócitos.......................................
1.4.4 Plaquetopenia...............................................................................
1.4.5 Plaquetopenia na malária.............................................................
1.4.6 Etiopatogenia da plaquetopenia na malária.................................
1.4.7 Relação entre plaquetopenia e citocinas produzidas durante a
infecção por malária.....................................................................
2 OBJETIVOS...........................................................................................
2.1
Objetivo geral...............................................................................
2.2
Objetivos específicos....................................................................
3 MATERIAL E MÉTODOS......................................................................
3.1
Local do estudo............................................................................
3.2
Tipo e tamanho da amostra........................................................
3.3
Critérios de elegibilidade..............................................................
3.4
Critérios de não inclusão.............................................................
3.5
Critérios de exclusão...................................................................
3.6
Seleção dos pacientes.................................................................
3.7
Coleta das amostras e procedimentos laboratoriais ................
3.8
Classificação de pacientes em plaquetopênicos e nãoplaquetopênicos...........................................................................
3.9
Cultura de células THP-1.............................................................
3.10
Isolamento de plaquetas..............................................................
3.11
Teste in vitro de fagocitose de plaquetas....................................
3.12
Avaliação da expressão de P-selectina........................................
3.13
Dosagem de citocinas..................................................................
3.14
Citometria de fluxo........................................................................
3.15
Análise estatística.........................................................................
3.16
Considerações éticas...................................................................
3.17
Limitações do estudo...................................................................
4 RESULTADOS.......................................................................................
4.1
Artigo publicado (resultado)........................................................
5 CONCLUSÃO........................................................................................
6 REFERÊNCIAS BIBLIOGRÁFICAS......................................................
7 ANEXOS................................................................................................
ANEXO A (Termo de Consentimento Livre e Esclarecido – TCLE) ...
ANEXO B (Ficha clínica do participante do estudo) ...........................
ANEXO C (Procedimento Operacional Padrão – POP) .......................
ANEXO D (Parecer do CEP da FMT-HVD..............................................
ANEXO E (Artigos publicados)..............................................................
ANEXO F (Normas da revista PLOS ONE.............................................
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Ficha Catalográfica
C672a
Coelho, Helena Cristina Cardoso.
Avaliação in vitro da fagocitose de plaquetas em pacientes
com malária vivax /. -- Manaus : Universidade do Estado do
Amazonas, Fundação de Medicina Tropical, 2012.
xiii. 141 f. : il.
Dissertação (Mestrado) apresentada ao Programa de PósGraduação em Medicina Tropical da Universidade do Estado do
Amazonas – UEA/FMT, 2012.
Orientador: Profº. Dr. Marcus Vinicius Guimarães de
Lacerda.
1.Malária 2.Plasmodium vivax 3.Plaquetopenia . Título
Ficha Catalográfica elaborada pela Bibliotecária Maria Eliana CDU:
N. Silva,
lotada na
616.928.5
Escola Superior de Ciências da Saúde - UEA
1
1 INTRODUÇÃO
1.1 Aspectos epidemiológicos da malária
Reconhecida como um grande problema para a saúde pública, a malária ocorre
em mais de 109 países e territórios. Segundo a Organização Mundial de Saúde
(OMS), em 2008, foram registrados aproximadamente 243 milhões de casos de
malária no mundo, com uma estimativa de 863 mil óbitos, principalmente em
crianças menores de cinco anos e mulheres grávidas do Continente Africano (1, 2).
As áreas de transmissão da malária no mundo são demonstradas na Figura 1.
Dados da OMS estimam que há entre 70 e 80 milhões de casos por ano de
malária decorrentes da infecção pela espécie Plasmodium vivax. As maiores
prevalências são observadas no sul e leste da Ásia (52%), leste do Mediterrâneo
(15%) e América do Sul (13%). No leste e sul da África, 5% das infecções por
malária são atribuídas a essa espécie, que pode representar entre seis e quinze
milhões dos casos por ano (3, 4).
Na região das Américas, a transmissão ocorre em 21 países, estimando-se que
três em cada 10 pessoas vivem em áreas com diferentes níveis de transmissão. A
malária pelo P. vivax representou 80% dos casos registrados em 2009 nesse
continente. Em conjunto, Brasil, Colômbia, Haiti e Peru foram responsáveis por 90%
dos casos no ano de 2009 (5).
Figura 1. Áreas de transmissão da malária (6).
2
No Brasil, a Amazônia Legal, composta pelos estados do Acre, Amapá,
Amazonas, Pará, Rondônia, Roraima, Maranhão, Mato Grosso e Tocantins, é a
principal região de ocorrência da malária. Em 2010, foram registrados 132.179 casos
nessa região, sendo o Amazonas (33.948 casos), Pará (30.065 casos), Rondônia
(16.401 casos) os responsáveis por 61% deste total (7).
Em Manaus, a Fundação de Medicina Tropical Doutor Heitor Vieira Dourado
(FMT- HVD), que funciona como um centro de referência para as pesquisas
científicas, cuidados de saúde e diagnósticos de doenças tropicais no estado do
Amazonas, atendeu no período entre janeiro de 2007 e dezembro de 2009, 114.404
pessoas que apresentavam quadro clínico febril, tidas como casos suspeitos de
malária. Dessas, por meio do exame da gota espessa a partir de sangue periférico,
27.029 (23,6%) apresentaram a presença do protozoário, o que confirmou a doença
(8).
A espécie P. vivax é responsável pela maioria dos casos no país. No entanto,
nem sempre essa espécie foi predominante. P. falciparum foi a espécie mais
prevalente até a década de 80, quando o número de casos de P. vivax começou a
aumentar relativamente. Em 1988, a incidência relativa dessas duas espécies era de
aproximadamente 50% em cada uma. Essa relação mudou a partir de 1990, quando
P. vivax começou a predominar (44,3% dos casos eram de P. falciparum). Em dados
de 2008, observa-se que aproximadamente 85% dos casos de malária no Brasil
foram causados por P. vivax (1, 9).
Alguns autores consideram que essa inversão de predominância entre as
espécies pode ser em parte, devida à implantação do Plano Nacional de Controle da
Malária (PNCM), que visa ao rápido diagnóstico e tratamento dos casos de malária.
Essas medidas podem controlar a transmissão do P. falciparum de uma forma mais
eficiente que no P. vivax, já que na malária falciparum o surgimento dos gametócitos
na corrente sanguínea leva um maior tempo (de oito a dez dias após a infecção)
comparado com a malária vivax (até três dias após a infecção), facilitando, com o
diagnóstico e tratamento precoces, a eliminação da infecção antes que o doente
transmita P. falciparum para o mosquito (9).
3
Além disso, um outro fator que limita o controle de P. vivax é a sua capacidade de
formar hipnozoítos no fígado, que após um período de tempo variável, transformamse em merozoítos que passam a circular novamente na circulação periférica,
tornando o hospedeiro novamente uma fonte de infecção (4).
Um terceiro ponto a ser considerado é a resistência do P. vivax ao tratamento
com a cloroquina, relatada em diversas partes do mundo (10), inclusive no Brasil.
Em um estudo realizado em Manaus demonstrou-se que a resistência ocorre em até
10% dos casos estudados (11).
A distribuição dos casos de malária no Brasil pode ser observada na Figura 2, na
qual são identificadas as áreas livres de malária (0 casos/1.000 habitantes), áreas
com baixa transmissão (0 - 1 casos/1.000 habitantes), áreas com alta transmissão
(1-100 casos e > 100 casos/1.000 habitantes).
Figura 2. Mapa do Brasil com distribuição de casos por 1.000 habitantes no
Brasil(12).
4
1.2 Aspectos clínicos da malária
A doença clínica está associada a sintomas inespecíficos como calafrios,
vômitos, mal-estar, cefaleia, febre, náuseas, vômitos, mialgia e icterícia. A anemia,
plaquetopenia, hepatomegalia e esplenomegalia também podem ser observadas
(13).
Na literatura são descritos relatos de gravidade associados ao P.vivax,
apresentando síndrome da angústia respiratória aguda (14), disfunção hepática,
icterícia, malária cerebral, insuficiência renal, anemia grave (15, 16), rabdomiólise
(17) ruptura do baço (18) e plaquetopenia grave (19-24).
O desenvolvimento da forma grave da malária provavelmente é resultado de uma
combinação de diversos fatores que envolvem o parasita e o hospedeiro (25), tais
como a variação genética do hospedeiro, idade (crianças são mais acometidas),
resistência a cloroquina, fatores de virulência do plasmódio, infecções mistas, além
dos fatores geográficos e sociais (26).
1.3 Aspectos imunológicos da malária: produção de citocinas
Na malária vivax, a liberação de endotoxinas durante a ruptura das hemácias
parasitadas estimulam as células do sistema imune, culminando com o aumento de
TNF-α e IL-6 (27, 28). Um estudo realizado na Turquia demonstrou um aumento de
IL-1 beta, IL-6 e IL-12, bem como uma correlação positiva entre IL-10 e IL-12 e a
parasitemia e uma correlação negativa entre a IL-8 com a parasitemia no soro de
pacientes infectados com P. vivax (29). Altas concentrações de IFN-γ e IL-10 foram
detectados no soro de crianças com malária falciparum grave (30). Por outro lado,
comparando indivíduos assintomáticos e casos descritos como malária grave, um
estudo realizado no Brasil mostrou que a razão de IFN-γ/IL-10 foi alta em casos
graves, enquanto IL-10 foi elevado em indivíduos assintomáticos (31).
5
1.4 Plaquetas
1.4.1 Formação das plaquetas
As plaquetas possuem um tamanho de 2 a 5 µm e um volume corpuscular médio
(VPM) de 6 a 10 fentolitros e se originarem da fragmentação do citoplasma dos
megacariócitos (19, 32).
Os megacariócitos são células grandes (>60µm), de linhagem mielóide, com um
núcleo poliplóide e diferem de outras células por permanecerem na medula após sua
maturação. Essas células possuem mecanismos especializados em produzir e
liberar as plaquetas na circulação sanguínea através da formação de extensões
citoplasmáticas, denominadas pró-plaquetas. Por meio de brotamento dessas
extensões citoplasmáticas ocorre a formação das plaquetas que são posteriormente
levadas pela corrente sanguínea (Figura 3) (33).
Figura 3. Imagem da formação das plaquetas. Patel et al, 2005, modificado (34).
A produção de plaquetas é estimulada por diversos fatores de crescimento
hematopoiético como o fator estimulador de colônias de granulócitos-monócitos
(GM-CSF, do inglês granulocyte-macrophage colony-stimulating factor), IL-3, IL-6,
IL-11 e trombopoietina (TPO), sendo esse último o mais importante. A TPO é uma
glicoproteína produzida pelo fígado e durante a plaquetopenia seus níveis
apresentam–se elevados (35).
6
1.4.2 Aspectos estruturais e funcionais das plaquetas
A membrana plasmática das plaquetas possui uma aparência rugosa (Figura 4-A),
com pequenas aberturas conectadas ao sistema canicular aberto (Figura 4-B).
Essas características permitem o aumento da superfície e a mudança da forma da
plaqueta durante sua ativação. O sistema canicular funciona como um caminho para
liberação do conteúdo dos grânulos presentes no citoplasma das plaquetas (32).
A
B
C
Figura 4. A- Imagem de uma plaqueta quiescente, apresentando forma discoide,
com superfície rugosa (aumento 30.000 X). B- Aberturas na superfície da plaqueta
conectadas ao sistema canicular aberto (aumento 26.000 X).C- Grânulos α (G) e
grânulos densos (D) no citoplasma de uma plaqueta (aumento32.000 X)(32).
Tanto a superfície das plaquetas quanto o sistema canicular aberto são revestidos
por diversos receptores que possuem a função de facilitar a adesão das plaquetas a
uma superfície danificada, desencadear a ativação plena da plaqueta, promover a
agregação plaquetária e interação com outros elementos celulares. Os principais
receptores envolvidos na hemostasia são os do complexo glicoproteico (GP): Ib-IX-V
(receptor para o fator Von Willebrand-vWF) e IIb-IIIa (receptor para fibrinogênio) (32).
O citoplasma das plaquetas possui dois tipos de grânulos essenciais para as
funções plaquetária: os grânulos α, maiores e mais abundantes no citoplasma e
grânulos densos (Figura 4-C). Proteínas e receptores interiorizados nestes grânulos
são expressos na membrana externa da plaqueta no momento de sua ativação,
como a P-selectina, uma das proteínas responsável pela interação entre plaquetas,
leucócitos e células endotelias, através da ligação com o ligante glicoproteico de Pselectina (PSGL-1). A tabela 1 descreve os principais constituintes dos grânulos
plaquetários (36).
7
As
plaquetas
são
desprovidas
de
moléculas MHC classe II. No
entanto,
elas contêm níveis significativos de moléculas MHC classe I em sua superfície (37).
Outra molécula importante expressa pelas plaquetas é o CD40L, que pode se ligar
ao CD40 expresso tanto no endotélio como em leucócitos (38, 39).
Tabela 1. Grânulos plaquetários e seus principais constituintes (36).
Grânulos α
Fator Plaquetário 4
Fibrinogênio
Fator V
Fator de vonWillebrand
Trombospondina
Fator de crescimento derivado
de plaquetas (PDGF)
Grânulos densos
ATP
ADP
Cálcio
Serotonina
P-selectina
Catecolaminas
As plaquetas quando ativadas também produzem citocinas, como IL-1 β, a qual
pode levar a um aumento da expressão de ICAM1 e IL-6 por células endoteliais
promovendo a adesão de leucócitos ao endotélio vascular (40, 41).
O citoesqueleto é composto por microtúbulos, actomiosina e espectrina e é
responsável por manter a forma da plaqueta quiescente e ativada (42).
As plaquetas possuem uma meia vida de 7 a 10 dias e caso não participem da
coagulação, são removidas da circulação pelo sistema reticuloendotelial (32). À
medida que as plaquetas envelhecem, perdem sua capacidade hemostática (43).
Plaquetas danificadas ou esgotadas são sequestradas e destruídas pelo baço. Além
de ser um importante sítio de sequestro de plaquetas, o baço constitui um
importante reservatório fisiológico de plaquetas (42).
1.4.3 Fagocitose de plaquetas por monócitos
Monócitos são leucócitos que se originam a partir de células progenitoras
presentes na medula óssea e são liberados para o sangue periférico, circulando por
alguns dias antes de se deslocarem para os tecidos, onde são denominados
8
macrófagos. Os monócitos constituem 5 a 10% dos leucócitos presentes no sangue
periférico em humanos e são importantes células efetoras tanto na resposta imune
inata, quanto na adaptativa. À medida que essas células se diferenciam em
macrófagos, adquirem receptores específicos e mecanismos para reconhecer e
fagocitar microrganismos, partículas, células infectadas e células apoptóticas (44).
Os macrófagos possuem diversos receptores, como Toll-like receptors (TLRs), os
receptores acoplados à proteína G, os receptores Fc e C3 e os receptores de
citocinas que atuam para ativar essa célula e induzir a resposta contra
microrganismos fagocitados. A estimulação dos TLRs é o primeiro passo para a
transição do macrófago para célula efetora do sistema imune.
A fagocitose por macrófago é mediada por pelo menos quatro mecanismos que
respectivamente envolve o receptor Fc de IgG, receptor do tipo scavenger, receptor
do tipo lectina e receptores de complemento (44).
O mecanismo de remoção das plaquetas da circulação sanguínea pelos
macrófagos esplênicos ainda não é claramente explicado na literatura. O processo
de ativação das plaquetas envolve diversas mudanças na superfície dessas
estruturas, incluindo a expressão de P-selectina e a perda da assimetria da
membrana. Essas mudanças na membrana das plaquetas podem gerar sinais
moleculares para os macrófagos que desencadeiam a fagocitose (45-47). Dados
demonstram que a ligação das plaquetas aos macrófagos é regulada principalmente
por receptores Fc presentes nos macrófagos que reconhecem IgG ligados a
superfície de plaquetas (48), pela ligação da P-selectina expressa em plaquetas
ativadas ao ligante da P-selectina 1 (PSGL-1) (49), pela
exposição de
fosfatidilserina pelas plaquetas, que se liga a receptores responsáveis por
reconhecer células apoptóticas (49-51) e pela exposição de glicoproteínas Ibα
(CD42b) pelas plaquetas que se ligam aos macrófagos através do Mac-1 (também
chamado de αMβ2 ou CD11b/CD18) (49).
9
1.4.4 Plaquetopenia
Define-se como plaquetopenia a redução do número de plaquetas circulantes no
sangue abaixo de 150.000/µL. A plaquetopenia resulta basicamente de três
processos: produção plaquetária deficiente, destruição plaquetária maior do que sua
taxa de reposição e distribuição anormal de plaquetas no organismo. A classificação
da plaquetopenia baseada em critérios fisiopatológicos está apresentada no quadro
1.
Quadro 1. Classificação fisiopatológica da plaquetopenia (52).
I. Produção plaquetária deficiente
1. Hipoplasias ou supressão de megacariócitos
1.1. Agentes físicos e químicos;
1.2. Anemia aplásica (hipoplasia megacariocítica congênita, síndrome de Fanconi);
1.3. Processos mieloptísicos, infecções virais;
2. Trombopoese ineficaz (distúrbios devidos a deficiência de vitamina B12 ou ácido fólico,
hemoglobinúria paroxística noturna; formas hereditárias);
3. Mecanismos de controle alterados (deficiência de trombopoetina, trombocitopenia
cíclica);
4. Miscelânea (muitas formas hereditárias);
II. Destruição plaquetária acelerada
1.Processos imunológicos
1.1. "Auto-anticorpos" (púrpura trombocitopênica idiopática, produção de anticorpos
induzidos por droga, anemias hemolíticas, lúpus eritematoso sistêmico, distúrbios
linforreticulares, hipertireoidismo);
1.2. Isoanticorpos (devido à incompatibilidade maternofetal; pós transfusão);
1.3. Outros processos imunológicos (alergias, eritroblastose fetal, reações
anafiláticas, complexos imunes relacionados ao HIV);
2. Processos não imunológicos
2.1. Coagulação intravascular disseminada (CID) (complicações obstétricas,
neoplasias, síndrome de Kasabach-Aldrich, infecções);
2.2. Processos microangiopáticos (púrpura trombocitopênica trombótica, válvulas
cardíacas prostéticas);
2.3. Miscelânea (infecções, transfusões maciças, aparelhos de circulação
extracorpórea, ristocetina, algumas formas hereditárias);
III. Distribuição plaquetária anormal
1. Distúrbio esplênico (neoplasias, processos congestivos, infiltrativos e infecciosos e
outras causas desconhecidas);
2. Anestesia hipotérmica;
3. Síndrome Kasabach-Merritt;
10
1.4.5 Plaquetopenia na malária
A plaquetopenia em pacientes com malária é comumente relatada por diversos
autores. Em uma revisão sistemática da literatura, a frequência da plaquetopenia
nesses pacientes variou de 24% a 94% (53).
A plaquetopenia grave (contagem de plaquetas <50.000/µL de sangue) também
foi relatada na malária. Um estudo realizado na Venezuela constatou que 32 dos 75
pacientes com malária e plaquetopênicos (43%) apresentavam grave plaquetopenia
(54). Em um estudo realizado no Brasil, a plaquetopenia foi encontrada em 70,8%
(n=168) dos pacientes com malária atendidos na FMT-HVD, entre 2004 e 2006.
Desses pacientes, 8,9% apresentavam uma contagem de plaquetas <50.000/µL
(19).
Em 124 pacientes com malária (64 pacientes por P. falciparum e 60 por P. vivax),
80,6% (n=100) apresentaram contagem de plaquetas abaixo de 150.000/µL,
constatando a alta frequência de plaquetopenia, principalmente no grupo dos
infectados por P. vivax (93,33%, contra 71,87% nos infectados por P. falciparum)
(21).
A plaquetopenia é um fato tão comum em pacientes com malária vivax, que
alguns autores descrevem que essa alteração hematológica juntamente com a febre
pode ser indicativo de infecção por malária (55-57).
1.4.6 Etiopatogenia da plaquetopenia na malária
Estudos envolvendo a etiopatogênese da plaquetopenia na malária são
realizados há pelo menos quatro décadas sendo atribuído como causas de
plaquetopenia: destruição imunomediada, sequestro esplênico e não-esplênico,
ativação plaquetária e ativação da cascata de coagulação, apoptose de plaquetas,
estresse oxidativo e alteração na produção pela medula óssea. Entretanto, até o
momento pouco se conhece sobre os mecanismos exatos que causam essa
alteração hematológica e há algumas controvérsias entre os estudos já realizados.
11
a) Destruição imunomediada, sequestro esplênico e não esplênico de
plaquetas
Alguns autores acreditam que durante a infecção na malária há uma produção de
anticorpos
antiplaquetários
e
formação
de
imunocomplexos
levando
à
plaquetopenia. Segundo os autores, isso ocorre provavelmente devido a ligação
dessas partículas às plaquetas. Posteriormente, essas plaquetas são fagocitadas
por macrófagos localizados no baço (56).
Especula-se que a plaquetopenia na malária aguda causada por P. falciparum é
resultante do sequestro das plaquetas pelo baço (60). De fato, é sabido que a
esplenomegalia é um achado comum em pacientes com malária e análises
histológicas revelaram um acúmulo de macrófagos no baço desses pacientes.
Observa-se que esses mesmos macrófagos esplênicos estão envolvidos na
fagocitose de eritrócitos normais e parasitados (61).
Um estudo experimental, realizado em ratos infectados com P. chabaudi,
demonstrou que os animais esplenectomizados não apresentavam redução no
número de plaquetas, ao contrário dos animais não-esplenectomizados, salientando
que a plaquetopenia estava associada ao sequestro pelo baço (62).
Dados
adicionais
foram
apresentados
em
um
estudo
com
pacientes
plaquetopênicos com malária vivax e falciparum não-complicada, os quais
evidenciaram que o sequestro de plaquetas na malária não-complicada parece ser
difuso, não apenas no baço ou no fígado. Esse trabalho também demonstrou que há
uma redução na vida média das plaquetas (de dez para dois dias) e que essa
redução foi menos intensa no paciente com malária vivax (4 dias) comparado com
os pacientes com malária falciparum (0,60 a 1,66 dias) (61).
Um trabalho de fundamental importância, apesar de antigo, demonstrou que 80%
dos monócitos do esfregaço sanguíneo de um paciente com malária falciparum
apresentavam fagocitose de plaquetas, sugerindo que esta fagocitose poderia ser
um importante mecanismo envolvido no desenvolvimento da plaquetopenia nos
casos de malária (63).
12
O alto nível do fator estimulador de colônia de macrófagos (M-CSF do inglês
Macrophage colony-stimulating factor) em pacientes com malária tanto vivax quanto
falciparum está associado à plaquetopenia, reforçando que os macrófagos teriam
um papel fundamental na destruição dessas partículas, visto que esse fator é
responsável pelo aumento da atividade de macrófagos (64).
Outro trabalho relevante sobre a fagocitose plaquetária, apesar de não ser
desenvolvido com pacientes com malária, demonstrou que o percentual de
fagocitose e os níveis de PAIgG (anticorpos da classe IgG associados a plaquetas)
aumentaram significativamente em indivíduos com dengue na fase aguda
comparando com voluntários saudáveis. Os autores encontraram ainda uma
correlação inversa entre a contagem de plaquetas e a porcentagem de fagocitose
(P=0,011) e os níveis de PAIgG (P=0,041) (65).
Estudos prévios realizados por esse mesmo grupo de pesquisadores
encontraram tanto IgG anti vírus da dengue quanto RNA do vírus do dengue em
plaquetas de pacientes infectados com dengue, sugerindo que imunocomplexos
ligados às plaquetas podem contribuir para o aumento de fagocitose dessas
partículas nesses pacientes (66).
Na malária vivax e falciparum observa-se uma correlação inversa entre a
contagem de plaquetas e a parasitemia, como se de alguma forma a quantidade de
antígenos contribuíssem para o desenvolvimento da plaquetopenia (19, 56).
Entretanto, existem alguns estudos contraditórios onde essa correlação não foi
encontrada (54, 67).
Ensaios de fagocitose utilizando imunocomplexos circulantes de pacientes com
malária vivax, demonstraram que a fagocitose de plaquetas normais por células
THP-1 (linhagem comercial de monócitos), estimuladas com PMA (Phobol Miristate
Acetate), ocorre de forma satisfatória, entretanto, a hipótese de que os
imunocomplexos poderiam aumentar a fagocitose de plaquetas na malária não foi
confirmada.
Nesse
trabalho,
verificou-se
também que
IgG
extraídas
dos
imunocomplexos circulantes não reconheceram antígenos da superfície plaquetária,
nem induziram a plaquetopenia, em camundongos, sugerindo que não há formação
de auto-anticorpos anti-plaquetários. Um outro aspecto abordado pelo autor é que os
13
pacientes recuperam sua contagem de plaquetas logo após a negativação da
parasitemia, e isso não aconteceria, caso houvesse a presença de auto-anticorpos,
devido à prolongada meia-vida da IgG que pode chegar a meses ou anos (19).
Os níveis de PAIgG foram previamente estudados em um trabalho no qual 16 de
17 pacientes plaquetopênicos com malária apresentaram um aumento desses
anticorpos e tanto os níveis de PAIgG quanto o número de plaquetas retornaram ao
normal após ausência do parasito na circulação. Além disso, foi confirmado que
ligações de IgG e ligações de imunocomplexos às plaquetas seriam improváveis de
causarem a plaquetopenia na malária. Segundo o autor, o que possivelmente
acontece na malária é a ligação de antígenos parasitários à superfície das plaquetas
durante a infecção aguda da malária e posteriormente a ligação secundária de
anticorpos antimaláricos a esses antígenos (68).
Um relato de dois casos de pacientes com malária vivax, plaquetopênicos, com
número normal de megacariócitos e sem evidências de coagulação vascular
dissemianda (CID), também demonstrou uma relação inversa entre os níveis de
PAIgG e a contagem de plaquetas. Essa associação foi relatada antes da detecção
de anticorpos contra a malária, sugerindo que os primeiros anticorpos produzidos
contra o parasito devem se ligar às plaquetas. De fato o aumento de PAIgG pode ser
devido a vários fatores, como aumento da expressão de IgG da superfície ou dos αgrânulos das plaquetas, aumento de anticorpos contra as plaquetas, aumento de
imunocomplexos circulantes e aumento de IgG não específicos ou anticorpos contra
o parasito. No entanto, os autores também excluem o aumento de PAIgG pelos
imunocomplexos circulantes e IgG não específicos, já que estes estavam normais no
sangue dos pacientes estudados e excluem anticorpos contra plaquetas devido ao
fato dos níveis de PAIgG retornarem ao normal sem uma terapia imunossupressora
(69).
A destruição auto-imune de plaquetas foi relatada como possível mecanismo em
um paciente procedente do Senegal, visto que foi identificado por citometria de fluxo
a presença de auto-anticorpos plaquetários (ligados a GPIb/IIIa). Porém, além do
estudo descrever apenas um paciente, não excluiu outras causas concomitantes da
plaquetopenia, como o vírus HIV (70).
14
Em um artigo de revisão sobre a malária falciparum, os autores acreditam que os
mecanismos imunes que causam a plaquetopenia surgem tardiamente no curso da
infecção e não pode ser a explicação de plaquetopenia leve ou moderada observada
no início da infecção malárica. Desta forma, os autores pressupõem que as
principais causas que iniciam a plaquetopenia na malária aguda parecem estar mais
relacionadas ao consumo generalizado de plaquetas no endotélio danificado (71).
b) Ativação plaquetária e ativação da cascata de coagulação
Marcadores de ativação plaquetária como o tromboxano, P-selectina e
micropartículas de plaquetas foram descritos em níveis elevados em pessoas
infectadas pelo P. falciparum e em modelos animais com P. berghei (72). Essa
ativação acaba levando a adesão das plaquetas ao endotélio, reduzindo seu número
na circulação (71).
Alguns autores acreditam que na fase inicial da malária parece haver um
aumento da ativação e agregação plaquetária. Um dos prováveis motivos para isso
ocorrer é a liberação da adenosina difosfato pelas hemácias parasitadas. Dessa
forma, as plaquetas agregadas são removidas da circulação, o que certamente
contribui com a redução de plaquetas na circulação sanguínea (71).
A ativação de plaquetas possivelmente acontece pela ligação da proteína
PfEMP1 do plasmódio nos receptores CD36 das plaquetas. Essa ligação leva a duas
potenciais consequências: formação de microagregados de hemácias infectadas
com as plaquetas ativadas, podendo levar a uma oclusão dos vasos sanguíneos e
adesão desses agregados no endotélio, ativando ainda mais as células endoteliais e
plaquetas (72).
No entanto, não é possível afirmar que esse evento isoladamente seja
responsável pela agregação plaquetária. A ativação das plaquetas pode ser causada
por outros diversos fatores, muitas vezes comuns na malária, como por exemplo,
presença de imunocomplexos, citocinas, sistema complemento, fator tecidual
liberado por lesão endotelial, entre outros (56, 71, 72).
15
Em uma pesquisa experimental, na qual voluntários foram infectados por P.
falciparum, verificaram que o número de plaquetas diminuiu entre 7 a 9 dias após a
infecção. Proporcionalmente a essa redução ocorreu o aumento do fator de von
Willebrand (marcador da ativação crônica das células endoteliais), propeptídeo von
Willebrand (marcador de ativação aguda das células endoteliais) e atividade do fator
de von Willebrand reforçando a hipótese de que a aglutinação de plaquetas pode ser
responsável pela plaquetopenia na malária falciparum, porém esse mecanismo não
foi comprovado com pacientes infectados por P. vivax (73).
Apesar da elevada concentração de citocinas pró-coagulantes, como TNF-α
serem encontrados nas infecções por malária vivax e falciparum, o distúrbio de
coagulação é consistentemente detectável somente em falciparum, sendo a ativação
da cascata de coagulação e depuração esplênica mecanismos responsáveis pela
redução de plaquetas na malária por essa espécie. Já na malária vivax, a
plaquetopenia possivelmente seria causada por um mecanismo imunológico (74).
Contudo, em um estudo com um grupo de pacientes infectados por P. vivax e outro
por P. falciparum não houve diferença na frequência de plaquetopenia ou
plaquetopenia grave entre esses pacientes, sugerindo mecanismos semelhantes de
destruição plaquetária pelos dois parasitos (19).
Estudos prévios associam a plaquetopenia da malária falciparum com CID (7577). No entanto, em muitos casos essa alteração hematológica não demonstrou
associação com aumento de produtos da degradação de fibrina, sugerindo que a
plaquetopenia poderia estar mais associada com uma lesão endotelial e consumo
isolado de plaquetas (78).
Dados comprobatórios foram demonstrados em um trabalho na Tailândia, onde a
maioria dos pacientes com malária e plaquetopenia grave (plaquetas <50.000/µL)
não estavam associados à CID (79).
Há evidências de que a ativação plaquetária medida pela P-selectina pode
intensificar a plaquetopenia na malária falciparum grave, mas parece não contribuir
para a plaquetopenia na malária falciparum não-grave e na malária vivax, visto que o
16
aumento da P-selectina não foi observado nesses pacientes em um ensaio clínico
(64).
c) Apoptose de plaquetas
Apesar das plaquetas serem desprovidas de núcleo, a apoptose de plaquetas
tem sido relacionada com a plaquetopenia em doenças infecciosas (80, 81). Existem
diversas maneiras de avaliar a apoptose em plaquetas, dentre elas a ativação das
caspases e expressão de fosfatildilserina na superfície plaquetária. Após a
apoptose, essas plaquetas são reconhecidas por macrófagos e fagocitadas.
Em um estudo realizado com pacientes com infecção secundária pelo vírus da
dengue demonstrou uma correlação positiva entre a apoptose e a fagocitose de
plaquetas (80). Em um modelo de malária experimental em camundongos, a
plaquetopenia foi acompanhada por um aumento na ativação de caspases
plaquetárias e uma aumento na liberação de micropartículas plaquetárias. Essas
caspases tiveram um papel importante na queda nos níveis de plaquetas. Ao
usarem um inibidor de caspases, a plaquetopenia nos animais infectados foi
atenuada e houve uma redução nos níveis de micropartículas plaquetárias (Piguet,
2002).
d) Estresse Oxidativo
Existem hipóteses de que o estresse oxidativo pode estar relacionado com a
plaquetopenia na malária. Espécies reativas de oxigênio podem ter um papel
importante na alteração estrutural e funcional das plaquetas. Em pacientes com
malária vivax, demonstrou-se uma correlação negativa entre a contagem de
plaquetas e peroxidação de lipídios plaquetários (82). Em adição a esses dados,
observou-se uma redução sérica do colesterol total e da concentração de HDL e
LDL em indivíduos com malária, o qual pode ser justificado pela peroxidação lipídica
(83).
Em um estudo transversal realizado em 2006 na FMT-HVD foram avaliados
marcadores de estresse oxidativo como o malondialdeído (MDA) e antioxidantes em
pacientes infectados por P. vivax com e sem plaquetopenia. Este estudo verificou
17
que os níveis de MDA tanto no plasma quanto nas plaquetas foram mais altos em
pacientes com plaquetopenia (<150.000/µL). Levando em consideração as
informações que as membranas de plaquetas são pouco resistentes ao estresse
oxidativo e que são mais delgadas que as membranas de hemácias, se houver uma
lise dos eritrócitos por estresse oxidativo, a lise das plaquetas por esse mesmo
mecanismo será inevitável. Outro resultado interessante demonstrado nesse
trabalho foi a forte correlação inversa entre a contagem de plaquetas e os níveis de
glutationa-peroxidase plaquetária, uma enzima responsável pela redução de
peróxido de lipídios. O aumento dessa enzima em pacientes plaquetopênicos na
malária vivax pode ser um mecanismo de compensação antioxidante pelas
plaquetas expostas ao estresse oxidativo (84).
e) Alteração na produção de plaquetas
Não há uma clara evidência que a plaquetopenia seja causada pelo
comprometimento da medula óssea. Por um período, acreditava-se que o plasmódio
pudesse invadir a medula óssea e alterar a produção de plaquetas. Um relato
isolado desse fato menciona o encontro de trofozoítos de P. vivax no interior de
plaquetas sugerindo que a invasão ocorra na circulação periférica e não na medula
posto que parasitas não foram encontrados em megacariócitos (85).
Exames da medula óssea de 89 crianças com malária falciparum revelaram
megacariócitos menos lobulados e imaturos, além de serem encontrados em
números elevados indicando provavelmente uma produção acelerada de plaquetas
(86).
A regulação e a produção de TPO foram estudadas em pacientes com malária e
demonstrou estar normal, o que deixa claro que não há inibição na produção de
plaquetas (87).
Há também a hipótese que citocinas produzidas no momento da infecção da
malária possam comprometer a medula. Em um trabalho realizado com crianças
com malária falciparum grave no Quênia, verificou-se que pacientes com a
contagem de plaquetas menor que 150.000/µL apresentavam elevada concentração
18
de IL-10 no plasma comparado com pacientes com número de plaquetas acima de
150.000/µL. No entanto, o trabalho não menciona a avaliação da medula óssea (67).
Estudos anteriores já tinham evidenciado essa participação da IL-10 na
plaquetopenia. A administração de baixa dose de IL-10 recombinante humana
(8µg/kg/dia) diminuiu a produção plaquetária em voluntários saudáveis. Nesse
mesmo estudo, houve uma redução no sequestro de plaquetas pelo baço desses
voluntários. Além disso, observou uma diminuição do número de colônias
formadoras de megacariócitos, comparado com os pacientes que receberam
placebo ao invés de IL-10. Assim, esses resultados sugerem que a plaquetopenia
pode ser causada, em parte, por uma redução na produção de plaquetas na medula
óssea (88).
1.4.7 Relação entre plaquetopenia e citocinas produzidas durante a infecção
por malária
Além da citocina IL-10, como foi mencionado no item anterior, outras citocinas
têm sido relacionadas com a plaquetopenia nos pacientes com malária. A dosagem
sérica de TNF-α foi inversamente correlacionado com a plaquetimetria em um
estudo realizado com 83 pacientes com malária vivax, na cidade de Belém (Pará Brasil) (89). No entanto, essa correlação não foi encontrada em outro estudo
realizado no Brasil (90).
Dados contraditórios sobre a relação da IL-1 com a plaquetimetria também são
encontrados. Avaliando a concentração da IL-1 em pacientes com malária vivax e
falciparaum, não se detectou correlação com a contagem de plaquetas (90). Em
outro estudo o aumento da citocina IL-1 estava associado com a plaquetopenia em
pacientes com malária vivax. Além da IL-1, esse estudo verificou que o aumento de
IL-6, IL-10 e TGF-β também apresentavam uma associação com a redução no
número de plaquetas (91).
É possível evidenciar que há alguns mecanismos postulados como a causa da
plaquetopenia na malária, porém poucos são os estudos que avaliam o papel da
fagocitose de plaquetas nessa alteração hematológica.
19
O desenvolvimento de estudos esclarecedores sobre a patogênese da
plaquetopenia na malária possivelmente contribuirá para o entendimento dos
mecanismos determinantes da malária, inclusive dos casos complicados, visto que
existe uma forte associação entre plaquetopenia e malária grave. Além disso,
estudos sobre esse assunto poderão auxiliar no desenvolvimento de um tratamento
de suporte da plaquetopenia na malária, menos empírico, deduzindo que seja mais
fácil de tratar ou até evitar essa alteração hematológica conhecendo-se a sua causa.
20
2 OBJETIVOS
2.1 Objetivo geral
Avaliar o papel da fagocitose na plaquetopenia da malária causada pelo P. vivax.
2.2 Objetivos específicos
2.2.1 Determinar o índice de fagocitose de plaquetas em pacientes com malária
vivax, com e sem plaquetopenia;
2.2.2 Verificar a correlação entre o índice da fagocitose e a contagem de plaquetas
no sangue periférico dos pacientes com malária vivax;
2.2.3 Avaliar o perfil de citocinas (IL-2, IL-4, IL-6, IL-10, IL17, IFN-γ e TNF-α) no soro
dos pacientes com malária vivax;
2.2.4 Verificar se existe correlação entre a contagem de plaquetas no sangue
periférico e fagocitose de plaquetas com concentrações séricas de citocinas em
pacientes com malária vivax;
2.2.5 Verificar a ativação de plaquetas de pacientes com malária vivax.
21
3 MATERIAL E MÉTODOS
3.1 Local do estudo
O estudo foi realizado em três instituições, todas localizadas na cidade de
Manaus, estado do Amazonas: (1) Fundação de Medicina Tropical Dr. Heitor Vieira
Dourado (FMT- HVD), onde ocorreu a seleção, avaliação da história clinica do
paciente, coleta de material biológico, avaliação laboratorial do paciente e testes de
fagocitose; (2) Fundação de Hematologia e Hemoterapia do Amazonas (HEMOAM),
local escolhido para realizar as análises dos ensaios de fagocitose e ativação
plaquetária; (3) Centro de Pesquisa Leônidas Maria Deane (CPqLMD) - Fundação
Oswaldo Cruz (FIOCRUZ), onde ocorreu a análise das concentrações de citocinas.
A FMT- HVD é o centro de referência para o diagnóstico e tratamento da malária
no Amazonas, além de ser também referência no ensino, pesquisa e assistências
nas doenças infecciosas e parasitárias do estado. Conta com uma Unidade
Ambulatorial, Unidade de Internação Hospitalar Dr. Nelson Antunes, Laboratório de
Análises Clínicas e as Gerências de Pesquisas, dentre as quais a Gerência de
Malária que possui laboratórios especializados para o diagnóstico de rotina e
pesquisa em malária (Microscopia, Sorologia, Biologia Molecular, Cultura in vitro de
plasmódio). A instituição conta também com uma enfermaria de pesquisa clínica
(PESCLIN),
setor
com
10
(dez)
leitos
exclusivamente
destinado
para
acompanhamento de indivíduos incluídos em projetos de pesquisa.
O HEMOAM, além de ser o centro referencial de diagnóstico e tratamento de
doenças hematológicas na região Norte, é uma Instituição de Pesquisa cadastrada
junto ao CNPq e à FAPEAM. Possui uma estrutura laboratorial para fins de pesquisa,
que conta com um citômetro de fluxo FACS Calibur – BD.
O CPqLMD possui uma estrutura laboratorial com uma sala específica de
citometria, equipada com um citômetro de fluxo FACSCanto™ II – BD.
22
3.2 Tipo e tamanho da amostra
Trata-se de amostragem não probabilística, dos pacientes da demanda
espontânea da FMT-HVD. Foram selecionados 35 pacientes para os ensaios de
fagocitose e 8 indivíduos que formaram o grupo dos controles, com a finalidade de
se estabelecer uma prova de conceito das alterações de fagocitose plaquetária na
malária.
3.3 Critério de elegibilidade
Pacientes com diagnóstico microscópico de malária por P. vivax.
3.4 Critérios de não-inclusão
4.4.1 Gestantes;
3.4.2 Pacientes menores de 18 anos;
3.4.3 Pacientes na vigência de tratamento antimalárico;
3.4.4 Pacientes com infecção mista (P. vivax e P. falciparum);
3.4.5 Pacientes com doenças de natureza imunológica conhecidas;
3.4.6 Pacientes com história de sangramento espontâneo frequente;
3.4.7 Pacientes com história de comorbidade com potencial de alterar o número das
plaquetas;
3.4.8 Pacientes em uso crônico de medicamentos (Quadro 2) com o potencial de
causar plaquetopenia;
A não-inclusão de pacientes gestantes se deve a ocorrência de plaquetopenia de
etiologia adversa daquela estudada neste trabalho. A não-inclusão de pacientes
menores de 18 anos se justificou pelo grande volume de sangue necessário para os
exames laboratoriais e para a realização dos experimentos estabelecidos no estudo.
Medicamentos
antimaláricos,
como
o
quinino,
podem
causar
alterações
hematológicas, motivo pelo qual pacientes na vigência de tratamento por esses
medicamentos não foram incluídos no estudo (92).
23
Pacientes com infecção mista pelo P. falciparum e P. vivax também não foram
incluídos, visto que o objetivo do proposto estudo é avaliar essa alteração
hematológica na espécie P. vivax.
3.5 Critérios de exclusão
3.5.1 Pacientes com exame sorológico positivo para vírus da imunodeficiência
humana (HIV), vírus da hepatite B (HBV), vírus da hepatite C (HCV) ou vírus do
dengue;
3.5.2 Pacientes com exame de PCR positivo para infecção mista P. vivax e P.
falciparum.
Quadro 2. Medicamentos que podem causar plaquetopenia (93).
Abciximab
Ácido aminossalicílico
Ácido nalidíxico
Ácido valpróico
Aminoglutetimida
Amiodarona
Anfotericina B
Captopril
Carbamazepina
Cimetidina
Clorpromazina
Clorotiazida
Clorpropamida
Danazol
Deferroxamina
Diatrizoato de meglumina
Diazepam
Diazóxido
Diclofenaco
Dietiletilbestrol
Digoxina
Eptifibatide
Fenitoína
Fluconazol
Furosemida
Haloperidol
Heparina
Hidroclorotiazida
Ibuprofeno
Interferon-α
Isoniazida
Levamisol
Lítio
Metildopa
Minoxidil
Nafazolina
Ouro
Oxipenbutazona
Oxitetraciclina
Paracetamol
Penicilina
Piperacilina
Procainamida
Quinidina
Quinino
Ranitidina
Rifampicina
SulfametaxozolTrimetoprima
Sulfassalazina
Sulfissoxazol
Sulindac
Tamoxifeno
Tiotixeno
Tirofiban
Vancomicina
A exclusão dos pacientes relacionados nos itens 4.6.1 e 4.6.2 foi realizada com o
objetivo de afastar outras causas de plaquetopenia que não seja a infecção por
malária vivax.
24
3.6 Seleção dos pacientes
A Seleção dos pacientes ocorreu de dezembro de 2011 a junho de 2012.
Os indivíduos foram selecionados para pesquisa após a confirmação do diagnóstico
de malária, por meio do exame de gota espessa, no Laboratório de Microscopia da
Gerência de Malária da FMT-HVD. A história clínica desses pacientes foi avaliada
através de um questionário presente na Ficha Clínica do Participante do Estudo
(Anexo E) e em seguida amostras de sangue foram coletadas para a avaliação
laboratorial e coleta de plaquetas.
Os indivíduos saudáveis foram selecionados obedecendo aos mesmos critérios
dos pacientes infectados por malária.
3.7 Coleta das amostras e exames laboratoriais
Após a avaliação da história clínica, os pacientes foram encaminhados a sala de
coleta na FMT-HVD. Coletou-se a vácuo, aproximadamente 20 mL de sangue
venoso periférico: em tubos contendo K2EDTA para a realização do hemograma e
ensaios de PCR para malária; e em tubos contendo citrato de sódio 3,8% para a
contagem de plaquetas e separação do plasma rico em plaquetas. O processamento
das amostras ocorreu até 2 horas após a coleta. Os resultados dos exames também
foram registrados na Ficha Clínica do Participante do Estudo (Anexo E).
Além do uso de tubos com anticoagulantes,
utilizaram-se tubos sem
anticoagulantes, para a coleta de sangue venoso periférico, com o objetivo de
separar o soro para a realização dos exames sorológicos para HIV, hepatite B,
hepatite C; dengue; leptospirose e dosagem de citocinas. O soro foi armazenado em
temperatura de -80 °C até o momento da realização dos exames.
Os ensaios de fagocitoses foram realizados imediatamente após a coleta de
plaquetas, ou seja, utilizando plaquetas frescas.
25
Os hemogramas e as contagens de plaquetas foram realizados no laboratório de
recepção de amostras de pesquisa da Gerência da Malária da FMT-HVD, em
aparelho automatizado do tipo SYSMEX KX-21N.
Os exames sorológicos para HIV, hepatite B, hepatite C e dengue foram
realizados no Laboratório de Virologia da FMT- HV.
Para o teste anti-HIV foi
utilizado o kit comercial Rapid Check HIV 1&2 ™. Para determinação das hepatites
virais Kits foram utilizados: a) para Hepatite B, foi verificado a presença do antígeno
de superfície HbsAg pelo kit imunoenzimático HBsAg ELISA Bioeasy®; b) para
Hepatite C, utilizou o teste imunoenzimático para determinação de anticorpo antiHCV pelo kit HCV ELISA Bioeasy®. Para o diagnóstico de dengue foi realizado a
pesquisa da proteína NS1 pelo Kit Dengue EDEN test Bioeasy®. Para o diagnóstico
de Leptospirose utilizou o teste imunocromatográfico Leptospira IgG/IgM (SD
BioLine®), que detecta anticorpo IgG e IgM contra a Leptospira interrogans.
Para a confirmação da infecção malárica por P. vivax foi realizado o diagnóstico
molecular. Para extração de DNA foi utilizado o kit QIAamp®
Blood Mini Dit
(Qiagen, Hilden, Alemanha), usando 200µL de sangue total, conforme instrução do
fabricante. Na realização de PCR em tempo real, o DNA foi amplificado usando
Applied Biosystems 7500 Fast System com primers e sondas TaqMan (94).
3.8 Classificação de pacientes em plaquetopênicos e não-plaquetopênicos
A tabela 2 demonstra classificação dos pacientes em plaquetopênicos (grave e
não-grave) e não-plaquetopênicos conforme a contagem de plaquetas.
Tabela 2. Classificação dos pacientes de acordo com a contagem de plaquetas.
Contagem de plaquetas/µL
Classificação
<50.000
Plaquetopênicos Graves
50.000 a 149.000
Plaquetopênicos Não-Graves
= ou > 150.000
Não-plaquetopênicos
26
3.9 Cultura de células THP-1
As células THP-1 foram cultivadas em meio RPMI suplementado com 10% de
soro bovino fetal (FBS) e 0,1% de gentamicina. Posteriormente as células THP-1
foram ativadas com PMA conforme descrito no POP_MAL_LB_011_v01_PT- Anexo
C.
3.10 Isolamento de plaquetas
As plaquetas
foram isoladas a
partir de
PRP
conforme
descrito
no
POP_MAL_LB_010_v01_PT – Anexo C.
3.11 Teste in vitro de fagocitose de plaquetas
O
procedimento
do
teste
de
fagocitose
está
detalhado
no
POP_MAL_LB_011_v01_PT– Anexo C. A figura 5 demonstra as etapas do
experimento de fagocitose de plaquetas.
3.12 Avaliação da expressão de P-selectina
A expressão da P-selectina foi avaliada utilizando anticorpo anti CD62P humano
conjugado
com
PE
(BD
Pharmingen™),
conforme
descrito
no
POP
POP_MAL_LB_010_v01_PT – Anexo C.
3.13 Dosagem de citocinas
A dosagem de citocinas (IL-2, IL-4, IL-6, IL-10, TNF-α, IFN-γ e IL-17) foi realizada
no CPqLMD-FIOCRUZ, utilizando o kit da BD Cytometric Bead Array (CBA) Human
Th1/Th2/Th17 Citokine e o citômetro de fluxo BD FACS Calibur, conforme instruções
do fabricante.
27
Figura 5. Etapas do experimento de fagocitose de plaquetas.
3.14 Citometria de fluxo
Em citômetro de fluxo, as células THP-1 foram previamente selecionadas (gate)
pelo tamanho (forward scatter/FSC-H) e pela densidade interna (side scatter/SSCH), excluindo-se, portanto, as plaquetas marcadas não fagocitadas. Posteriormente,
avaliou-se a mediana de intensidade de fluorescência (MIF) emitida no canal FL1-H
por cada célula THP-1 contada no gate. A frequência de fagocitose de plaquetas
(FFg) foi determinada pela contagem de células positivas para CMFDA nesse canal.
Possivelmente o tamanho das plaquetas pode interferir na intensidade de
fluorescência, pressupondo que plaquetas maiores terão maior quantidade de
28
CMFDA e desta forma irão emitir uma maior intensidade de fluorescência. Dados
demonstram que o VPM dos pacientes com plaquetopenia é maior comparado com
os pacientes sem plaquetopenia (19, 67). Assim, para padronização dos valores de
fagocitose de plaquetas foi criado um índice de fagocitose (IFg) utilizando a fórmula
IFg = MIF x FFg/100 x VPM.
3.15 Análise estatística
As análises estatísticas foram realizadas utilizando o programa GraphPad Prism
versão 5.00 (GraphPad Software, CA,US), também utilizado para a construção dos
gráficos. Teste de Kolmogorov-Smirnov foi utilizado para determinar a normalidade
dos dados. A correlação entre variáveis foi analisada através do coeficiente de
correlação de Spearman. As comparações entre 2 grupos foram analisadas usando
o teste U de Mann–Whitney. Teste de Wilcoxon foi utilizado nas análises pareadas.
Para comparar frequência foi aplicado o teste Qui-quadrado ou teste exato de
Fisher. A significância foi considerada em caso de p<0,05.
3.16 Considerações éticas
O projeto foi submetido à apreciação do Comitê de Ética em Pesquisa (CEP) da
FMT-HVD, no dia 13 de maio de 2011, e foi aprovado no dia 29 de julho de 2011 sob
registro no CEP n. 1610-11, CAAE- 0029.0-114.114-11 (Anexo D).
Todos os voluntários que aceitaram a participar do estudo assinaram o TCLE
(Anexo A) após o devido esclarecimento verbal dos objetivos e métodos do projeto.
Uma cópia do TCLE foi entregue ao paciente e outra cópia foi arquivada sob
responsabilidade do pesquisador responsável.
O presente projeto não ofereceu risco para o paciente, já que o a única técnica
utilizada foi a punção venosa de sangue periférico utilizando material descartável,
sendo realizada por profissionais de saúde que compõem a equipe de pesquisa. A
identidade do indivíduo será mantida em sigilo em qualquer publicação futura
resultante do estudo, e ainda, não houve nenhum custo para o paciente em relação a
qualquer exame.
29
Todos pacientes do estudo receberam o devido tratamento antimalárico gratuito
preconizado pelo Ministério da Saúde do Brasil (95), inclusive os que não aceitaram
participar da pesquisa.
3.17 Limitações do estudo
Através da metodologia desse estudo, não foi possível diferenciar se a
fluorescência medida pela citometria de fluxo nos monócitos corresponde apenas às
plaquetas internalizadas (fagocitadas) ou também às plaquetas aderidas à superfície
dos monócitos. No entanto, considera-se que se as plaquetas aderiram à superfície
dos monócitos, em algum momento essas partículas iriam ser internalizadas.
Não se pode garantir que os achados da fagocitose in vitro utilizando monócitos
de uma linhagem comercial possam ser extrapolados para o que acontece com os
macrófagos esplênicos.
Os participantes selecionados poderão ter alguma doença não diagnosticada
pelos exames laboratoriais ou história clínica, que possa alterar a função ou a
contagem das plaquetas.
30
4 RESULTADOS
Os resultados e discussão deste trabalho estão apresentados na forma de
artigo científico, apresentado a seguir, segundo as normas de publicação da Plos
One.
Thrombocytopenia in Plasmodium vivax Malaria Is
Related to Platelets Phagocytosis
Helena Cristina C. Coelho1,2, Stefanie C. P. Lopes3, João Paulo D. Pimentel4,6, Paulo A. Nogueira4,
Fábio T. M. Costa3, André M. Siqueira1,2, Gisely C. Melo1, Wuelton M. Monteiro2,5, Adriana Malheiro5,6,
Marcus V. G. Lacerda1,2*
1 Universidade do Estado do Amazonas, Manaus, Amazonas, Brazil, 2 Fundação de Medicina Tropical Dr. Heitor Vieira Dourado, Manaus, Amazonas, Brazil, 3 Universidade
Estadual de Campinas, Campinas, São Paulo, Brazil, 4 Instituto Leônidas e Maria Deane, Fiocruz, Manaus, Amazonas, Brazil, 5 Universidade Federal do Amazonas, Manaus,
Amazonas, Brazil, 6 Fundação de Hematologia e Hemoterapia do Amazonas, Manaus, Amazonas, Brazil
Abstract
Background: Although thrombocytopenia is a hematological disorder commonly reported in malarial patients, its
mechanisms are still poorly understood, with only a few studies focusing on the role of platelets phagocytosis.
Methods and Findings: Thirty-five malaria vivax patients and eight healthy volunteers (HV) were enrolled in the study.
Among vivax malaria patients, thrombocytopenia (,150,000 platelets/mL) was found in 62.9% (22/35). Mean platelet
volume (MPV) was higher in thrombocytopenic patients as compared to non- thrombocytopenic patients (p = 0.017) and a
negative correlation was found between platelet count and MPV (r = 20.483; p = 0.003). Platelets from HV or patients were
labeled with 5-chloromethyl fluorescein diacetate (CMFDA), incubated with human monocytic cell line (THP-1) and platelet
phagocytosis index was analyzed by flow cytometry. The phagocytosis index was higher in thrombocytopenic patients
compared to non-thrombocytopenic patients (p = 0.042) and HV (p = 0.048). A negative correlation was observed between
platelet count and phagocytosis index (r = 20.402; p = 0.016). Platelet activation was assessed measuring the expression of
P-selectin (CD62-P) in platelets’ surface by flow cytometry. No significant difference was found in the expression of Pselectin between thrombocytopenic patients and HV (p = 0.092). After evaluating the cytokine profile (IL-2, IL-4, IL-6, IL-10,
TNF-a, IFN-c and IL-17) in the patients’ sera, levels of IL-6, IL-10 and IFN-c were elevated in malaria patients compared to HV.
Moreover, IL-6 and IL-10 values were higher in thrombocytopenic patients than non-thrombocytopenic ones (p = 0.044 and
p = 0.017, respectively. In contrast, TNF-a levels were not different between the three groups, but a positive correlation was
found between TNF-a and phagocytosis index (r = 20.305; p = 0.037).
Conclusion/Significance: Collectively, our findings indicate that platelet phagocytosis may contribute to thrombocytopenia
found in vivax malaria. Finally, we believe that this study opens new avenues to explore the mechanisms involved in platelet
dysfunction, commonly found in vivax malaria patients.
Citation: Coelho HCC, Lopes SCP, Pimentel JPD, Nogueira PA, Costa FTM, et al. (2013) Thrombocytopenia in Plasmodium vivax Malaria Is Related to Platelets
Phagocytosis. PLoS ONE 8(5): e63410. doi:10.1371/journal.pone.0063410
Editor: Luzia Helena Carvalho, Centro de Pesquisa Rene Rachou/Fundação Oswaldo Cruz (Fiocruz-Minas), Brazil
Received February 18, 2013; Accepted April 2, 2013; Published May 24, 2013
Copyright: ß 2013 Coelho et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits
unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
Funding: HCCC received a fellowship from CAPES and SCPL was sponsored by a FAPESP fellowship. MVGL and FTMC are CNPq fellows. FTMC is also a fellow from
Programa Estratégico de Ciência, Tecnologia & Inovação nas Fundações Estaduais de Saúde (PECTI/AM-Saúde) from Fundação de Amparo à Pesquisa do Estado
do Amazonas (FAPEAM, Amazonas - Brazil). This work was supported by CNPq and FAPEAM grants. The funders had no role in study design, data collection and
analysis, decision to publish, or preparation of the manuscript.
Competing Interests: The authors have declared that no competing interests exist.
* E-mail: [email protected]
[7]. High frequency of thrombocytopenia in patients with malaria
has been well-documented in several studies [8], including reports
from Manaus in the Brazilian Amazon [8,9]. Indeed, Kochar and
colleagues have recently shown that severe thrombocytopenia
(platelet count ,206103/mm3) is a common manifestation in
patients with vivax mono-infection confirmed by PCR [10,11].
Research on the pathogenesis of malaria thrombocytopenia has
been conducted for more than four decades, however the exact
mechanism underlying this phenomenon remains not elucidated.
Nevertheless, thrombocytopenia in malaria seems to be a
multifactorial phenomenon and probably involves an increase in
platelets destruction and consumption [12]. Moreover, although
some studies showed bleeding associated with thrombocytopenia
Introduction
Plasmodium infections are still a major public health problem,
resulting in millions of deaths annually worldwide [1]. Although P.
falciparum is responsible for the majority of severe complications
cases and malaria-associated mortality [2]; vivax malaria has now
clearly emerged as a potentially lethal condition [3,4], despite of
having previously been considered a benign disease. P. vivax is
more widely distributed than P. falciparum and has potential to
cause morbidity and mortality amongst the 2.85 billion people
living at risk of infection [5]. In Brazil, P. vivax accounts for up to
80% of the malaria cases [6].
Thrombocytopenia and anemia are the most common malariaassociated hematological complications in P. vivax and P. falciparum
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Platelets Phagocytosis in Vivax Malaria
in malaria [11,13], low platelet counts were not commonly
accompanied by severe bleeding [8].
Several mechanisms have been proposed to explain malaria
thrombocytopenia [12,14–20]. Some studies suggest that the low
platelet counts in malaria might be caused by activation [20] and/
or apoptosis of platelets [14], thus leading to its removal by the
immune system [12,15]. Nonetheless, it has also been proposed
that immune complexes generated by malarial antigen could lead
to sequestration of the injured platelets in the spleen followed by
phagocytosis by splenic macrophages [16–19].
Recently, Klein and Ronez [21] showed a blood smear from a
P. falciparum patient compatible with peripheral hemophagocytosis.
This patient presented marked thrombocytopenia and platelet-like
particle inside the monocytes [21]. Indeed, platelet phagocytosis in
malaria was shown more than 20 years ago in a patient report with
80% of circulating monocytes presenting platelets inside [22].
Although there are some evidences of phagocytosis involvement
in malaria thrombocytopenia, information regarding the mechanisms responsible for this phenomenon is scarce. Herein, we
investigate the role of platelets phagocytosis in malaria vivax
thrombocytopenia, after establishing an in vitro phagocytosis assay
based on flow cytometry in the presence of platelets from patients
and healthy donors and THP-1 cells.
acidified with citric acid 0,15 M until the PRP pH reached 6.4 and
then 1 mg/mL of prostaglandin E-1 (PGE-1) was added to avoid
platelet stimulation. PRP was pelleted by centrifugation for 10 min
at 1,6006g and the platelets pellet was re-suspended in phosphate
buffered saline (PBS) supplemented with 0.5% bovine serum
albumin, 2mM EDTA, 0.1% sodium azide and 1 mg/mL PGE-1.
For phagocytosis experiments, platelets were fluorescently labeled
with 5 mg of CellTrackerH Green CMFDA (InvitrogenH) by
60 min incubation at 37uC, followed by two washes in supplemented PBS. The number of platelets was determined and the
solution was adjusted to 506106 platelets/mL. The efficiency of
platelet labeling with CMFDA was determined to be above 95%
using flow cytometry (FACS CaliburH, BD BiosciencesH, San Jose,
CA).
P-selectin Expression
P-selectin expression in platelets (chosen as a surrogate of
platelet activation) was measured in two moments, in the PRP and
after platelet isolation. For this purpose 100 mL of PRP or platelet
solution (56106 platelets/mL) were incubated with 4 mL of PE
mouse anti-human CD62-P (BD PharmingenH) for 30 min at
37uC. After two washes in supplemented PBS, P-selectin
expression was measured on a FACScaliburH (BD BiosciencesH,
San Jose, CA).
Materials and Methods
THP-1 Cells
Ethics Statement
Human monocytic THP-1 cells (ATCCH TIB-20H) were
cultured in RPMI-1640 medium (GibcoH) supplemented with
10% fetal calf serum (FCS) and gentamicin (40 mg/L) at 37uC.
THP-1 cells were counted in a Neubauer chamber and 16106
cells per well were added in a 24 wells plate. Maturation was
induced by incubation with 60 gg/mL of Phorbol 12-Myristate
13-Acetate (PMA) (CalbiochemH, San Diego, CA) for 2 hours at
37uC. After this period, the supernatant was removed and the
THP-1 cells were washed twice with RPMI medium.
All protocols and consents forms were approved by the Ethics
Review Board of the Fundação de Medicina Tropical Dr. Heitor Vieira
Dourado (FMT-HVD) (approval number 1610–11). A signed
informed consent was obtained from each subject enrolled in this
study.
Study Area and Subjects
Patients were recruited and examined at FMT-HVD, a tertiary
care center for infectious diseases in Manaus, the capital of the
Amazonas State, Brazil. Up to 20 mL of peripheral blood was
collected immediately after confirmation of P. vivax infection by
thick blood smear (n = 35). Afterwards, patients were treated with
chloroquine and primaquine, according to the standard protocol
recommended by the Brazilian Malaria Control Program. P. vivax
mono-infection was subsequently confirmed by polymerase chain
reaction (PCR) analysis [23]. Peripheral blood was also collected
from eight healthy volunteers (HV) living in Manaus, negative for
P. vivax infection by thick blood film and PCR and with no
previous history of malaria.
Clinical and demographical data were acquired through a
standardized questionnaire, and the hematological profile,
including peripheral platelet count and MPV, were determined
using a cell counter (Sysmex KX-21NH). Patients presenting any
other co-morbidity related to thrombocytopenia that could be
traced were excluded from the study, as well as HVs. The comorbidities investigated were human immunodeficiency virus
(HIV) (Rapid Check HIV 1&2H), dengue (Dengue Eden Test
BioeasyH, MG, Brazil), leptospirosis (SD Bioline Leptospira
IgG/IgM, Kyonggi-doH, Korea), hepatitis C (Anti-HCV BioeasyH, MG, Brazil) and hepatitis B (HBsAg ELISA BioeasyH, MG,
Brazil).
In vitro Platelet Phagocytosis
After cell maturation, phagocytosis of platelets by THP-1 cells
was measured by flow cytometry as previously described [24].
Briefly, 56106 fluorescently labeled platelets were added to each
well and then plates were centrifuged at 5006g for 5 min at room
temperature. After 60 min of incubation in 5% CO2 atmosphere
at 37uC, the THP-1 cells were harvested, washed three times in
PBS and fixed in paraformaldehyde 4% in cacodylate buffer for
flow cytometry analysis.
Flow Cytometry Analysis
The THP-1 cells were gated and 10,000 events were acquired
from each sample. The frequency of platelet phagocytosis (FP)
was determined by counting the CellTrackerH Green CMFDA
positive cells in FL1-H. The median intensity of fluorescence
(MIF) emitted for each cell was also evaluated. As larger platelets
have a greater amount of CellTrackerH Green CMFDA, the
mean platelet volume (MPV) may affect the intensity of
fluorescence. Then, to standardize the platelet phagocytosis for
each sample, we created a formula to calculate the Phagocytosis
index: PI = MIF6FP/1006MPV.
Platelets Isolation
Cytokine Measurements
Platelets were isolated from whole blood collected in sodium
citrate solution (3.8%) from vivax malaria patients or HVs and
centrifuged for 10 min at 2006g to generate platelet-rich plasma
(PRP). To avoid platelets aggregation and activation, PRP was
The levels of IL-2, IL-4, IL-6, IL-10, IL-17, IFN-c and TNF-a
were quantified in cryopreserved serum using the Cytometric Bead
Array kit (CBA, BD Biosciences PharmingenH) following manufacturer’s instructions. All the cytokine levels below detection limit
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Platelets Phagocytosis in Vivax Malaria
Table 1. Characteristics of patients with Plasmodium vivax (with and without thrombocytopenia).
Patients
Characteristics
Total
Age, years (mean 6SD)
Sex (%)
M
F
Duration of symptoms in days (mean 6SD)
Previous malaria episodes (%)
Yes
No
T
41.8613.6
40.1613.8
42.4613.9
0.918a
29/35 (82.9)
9/13(69.3)
19/22 (86.4)
0.383b
6/35 (17.1)
4/13(30.7)
3/22 (13.6)
5.564.0
5.363.5
5.364.9
0.605a
27/35 (77.2)
11/13 (84.6)
16/22 (72.7)
0.680b
8/35 (22.8)
2/13 (15.4)
6/22 (27.3)
3.663.6
3.162.9
3.964.0
0.876a
,6 months
13/27 (48.2)
7/16 (43.7)
6/11 (54.6)
0.581b
$6 months
14/27 (51.8)
9/16 (56.3)
5/11 (45.4)
Nu of previous malaria episodes (mean 6SD)
Last malaria (%)
P value*
NT
SD = standard deviation.
NT = non-thrombocytopenic; T = thrombocytopenic.
*Non-thrombocytopenic patients6thrombocytopenic patients.
a
Mann Whitney test.
b
Chi-square or Fisher’s exact test.
doi:10.1371/journal.pone.0063410.t001
were given half of the threshold value and those values above the
upper detection limit were excluded from the analysis.
Results
Patient’s Characteristics and Thrombocytopenia
Frequency
Statistical Analysis
According to Table 1, thrombocytopenia (,150,000 platelets/
All data were expressed as the mean 6 SD. Correlations
were analyzed using the Spearman correlation. Normal distribution of data was evaluated with the Kolmogorov-Smirnov
test. Comparisons between groups were analyzed using the
Mann-Whitney U test (two groups) or Kruskal Wallis test. Pselectin expression before and after platelets isolation were
compared by Wilcoxon signed rank test. Differences were
considered statistically significant when p#0.05. Statistical
analysis was performed using the GraphPad PrismH version
5.0 (GraphPad SoftwareH, CA, US).
mL) was found in 62.9% of the patients (22/35) enrolled in this
study. Amongst thrombocytopenic patients, 18.2% (4/22) presented severe thrombocytopenia (,50,000 platelets/mL). Moreover, no significant difference in duration of clinical malaria
symptoms and number of previous malaria episodes were observed
between thrombocytopenic and non-thrombocytopenic patients
(Table 1). Likewise, the frequencies of primary infection and past
malaria infection in the last six months were similar in both groups
(Table 1).
MPV and Thrombocytopenia
MPV was significantly elevated in patients with thrombocytopenia (Figure 1A). Moreover, a negative correlation was observed
Figure 1. Mean platelet volume (MPV). MPV comparisons between healthy volunteers (HV), non-thrombocytopenic (NT) and thrombocytopenic
(T) patients with vivax malaria (A).
doi:10.1371/journal.pone.0063410.g001
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Platelets Phagocytosis in Vivax Malaria
Figure 2. Parasitemia and thrombocytopenia. Comparisons of parasitemia (per mm3 of blood) between non-thrombocytopenic (NT) and
thrombocytopenic (T) patients with malaria (A). Correlation between platelet count and parasitemia (mm3 of blood) (B).
doi:10.1371/journal.pone.0063410.g002
between the MPV and the platelet count in malaria patients
(r = 20.483; p = 0.003) (Figure 1B).
P-selectin Expression
Parasitemia was similar in thrombocytopenic and non-thrombocytopenic patients (Figure 2A) and no correlation was found
between platelet count and parasitemia (Figure 2B).
P-selectin expression was similar between thrombocytopenic
patients and HVs in two time-points: immediately after harvesting
(PRP) or after washing and CMFDA labeling. According to
Figure 4, no significant increase in P-selectin expression was found
in platelet isolation process for either non-thrombocytopenic or
thrombocytopenic patients.
Phagocytosis Assay
Cytokine Profile in Patients’ Sera
The phagocytosis index was significantly higher in patients with
thrombocytopenia malaria than in patients without thrombocytopenia (p = 0.042) and HV (p = 0.048) (Figure 3A). Moreover,
significantly correlation was observed between platelet count and
phagocytosis index (r = 20.426; p = 0.016) (Figure 3B). Phagocytosis index not corrected by MPV was also analyzed and lead to
the same results (data not shown).
Of seven cytokines analyzed in this study, IL-6, IL-10 and IFNc were elevated in malaria patients sera, thrombocytopenic or not,
compared to HVs (Figure 5A, 5B and 5C). IL-6 and IL-10 were
higher in thrombocytopenic patients than in non-thrombocytopenic ones (Figure 5A and 5B). Indeed, negative correlations were
found between platelet counts and IL-6 and IL-10 values
Parasitemia and Thrombocytopenia
Figure 3. Phagocytosis index (PI). Comparisons of phagocytosis between healthy volunteers (HV), non-thrombocytopenic (NT) and
thrombocytopenic (T) patients with malaria (A). Correlation between platelet count and phagocytosis index (B).
doi:10.1371/journal.pone.0063410.g003
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Platelets Phagocytosis in Vivax Malaria
Discussion
Despite not being a criterion for severe malaria, thrombocytopenia is one of the most common complications of both P. vivax
and P. falciparum malaria. Recently, Kochar and colleagues have
shown that thrombocytopenia is more frequent and severe among
patients with P. vivax infection [10]. Nevertheless, only a limited
number of studies have addressed key questions on the pathogenesis of thrombocytopenia in malaria. Of those, two independent
studies have shown platelets phagocytosis in malaria thrombocytopenic patients [21,22], although a detailed investigation of this
phenomenon was not pursued. Herein, by means of an in vitro
phagocytosis assay, we evaluated the involvement of platelet
phagocytosis in vivax malaria thrombocytopenia.
In this study, thrombocytopenia was frequently detected
amongst vivax malaria patients (62.9%) as well as severe
thrombocytopenia (platelet counts under 50,000 platelets/mL)
(18.2%). Nevertheless, we did not observe association between
severe thrombocytopenia and bleeding in these patients, although
severe thrombocytopenia is occasionally associated with severity
[25,26] including severe vivax patients [27,28].
In this study, MPV was elevated in thrombocytopenic patients
and a negative correlation between platelet counts and MPV was
detected in malaria patients. Our findings corroborates previous
studies [11] and are in line with the rationale that larger platelets
observed in thrombocytopenic patients may be a manner to
Figure 4. P-selectin expression in healthy volunteers an
thrombocytopenic patients. The P-selectin expression in platelets
was measured in two moments, in the PRP and in isolated platelets.
doi:10.1371/journal.pone.0063410.g004
(Figure 6A and 6B). A positive correlation was found only between
phagocytosis index and TNF-a values (Figure 7).
Figure 5. Cytokines levels. Comparisons of IL-6 (A), IL-10 (B), and IFN-c (C) between healthy volunteers (HV), non-thrombocytopenic (NT) and
thrombocytopenic (T) patients.
doi:10.1371/journal.pone.0063410.g005
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Platelets Phagocytosis in Vivax Malaria
Figure 6. Correlation between cytokines levels and platelet count. Correlation between IL-6 (A) and IL-10 (B) and platelet count.
doi:10.1371/journal.pone.0063410.g006
platelet count in P. falciparum experimentally infected volunteers
[36]. In contrast, Lee and colleagues showed that circulating Pselectin in plasma was elevated in P. falciparum severe malaria but
not in P. vivax or P. falciparum non-severe infections [20]. As Pselectin expression levels were not augmented in the platelets from
thrombocytopenic patients in our study, we believe that this
molecule is not directly involved in platelet phagocytosis.
Cytokines released during an acute inflammatory response
could contribute to the pathogenesis of thrombocytopenia.
Recently, a study showed that the administration of IL-10 to
healthy volunteers was capable of inducing thrombocytopenia
[37]. This decrease in platelet counts in IL-10 treated group was
accompanied by reduction in the amount of megakaryocyte
colony-forming units, indicating the participation of this cytokine
in platelet production [37]. Actually, it has been shown that
thrombocytopenia in children with acute falciparum malaria is
strongly associated with plasma concentrations of IL-10, but not
with P. falciparum parasitemia or other plasma cytokines [38]. Park
and colleagues showed higher levels of IL-1, IL-6, IL-10 and TGFß in P. vivax thrombocytopenic patients compared to nonthrombocytopenic [39]. Indeed, similar to previous findings
[38,39], we observed that IL-6 and IL-10 levels are elevated in
thrombocytopenic patients serum compared to non-thrombocytopenic ones, and negative correlations between IL-6 and IL-10
levels and platelet count were found.
TNF-a has been associated with platelet consumption in mice
but not with platelet production [40]. In our study, TNF-a levels
were similar in malaria patients and HV but a positive correlation
between TNF-a levels in serum and phagocytosis index was found.
In contrast, IFN-c was elevated in thrombocytopenic patients as
compared to HV. In fact, high levels of IFN-c and TNF-aare often
correlated to severity in murine experimental models and in
humans infected with P. falciparum and P. vivax [28,41–44].
However, the relationship between thrombocytopenia and severe
malaria is nebulous [8,38], and further studies are needed to
understand the pathogenesis associated with thrombocytopenia.
compensate the low absolute number of platelets in the periphery;
therefore preserving primary hemostasis and avoiding severe
bleeding [8].
Negative correlation between parasitemia and thrombocytopenia has been shown elsewhere [29–31], and this correlation has
been attributed to platelets shortened lifespan due to immune
complexes sequestration in their surface [16–18]. Surprisingly, we
did not find any relation between parasitemia and platelet counts
in vivax malaria patients. Despite of our small sample size, findings
corroborate a large study conducted in Bikaner, India [32].
Indeed, despite the fact that circulating immune complexes are
elevated in vivax and falciparum malaria, their role in the
development of thrombocytopenia is not clear [33,34]. Nonetheless, we observed a negative correlation between platelet counts
and phagocytosis index, indicating that platelet phagocytosis may
be involved in thrombocytopenia pathogenesis in vivax malaria.
It has been proposed that platelet phagocytosis could be
mediated by the increase in P-selectin expression in the surface of
activated platelets [35]. However, only two studies evaluated Pselectin expression in malaria thrombocytopenia [20,36], and just
one in P. vivax malaria [20]. Recently, de Mast and colleagues
showed that P-selectin expression in platelets surface and
circulating P-selectin in plasma were not associated with low
Conclusion
Collectively, our findings demonstrate that platelet phagocytosis
is associated to thrombocytopenia and correlates with TNF-a, a
cytokine normally attributed to severity in malaria. Moreover, we
showed that this increase in phagocytosis has not been associated
with parasitemia or platelet activation. Importantly, our data
Figure 7. Correlation between TNF-a and phagocytosis index
(PI).
doi:10.1371/journal.pone.0063410.g007
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Platelets Phagocytosis in Vivax Malaria
brings new insights about the mechanisms involved in malaria
vivax thrombocytopenia and highlights the potential relevance of
this phenomenon.
Author Contributions
Conceived and designed the experiments: MVGL. Performed the
experiments: HCCC SCPL JPDP PAN AM. Analyzed the data: AMS
GCM WMM. Wrote the paper: HCCC FTMC MVGL.
Acknowledgments
This paper is dedicated to Prof. Maria Imaculada Muniz-Junqueira, who
introduced us all to the art of phagocytosis.
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Int J Environ Res Public Health 2: 123–131.
7
May 2013 | Volume 8 | Issue 5 | e63410
38
5. CONCLUSÃO
5.1 O índice de fagocitose dos pacientes plaquetopênicos com malária vivax foi
maior comparado com o índice de fagocitose dos pacientes não-plaquetopênicos
com malária e pessoas saudáveis, sugerindo que a plaquetopenia na malária vivax
está associada com a fagocitose de plaquetas;
5.2 A contagem de plaquetas no sangue periférico dos pacientes com malária esteve
inversamente correlacionada com o índice de fagocitose.
5.3 Concentrações séricas de IL-6 e IL-10 apresentaram-se mais elevadas nos
pacientes com malária vivax plaquetopênicos do que nos pacientes nãoplaquetopênicos e pessoas saudáveis. A concentração sérica de IFN-γ foi maior em
pacientes não-plaquetopênicos do que em pessoas saudáveis. TNF-α apresentou-se
mais elevado em pacientes plaquetopênicos com malária vivax do que em pessoas
saudáveis.
5.4 A contagem de plaquetas no sangue periférico nos pacientes com malária vivax
apresentou-se inversamente correlacionada com as concentrações séricas de IL-6 e
IL-10. O índice de fagocitose correlacionou-se apenas com TNF- α.
5.5 Não houve diferença na expressão de P-selectina pelas plaquetas entre os
pacientes plaquetopênicos e pessoas saudáveis, tanto no plasma rico em plaquetas,
quanto nas plaquetas isoladas, sugerindo que o evento de fagocitose acontece de
forma independente da ativação plaquetária.
39
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7. ANEXOS
ANEXO A: Termo de Consentimento Livre e Esclarecido – TCLE
TERMO DE CONSENTIMENTO LIVRE E ESCLARECIDO (TCLE)
1. DADOS DE IDENTIFICAÇÃO
Nome (Paciente):.....................................................................................................
Documento de Identidade:.......................................................................................
Endereço: .................................................................CEP: .....................................
Cidade: .............................Estado...................Fone:...............................................
2. DADOS SOBRE O ESTUDO
2.1 Titulo: Avalição in vitro da fagocitose de plaquetas em pacientes com malária vivax e falciparum.
2.2 Pesquisadores Principais: Helena Cristina Cardoso Coelho (Aluna do programa de pós-graduação
em Medicina Tropical da Universidade do Estado do Amazonas em Convênio com a Fundação de
Medicina Tropical Dr. Heitor Vieira Dourado) e Dr. Marcus Vinícius Guimarães de Lacerda (Orientador).
2.3 Aprovação pelo Comitê de Ética em Pesquisa (CEP): Nº 1610-11
3. EXPLICAÇÕES DO PESQUISADOR AO PACIENTE
A malária é uma doença muito comum na amazônia. Ela é transmitida pela picada do carapanã.
No Brasil existem dois tipos de malária: a malária vivax e a malária falciparum. Nos dois tipos, o
paciente pode ter só febre, mas também pode morrer se não for tratado. Umas das complicações que
a malária causa é a diminuição do número de uma célula chamada plaqueta. As plaquetas são
importantes para o seu corpo não sangrar, assim se essas células diminuirem no seu sangue, você
poderá sangrar pela pele nariz ou boca. Ainda não se sabe porque isso acontece na malária. Para
podermos saber o motivo de isso acontecer, precisamos estudar o sangue dos pacientes com
malaria.
Assim, precisamos colher 20 mL de sangue da veia do seu braço, além do exame da malária,
que foi colhido no dedo. Com esse sangue estudaremos as plaquetas e os glóbulos brancos além
de fazer um exame chamado hemograma. Podemos também realizar exames para excluir outras
causas que diminuem as plaquetas no seu sangue como: Teste de HIV, teste de hepatites e teste
para dengue.
Depois de colher o sangue do braço, ele pode doer um pouco na região da picada da agulha e
pode ficar com uma mancha roxa.
Além da colheita de sangue, o participante da pesquisa deverá responder um questionário
informando alguns dados clínicos importantes para a pesquisa.
A coleta será feita com material esterilizado e descartável sem risco algum para os pacientes.
Os resultados dos exames poderão ser visto pela pessoa que participar da pesquisa. O sangue
colhido que sobrar poderá ser guardado no freezer com um número (sem o nome da pessoa) e
poderá ser utilizado para outro estudo no futuro.
A participação nesse estudo será confidencial e os resultados dos exames serão mostrados
apenas às pessoas do Hospital Tropical que trabalham com malária ou pesquisadores de outras
cidades ou países, mas o nome da pessoa que participa será mantido em segredo.
Caso seja necessário a pessoa poderá ser consultada por um médico participante do grupo de
pesquisa.
A pessoa que aceitou participar da pesquisa tem todo o direito de dizer que não quer mais
participar em qualquer momento. Se isso acontecer, a pessoa será tratada com a medicação
adequada e terá o direito ao atendimento no Hospital Tropical sempre que precisar.
A pessoa que aceitar participar da pesquisa assinará duas cópias deste documento, uma cópia
ficará com o pesquisador, e outra ficará com o paciente.
Para obtenção de quaisquer informações o paciente poderá entrar em contato com o Presidente do
Comitê de Ética em Pesquisa do Hospital Tropical (grupo de pessoas que avalia os projetos de
pesquisa que são realizados em um hospital): Dr. Luiz Carlos de Lima Ferreira (Telefone: 3238-1711,
ramal 319) ou com a pesquisadora responsável Helena Cristina Cardoso Coelho (8142-6721).
Informamos que você não receberá qualquer benefício adicional, nem dinheiro, mas estará
contribuindo para o estudo da doença, que ainda mata muitas pessoas.
4. AFIRMAÇÕES DO PACIENTE
4.1 Fui esclarecido sobre os objetivos da pesquisa, os procedimentos, riscos e benefícios SIM.......
NÃO .......
4.2 Fui esclarecido sobre a liberdade de desistir de participar a qualquer momento, sem que isso traga
prejúizos ao meu atendimento e tratamento. SIM....... NÃO .......
4.3 Fui esclarecido de que não haverá remuneração financeira. SIM....... NÃO .......
4.4 Fui esclarecido de que não heverá indenização além das previstas pela lei, em reparação a dano
imediado ou tardio, causado pela pesquisa em questão. SIM....... NÃO .......
5. CONSENTIMENTO PÓS-INFORMADO
Declaro que recebi a explicação de que serei um dos participantes dessa pesquisa e entendo todas as
suas etapas e objetivos. Se eu não souber ler ou escrever, uma pessoa de minha confiança lerá este
documento para mim e depois escreverá nesta página o meu nome e a data do preenchimento.
E por estar devidamente informado e esclarecido sobre o conteúdo deste termo, livremente, sem
qualquer pressão por parte dos pesquisadores, expresso meu consentimento para minha inclusão
nesta pesquisa.
...........................................................................................
Assinatura do paciente
..............................................................................................
Assinatura do pesquisador que conversou com o paciente
....................................................
Local e Data
..........................
Data
51
ANEXO B: Ficha clínica do participante do estudo
AVALIAÇÃO IN VITRO DA FAGOCITOSE DE PLAQUETAS EM PACIENTES COM
MALÁRIA VIVAX
Mestrado do Programa de Pós-Grauduação UEA/FMT-HVD
Aluna responsável: Helena Cristina Cardoso Coelho
FICHA CLÍNICA DO PARTICIPANTE DO ESTUDO
Número:
Data de inclusão: ........ /........ /........
1 IDENTIFICAÇÃO
Nome:...............................................................................................................
Registro:..........................
Data de nascimento: ........ /........ / ........
Sexo: 1-M 2- F
Idade:....................
Endereço:................................................................................................................................................
Bairro:...............................................................
Município:..............................
Telefone residencial:.........................................................
Estado:........................
Telefone celular:.........................................
2 CARACTERÍSTICAS INDIVIDUAIS
2.1 Local provável de infecção:
1-Manaus 2-Outro município 3 -Outro estado
Qual?.......................................................................................................................................................
2.2 Sua exposição à área endêmica é eventual
1- sim 2-não
2.3 Se não, há quanto tempo vive em área endêmica?
1-<6m 2-6m 3-1a 4->2
2.4 Número de episódios prévios de malária:...............................................................................................
2.5 Última malária foi há quanto tempo ?......................................................................................................
2.6 Tipo da última malária
2.7 Usou alguma medicação antimalária nos últimos 60 dias?
1-V 2- F
3-F+V
4- não sabe
1- sim 2-não
Qual?.......................................................................................................................................................
2.8 Usou algum outro medicamento?
1- sim 2-não
Qual?.......................................................................................................................................................
Quanto tempo e Quando foi a última vez?...............................................................................................
2.9 Tem ou já teve alguma doença hematológica ou imunológica?
1- sim 2-não
Qual?.......................................................................................................................................................
2.10 Algum familiar já teve alguma doença hematológica ?
1- sim 2-não
Qual?.......................................................................................................................................................
2.11 Já precisou fazer alguma hemotransfusão?
1- sim 2-não
2.12 Há quanto tempo?...................................................................................................................................
2.13 Tem facilidade em sangrar após trauma?
1- sim 2-não
2.14 Já teve algum sangramento espontâneo?
1- sim 2-não
Que tipo de sangramento?.......................................................................................................................
2.15 Possui alguma outra doença?
1- sim 2-não
Qual?.......................................................................................................................................................
A doença está em atividade nos últimos dias?
2.16 Usa alguma medicação regular?
1- sim 2-não
1- sim 2-não
Qual?.......................................................................................................................................................
2.17 Está gestante?
1- sim 2-não
2,18 Tabagista?
1- sim 2-não
2,19 Uso regular de álcool?
1- sim 2-não
3 HISTÓRIA DA DOENÇA ATUAL
3,1 Doença atual há .......................... Dias
3,2 Sintomas:
Febre
1-sim 2-não
Dor abdominal 1-sim 2-não
Vômitos
1-sim 2-não
Náusea
1-sim 2-não
Mioartralgia 1-sim 2-não
Diarréia
1-sim 2-não
Cefaléia
1-sim 2-não
Sangramento 1-sim 2-não
Dispnéia
1-sim 2-não
Calafrios 1-sim 2-não
4 EXAMES LABORATORIAIS
4,1 Tipo de malária (Microscopia):
1-V
2- F
4,2 Parasitemia da malária vivax
4,3 Esquizontes ?
3-V+F
1- < 1/2+
2- 1/2+
1- sim 2-não
6
3
4,4 Hemácias.......................x10 /µL
MCV................../µm
4,5 Hematrócrito..................................%
3- +
4- ++
5- +++
4,6 Leucócitos .......................................x10 /µL
Gametócitos
1- sim 2-não
MCH...................pg
MCHCM...................g/dL
Leucócitos .....................................%
3
........................................x10 /µL
Linfócitos.....................................%
3
4,8 Monócitos........................................x10 /µL
Monócitos.....................................%
3
4,9 Neutrófilos........................................x10 /µL
Neutrófilos.....................................%
3
4,10 Eosinófilos........................................x10 /µL
Eosinófilos.....................................%
3
4,11 Plaquetas........................................x10 /µL
MPV........................................./µm3
4,12 IgG anti-HIV (ELISA)
1-positivo 2-negativo 3-não realizado
4,13 HBsAg (ELISA)
1-positivo 2-negativo 3-não realizado
4,14 Anti-HCV (ELISA)
1-positivo 2-negativo 3-não realizado
4,15 Proteína NS1 Dengue
1-positivo 2-negativo 3-não realizado
4,16 Anti-IgG/igM Leptospirose
1-positivo 2-negativo 3-não realizado
4,17 PCR malária
6- ++++
Hb .....................................................
3
4,7 Linfócitos
4 - Negativo
1-V
2- F
3-V+F
4 - Negativo
5 CLASSIFICAÇÃO CONTAGEM DE PLAQUETAS
5,1
1-Plaquetopênicos 2-Não plaquetopênicos
Teste de Fagocitose N.
54
ANEXO C: Procedimento Operacional Padrão - POP
POP_MAL_LB_009_v01_PT
Procedimento Operacional Padrão
Gerência de Malária
Código POP
POP_MAL_LB_009_v01_PT
Título
Congelamento e descongelamento de células THP-1
Idioma da versão original
Português
Escrito ou traduzido por: Revisado por:
Aprovado por:
Helena C. C. Coelho
Paulo Nogueira , João Paulo
Pimentel e Stefanie Lopes
Marcus V. G. Lacerda
Data & assinatura
Data & assinatura
Data & assinatura
Emenda
1
Data de aplicação:
Data da próxima
revisão:
Razão da emenda
OBJETIVOS
Padronizar o procedimento de congelamento e descongelamentos de células THP-1.
2
DEFINIÇÕES
RPMI: meio para cultura de células humanas desenvolvido no Roswell Park Memorial Institute.
SFB: Soro Fetal Bovino.
Células THP-1: linhagem celular de leucemia monocítica aguda humana.
3
APLICÁVEL A
As células THP-1 serão utilizadas em testes de fagocitose.
4
RESPONSABILIDADES
Gerente da unidade, coordenador da subunidade e pessoal técnico.
5
POP’S RELACIONADOS
POP_MAL_LB_011_v01_PT
POP_MAL_LB_009_v01_PT
6
Página 2 de 2
PROCEDIMENTOS
6.1 Recursos necessários para o procedimento:
6.1.1 Garrafas com células THP-1 mantidas em meio RPMI com 10% de SFB e gentamicina (meio RPMI
completo)
6.2 Materiais necessários para o procedimento:
6.2.1 Tubos de polipropileno de 15 mL
6.2.2 Gelo seco e Caixa térmica
6.2.3 Criotubos
6.2.4 Pipetas ajustáveis de canal único (10 µL, 200 µL, 1000 µL)
6.2.5 Ponteiras estéreis (10 µL, 200 µL, 1000 µL)
6.2.6 Pipetas sorológicas estéreis (10 mL)
6.2.7 Caixa StrataCooler
6.3 Equipamentos necessários para o procedimento:
6.3.1 Cabine de fluxo laminar
6.3.2 Banho-maria
6.3.3 Microscópio óptico com lente objetiva de aumento de 40x
6.3.4 Câmara de neubauer
6.4 Soluções necessárias para o procedimento:
6.4.1 Meio RPM
6.4.2 Meio RPMI completo: com 10% de SFB e 0,1% de gentamicina
6.4.3 Meio de congelamento: RPMI completo com 10% de Dimethyl Sulphoxide (DMSO)
6.4.4 Azul de Trypan (para contagem de células)
6.5 Procedimento para congelamento:
6.5.1 Retirar as células THP-1 da garrafa e transferir-las para um tubo falcon 15 mL;
6.5.2 Determinar a concentração celular utilizando o azul de trypan e a câmara de neubauer, conforme
descrito no item 6.7;
6.5.3 Centrifugar o tubo contendo as células THP-1 a 100xg por 6 minutos a 24ºC;
6.5.4 Identificar os criotubos e colocá-los na caixa StrataCooler que deve estar previamente resfriada a 4ºC;
6.5.5 No momento do congelamento, retirar a caixa StrataCooler e o meio de congelamento da geladeira e
destampar os criotubos mantendo as tampas viradas para cima;
6.5.6 Após o término da centrifugação, desprezar o sobrenadante e resuspender o pellet e com o auxílio de
uma pipeta estéril, aspirando o volume necessário de meio de congelamento para cada amostra (considere
6
1mL de meio de congelamento para cada 1x10 células);
6.5.7 Transferir o meio de congelamento para o tubo com células e homogeneizar bem com o auxílio da
POP_MAL_LB_009_v01_PT
Página 3 de 3
pipeta;
6.5.8 Transferir essa solução para os criotubos;
6.5.9 Tampar os criotubos e armazenar no freezer -80ºC.
6.6 Procedimento para descongelamento:
6.6.1 Separar um tubo de 15 mL previamente identificado e adicionar 10mL de meio de cultura RPMI;
6.6.2 Retirar os criotubos com as células THP-1 do freezer -80ºC e mantê-los no gelo seco até o momento do
descongelamento;
6.6.3 Descongelar uma amostra de cada vez rapidamente, com leve agitação no banho-maria a 37ºC até que
a amostra se desgrude do fundo do criotubo. Parar quando ainda houver um pedaço de gelo visível no
criotubo;
6.6.4 Adicionar 1 mL de meio de cultura RPMI gotejando lentamente no criotubo e transferir o meio com a
amostra para o tubo de 15 mL;
6.6.5 Repetir a operação até retirar toda a amostra do criotubo;
6.6.6 Lavar o criotubo com mais 1 mL do meio RPMI (principalmente as paredes do criotubo) e transferir para
o tubo de 15mL respectivo;
6.6.7 Centrifugar a 100xg por 6 minutos;
6.6.8 Desprezar o sobrenadante e lavar as células mais uma vez com meio RPMI.
6.6.9 Após a última lavagem, resuspender o pellet em 1 mL de meio RPMI completo
6.6.10 Fazer a contagem e a viabilidade das células, utilizando azul de trypan e câmara de Neubauer,
conforme descrito no item 6.7.
6.7 Contagem e viabilidade celular
6.7.1 Retirar uma alíquota de10µL da solução de células e misturar em 10µL de azul de trypan.
6.2.1 Contar as células em todos os quatro quadrantes da câmara de Neubauer como mostrado na Figura 1.
6.3.1 Multiplicar a média dos quatro quadrantes pelos fatores de diluição e pelo fator de correção da câmara
de Neubauer (104):
Células/ml = (média de células nos quatro quadrantes) x fatores de diluição x 104
6.2.16 Para determinar a viabilidade celular, contar as células de coloração azul (células mortas) na câmara
de Neubauer. A viabilidade é determinada pelo teste de exclusão do corante azul de trypan. Células viáveis
são impermeáveis a este corante, uma vez que sua penetração na célula indica a perda da integridade de sua
membrana:
Viabilidade (%) = Células vivas x 100 / Totais de Células (Células vivas e mortas).
Nota: Este procedimento deve ser realizado em cabine de fluxo laminar.
POP_MAL_LB_009_v01_PT
Página 4 de 4
Figura 1: Câmara de Neubauer. Em vermelho: os 4 quadrantes que devem ser contados para determinar o
número de células por mL.
7
8
REFERÊNCIAS
•
Protocol TBRU#8 “A Prospective Study of Shortening the Duration of Standar Short
Course Chemotherapy from 6 Months to 4 Months in HIV-non-Infected Patients with Fully
Drug-Susceptible, Non-cavitary Pulmonary Tuberculosis with Negative Sputum Cultures
after 2 Months of Anti-TB Treatment”.
•
John E. Coligan et al editors. Current Protocols in Immnology. John Wiley & Sans Inc,
1994. Volume 03, Appendix 3 G.
REGISTROS DOS ANEXOS
Não se aplica.
A
versão
atual
deste
POP
foi
traduzida
a
____________________ e a versão traduzida entra em efeito
em ___/___/_____.
dd
mm
aa
Assinatura do responsável: ______________________
POP_MAL_LB_010_v01_PT
Procedimento Operacional Padrão
Gerência de Malária
Código POP
POP_MAL_LB_010_v01_PT
Título
Separação, marcação, opsonização de plaquetas e avaliação da expressão
de P-selectina em plaquetas.
Idioma da versão original
Português
Escrito ou traduzido por: Revisado por:
Aprovado por:
Helena C. C. Coelho
Paulo Nogueira, João Paulo
Pimentel e Stefanie Lopes
Marcus V. G. Lacerda
Data & assinatura
Data & assinatura
Data & assinatura
Emenda
1
Data de aplicação:
Data da próxima
revisão:
Razão da emenda
OBJETIVOS
Este procedimento visa isolar, marcar e opsonizar plaquetas do sangue periférico.
2
•
•
•
•
3
DEFINIÇÕES
PBS - Phosphate buffered saline
PGE1 - Prostaglandina E1
CMFDA – Diacetato de 5-clorometilfluoresceína, marcador fluorescente verde lipofílico, que entra no
citoplasma das células e, por conta de ligações covalentes, não volta ao meio extracelular.
PE – Phycoerythrin.
APLICÁVEL A
O preparado por este procedimento será usado em testes de fagocitose (POP_MAL_LB_011_v01_PT).
4
RESPONSABILIDADES
Gerente da unidade, coordenador da subunidade e pessoal técnico.
5
POP’S RELACIONADOS
POP_MAL_LB_011_v01_PT
POP_MAL_LB_010_v01_PT
6
Página 2 de 2
PROCEDIMENTOS
6.1 Recursos necessários para o procedimento:
6.1.1 Aproximadamente 8 mL de sangue total com anticoagulante (em 2 tubos com citrato de sódio 3,2% de
4 mL).
6.2 Materiais necessários para o procedimento:
6.2.1 Tubos de polipropileno de 15 mL
6.2.2 Pipetas sorológicas estéreis (10 ml)
6.2.3 Pipetas ajustáveis de canal único (10 µL, 200 µL, 1000 µL)
6.2.4 Ponteiras estéreis (10 µL, 200 µL, 1000 µL)
6.2.5 Câmara de Neubauer ou aparelho automatizado para contagem de plaquetas
6.2.6 Contador de células
6.2.7 Microtubos de 2 mL
6.3 Equipamentos para o procedimento:
6.3.1 Cabine de fluxo laminar
6.3.2 Centrífuga refrigerada
6.3.3 Centrífuga Eppendorf
6.3.4 Microscópio óptico com lente objetiva de aumento de 40x
6.3.4 PHmetro
6.4 Reagentes necessários para o procedimento:
•
•
•
•
•
•
•
PBS (suplementado com 0,5% albumina bovina, 2 mM EDTA, e 0,1% azida sódica)
Ácido cítrico 0,15M
PGE1
CellTracker® Green CMFDA (Molecular Probes –Invitrogen®) 50 µg/frasco
Meio de cultura RPMI 1640
Anticorpo monoclonal IgG2a anti MHC classe I humano (W6/32)
Anticorpo anti CD62P humano conjugado com PE (BD Pharmingen™)
6.5 Preparação dos reagentes:
CMFDA Solução de Estoque
CMFDA.................................................................................................................................................................................................................................50 µg
DMSO................................................................................................................................................................................................................................100 µL
Deixar o frasco com 50 µg de CMFDA em temperatura ambiente por 20 minutos e reconstituir em DMSO
(dimetilsulfóxido anídrico). Separar alíquotas de 10 µL em tubos de Eppendorfs (solução de estoque).
POP_MAL_LB_010_v01_PT
Página 3 de 3
CMFDA Solução de Trabalho
CMFDA Solução de estoque............................................................................................................................................................................10 µL
RPMI......................................................................................................................................................................................................................................25 µL
Ácido Cítrico 0,15M
Acido Cítrico.........................................................................................................................................3g
Água destilada qsp..............................................................................................................................100mL
6.6 Armazenamento dos reagentes:
Ácido Cítrico – Armazenar à temperatura ambiente.
PBS – Armazenar à temperatura de 2 a 8º C.
PGE-1 – Armazenar à temperatura de -20 º C.
CellTracker® Green – Armazenar à temperatura de -20 º C.
Meio de cultura RPMI 1640 - Armazenar à temperatura de 2 a 8º C.
Anticorpo monoclonal IgG2a anti MHC classe I humano (W6/32) - Armazenar à temperatura de -20º C.
6.7 Procedimento para separação e marcação de plaquetas:
6.7.1 Centrifugar os tubos a 1.500 rpm (130 a 200 g) por 10 minutos a temperatura 24 º C;
6.7.2 Recuperar o sobrenadante (PRP) e transferir para um tubo falcon;
6.7.3 Acidificar o PRP com ácido cítrico 0,15 M a pH 6,4;
6.7.4 Acrescentar PGE-1 (concentração final de 0,3 µM);
6.7.5 Centrifugar a 3.000 rpm (1.600g) por 10 minutos a temperatura 24 º C;
6.7.6 Descartar o sobrenadante e resuspender em 1 mL de PBS e PGE-1 (concentração final de 0,3 µM);
6.7.7 Acrescentar 5 µg de CMFDA e deixar a temperatura ambiente, protegido da luz, por 60 minutos;
6.7.8 Centrifugar a 3.000 RPM (1.600g) por 10 minutos a temperatura 24 º C;
6.7.9 Descartar o sobrenadante e lavar as plaquetas com PBS e PGE-1 (duas vezes);
6.7.10 Após a última lavagem, resuspender as plaquetas em 1 mL de meio RPMI;
6.7.11 Contar as plaquetas e ajustar para 50x106/mL.
Nota: Estes procedimentos devem ser realizados em cabine de fluxo laminar.
6.8 Contagem de plaquetas na camâra de neubauer:
6.8.1.Retirar uma alíquota de 10µL da solução com plaquetas e diluir em 1000 µL de PBS;
6.8.2 Deixar a camâra de neubauer em repouso em camâra úmida por 10 minutos;
POP_MAL_LB_010_v01_PT
Página 4 de 4
6.8.3 Contar as plaquetas no quadrante central da câmara de Neubauer (área 5 da figura 1);
6.8.4 Multiplicar o valor encontrado por 1.000.
Plaquetas/µL = Quadrante central x 1.000.
6.9 Procedimento opsonização de plaquetas (para realizar o controle positivo):
6.9.1 Separar 400 µL da solução de plaquetas que foi previamente ajustada.
6.9.2 Acrescentar 3,5 µL de anticorpo monoclonal IgG2a anti MHC classe I humano (W6/32);
6.9.3 Incubar por 30 minutos á temperatura ambiente, protegido da luz;
6.9.4 Lavar as plaquetas com o PBS (duas vezes);
6.9.5 Resuspender em 400 µL de meio RPMI;
6.9.6 Armazenar a 20ºC até o teste de fagocitose.
6.10 Procedimento para avaliar a expressão de P-selectina em plaquetas
6.10.1 Incubar 100µl de PRP com 4µL anticorpo anti CD62P humano conjugado com PE (BD Pharmingen™)
por 30 min a 37°C.
6.10.2 Lavar duas vezes com PBS suplementado e PGE-1.
6.10.3 Acrescentar 200 µL de solução fixadora de células (paraformoldeído e cacodilato) e analisar a
expressão de CD62P (P-selectina) por citometria de fluxo.
6.11 Procedimento para avaliar a marcação de CMFDA e expressão de P-selectina no citômetro.
6.11.1 Seleciona (gate) as plaquetas pelo volume (forward scatter/FSC-H) e pela densidade interna (side
scatter/SSC-H) e avalia o porcentual de células positivas para CMFDA (no canal FL1-H) ou PE (no canal
FL2-H).
Nota: As células marcadas com CMFDA ficam fluorescentes e viáveis por até 24 horas. Proteger a amostra
da luz até a análise por citometria.
7
REFERÊNCIAS
•
Baker, G. R., et al. A simple, fluorescent method to internally label platelets suitable for
physiological measurements. Am J Hematology, v. 56, Sep, p. 15-25. 1997.
•
Lim, J., et al. Flow cytometric monocyte phagocytic assay for predicting platelet transfusion
outcome. Immunohematology, v. 42, Mar, p. 309-316. 2002.
•
Lacerda, M. V. G. Manifestações Clínicas e Patogênese da plaquetopenia na malária.
Universidade de Brasília, Brasília, 2007.
•
Honda, S., M. Saito, et al. Increased phagocytosis of platelets from patients with secondary
dengue virus infection by human macrophages. Am J Trop Med Hyg, v.80, n.5, May, p.841-5.
2009.
POP_MAL_LB_010_v01_PT
•
8
Página 5 de 5
Semple, J. W., et al. Platelet-bound lipopolysacharide enhances Fc receptor-mediated
phagocytosis of IgG opsonized platelets. Blood, v.109, n. 11, Jun, p. 4803-5.2007.
REGISTROS DOS ANEXOS
Não se aplica.
ANEXOS
Figura 1: Câmara de Neubauer. Número 5 é o quadrante central que deve ser contado para obter o número
de plaquetas.
A
versão
atual
deste
POP
foi
traduzida
a
____________________ e a versão traduzida entra em efeito
em ___/___/_____.
dd
mm
aa
Assinatura do responsável: ______________________
POP_MAL_LB_011_v01_PT
Procedimento Operacional Padrão
Gerência de Malária
Código POP
POP_MAL_LB_011_v01_PT
Título
Teste de fagocitose de plaquetas por células THP-1
Idioma da versão original
Português
Escrito ou traduzido por: Revisado por:
Aprovado por:
Helena C. C. Coelho
Paulo Nogueira, João Paulo
Pimentel e Stefanie Lopes
Marcus V. G. Lacerda
Data & assinatura
Data & assinatura
Data & assinatura
Emenda
1
Data de aplicação:
Data da próxima
revisão:
Razão da emenda
OBJETIVOS
Descrever o procedimento de fagocitose de plaquetas por células THP-1.
2
DEFINIÇÕES
Fagocitose: processo no qual partículas estranhas são envolvidas e destruídas por células especializadas ou
fagócitos.
CMFDA: diacetato de 5-clorometilfluoresceína, marcador fluorescente verde lipofílico, que entra no citoplasma
das células e, por conta de ligações covalentes, não volta ao extracelular.
Citometria de fluxo: técnica biofísica de análise qualitativa e quantitativa de partículas, biológicas ou não, em
suspensão monodispersa em meio líquido.
Celulas THP-1: linhagem celular de leucemia monocítica aguda humana.
PMA: Forbol 12-miristato 13-acetato (Phorbol Miristate Acetate).
3
APLICÁVEL A
O procedimento se aplica apenas à pesquisa experimental.
POP_MAL_LB_011_v01_PT
4
RESPONSABILIDADES
Gerente da unidade, coordenador da subunidade e pessoal técnico.
5
POP’S RELACIONADOS
POP_MAL_LB_010_v01_PT
6
6.1
PROCEDIMENTOS
Materiais necessários para o procedimento:
6.1.2 Células THP-1 mantidas em meio de cultura RPMI 1640 com SFB 10% a 37ºC
6.1.3 Placa de cultura de tecido com 24 escavações
6.1.4 Ponteiras para pipetas automáticas
6.1.5 Pipetas automáticas de 20 µL, 200 µL e 1000 µL
6.1.6 Gelo
6.1.7 Tubos de ensaio de polipropileno de 5 mL
6.1.8 Eppendorfs
6.1.9 Lâminas e lamínulas
6.1.10 Tubos para citometria
6.2
Equipamentos necessários para o procedimento:
6.2.1 Destilador de água
6.2.2 Balança semi-analítica
6.2.3 Estufa a 37ºC
6.2.4 Centrífuga refrigerada
6.2.5 Capela de fluxo laminar
6.2.6 Câmara de Neubauer ou aparelho automatizado para contagem de células.
6.2.7 Citômetro de fluxo
6.3 Reagentes necessários para o procedimento:
6.3.1 PBS 1x
6.3.2 Meio de cultura RPMI 1640
6.3.3 Soro fetal bovino (SFB)
6.3.4 Gentamicina
6.3.5 Solução para fixar as células (cacodilato e paraformaldeído)
6.3.6 PMA ( Forbol 12-miristato 13-acetato)
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POP_MAL_LB_011_v01_PT
Página 3 de 5
6.4. Preparação dos reagentes:
PBS 10x
Fosfato de sódio bibásico
Na2HPO4...................................................................................................................................11,94 g
Fosfato de sódio hidratado
NaH2PO4.H2O........................................................................................................................... 2,56 g
Cloreto de sódio
NaCl............................................................................................................................. .................87,66 g
Água destilada
H2O..........................................................................................................................................................1 L
Adicionar aproximadamente 900mL de água destilada estéril em um Erlenmeyer.
Colocar o fosfato de sódio e o fosfato de sódio hidratado até que se dissolvam completamente.
Ajustar o pH para 7,2-7,4 com NaOH 1 N ou HCl 1 N.
Depois de ajustar o pH, adicionar o NaCl até que se dissolva completamente.
Ajustar para 1 L com água destilada.
Fracionar em frascos para armazenamento de reagentes estéreis.
PBS 1x
Fazer uma diluição 1:10 com água destilada.
Ajustar o pH para 7,2-7,4 com NaOH 1 N ou HCl 1 N.
RPMI com 10% de SFB e 0,1% de gentamicina.
Soro fetal bovino ................................................................................................................................................................................5 mL
RPMI 1640........................................................................................................................................................................................................................ 45 mL
Gentamicina ......................................................................................................................................... 50 µL
6.5. Armazenamento dos reagentes:
Os reagentes utilizados nesse procedimento devem ser armazenados à temperatura de 2 –8º C.
6.6 Estimulação das células THP-1
6.6.1 Incubar 1x106 células THP-1 por poço (placa de cultura com 24 escavações) com RPMI 1640 e PMA
(60ng) por 2 horas, em estufa a 37º C e 5% de co2.
6.7 Procedimento para o teste de fagocitose:
6.7.1 Após 2 horas de incubação com PMA, aspirar o sobrenadante de cada poço da placa e lavá-los com
RPMI 1640 2 vezes.
6.7.2 Adicionar 900 µL de RPMI 1640.
6
6.7.3 Adicionar 100 µL da solução de plaquetas marcadas com CMFDA (50x10 /mL), com (controle positivo)
ou sem (pacientes e controle negativo) anticorpo monoclonal IgG2a anti MHC classe I humano (W6/32) em
cada poço.
6.7.5 Centrifugar a placa 500 g por 5 minutos à temperatura de 37ºC.
POP_MAL_LB_011_v01_PT
Página 4 de 5
6.7.4 Incubar a placa em estufa à 37ºC com 5% de Co2 por 60 minutos.
6.7.5 Interromper a fagocitose colocando a placa em uma vasilha com gelo.
6.7.6 Retirar o sobrenadante.
6.7.7 Acrescentar 200 µL de PBS 1X em cada poço e retirar as células com o auxílio da ponteira.
6.7.8 Passar as células para os tubos de citometria.
6.7.9 Lavar as células 3 vezes com PBS 1X, centrifugando os tubos a uma velocidade de 1.300 RPM por 7
minutos.
6.7.10 Descartar o sobrenadante e acrescentar 200 µL de solução fixadora de células (paraformoldeído e
cacodilato).
6.7.11 Armazenar à temperatura 2-8 ºC até o momento da leitura no citômetro.
6.8 Procedimento para leitura da fagocitose por citometria de fluxo:
6.8.1 Em citômetro de fluxo, fazer uma seleção prévia (gating) das células THP-1, pelo volume (forward
scatter/FSC-A) e pela densidade interna das células contidas no tubo de ensaio (side scatter/SSC-A),
excluindo-se, portanto, as plaquetas marcadas não fagocitadas.
6.8.2 Fazer e leitura no canal de fluorescência 1 (FL1-H).
6.8.3 A variável analisada foi a mediana de intensidade fluorescência emitida em FL1-H por cada célula THP1 contadas no gate e a porcentagem de células THP-1 positivas para CMFDA.
Nota: A fluorescência medida pela citometria de fluxo nas células THP-1 corresponde não apenas às
plaquetas internalizadas durante a fagocitose, mas também às plaquetas aderidas à superfície dos
monócitos.
7
8
REFERÊNCIAS

Auwerx J. The human leukemia cell line, THP-1: a multifacetted model for the study of
monocyte-macrophage differentiation. Experientia 1991;47:22-31.

Honda, S., M. Saito, et al. Increased phagocytosis of platelets from patients with secondary
dengue virus infection by human macrophages. Am J Trop Med Hyg, v.80. 2009.

Lim, J., et al. Flow cytometric monocyte phagocytic assay for predicting platelet transfusion
outcome. Immunohematology, v. 42, Mar, p. 309-316. 2002.

Lacerda, M. V. G. Manifestações Clínicas e Patogênese da plaquetopenia na malária.
Universidade de Brasília, Brasília, 2007.

Richmond JY, Mckinney RW. Biossegurança em laboratórios biomédicos e de
microbiologia. Brasília: Ministério da Saúde: Fundação Nacional de Saúde; 2001.
REGISTROS DOS ANEXOS
Não se aplica.
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A versão atual deste POP foi traduzida a ____________________ e a
versão traduzida entra em efeito em ___/___/_____.
dd
mm
aa
Assinatura do responsável: ______________________
69
ANEXO D: Parecer do CEP da FMT-HVD
91
ANEXO E: Artigos publicados
Plasma Circulating Nucleic Acids Levels Increase
According to the Morbidity of Plasmodium vivax Malaria
Bernardo S. Franklin1*, Barbara L. F. Vitorino1, Helena C. Coelho2, Armando Menezes-Neto1, Marina L. S.
Santos1, Fernanda M. F. Campos1, Cristiana F. Brito1, Cor J. Fontes3, Marcus V. Lacerda2, Luzia H.
Carvalho1*
1 Laboratório de Malária, Centro de Pesquisa René, Fundação Oswaldo Cruz, Belo Horizonte, Minas Gerais, Brazil, 2 Gerência de Malária, Fundação de Medicina Tropical Dr.
Heitor Vieira Dourado, Manaus, Amazonas, Brazil, 3 Departamento de Clı́nica Médica, Universidade Federal de Mato Grosso, Cuiaba, Mato Grosso, Brazil
Abstract
Background: Given the increasing evidence of Plasmodium vivax infections associated with severe and fatal disease, the
identification of sensitive and reliable markers for vivax severity is crucial to improve patient care. Circulating nucleic acids
(CNAs) have been increasingly recognized as powerful diagnostic and prognostic tools for various inflammatory diseases
and tumors as their plasma concentrations increase according to malignancy. Given the marked inflammatory status of P.
vivax infection, we investigated here the usefulness of CNAs as biomarkers for malaria morbidity.
Methods and Findings: CNAs levels in plasma from twenty-one acute P. vivax malaria patients from the Brazilian Amazon
and 14 malaria non-exposed healthy donors were quantified by two different methodologies: amplification of the human
telomerase reverse transcriptase (hTERT) genomic sequence by quantitative real time PCR (qPCR), and the fluorometric
dsDNA quantification by Pico Green. CNAs levels were significantly increased in plasma from P. vivax patients as compared
to healthy donors (p,0.0001). Importantly, plasma CNAs levels were strongly associated with vivax morbidity (p,0.0001),
including a drop in platelet counts (p = 0.0021). These findings were further sustained when we assessed CNAS levels in
plasma samples from 14 additional P. vivax patients of a different endemic area in Brazil, in which CNAS levels strongly
correlated with thrombocytopenia (p = 0.0072). We further show that plasma CNAs levels decrease and reach physiological
levels after antimalarial treatment. Although we found both host and parasite specific genomic sequences circulating in
plasma, only host CNAs clearly reflected the clinical spectrum of P. vivax malaria.
Conclusions: Here, we provide the first evidence of increased plasma CNAs levels in malaria patients and reveal their
potential as sensitive biomarkers for vivax malaria morbidity.
Citation: Franklin BS, Vitorino BLF, Coelho HC, Menezes-Neto A, Santos MLS, et al. (2011) Plasma Circulating Nucleic Acids Levels Increase According to the
Morbidity of Plasmodium vivax Malaria. PLoS ONE 6(5): e19842. doi:10.1371/journal.pone.0019842
Editor: Fabio T. M. Costa, State University of Campinas, Brazil
Received December 2, 2010; Accepted April 18, 2011; Published May 17, 2011
Copyright: ß 2011 Franklin et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits
unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
Funding: This work was supported by the Research Foundation of Minas Gerais State (FAPEMIG), The Brazilian National Research Council (CNPq), and Oswaldo
Cruz Foundation (FIOCRUZ, PAPES V), Pronex Malaria, CNPq/DECIT/MS; scholarships from FAPEMIG (FMFC), CNPq-FIOCRUZ (BSF), and CNPq (LHC, ATC, and CFAB)
are also acknowledged. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.
Competing Interests: The authors have declared that no competing interests exist.
* E-mail: [email protected] (BSF); [email protected] (LHC)
true spectrum of clinical disease in endemic areas remains
unknown [8]. The few studies that have addressed the pathogenesis of vivax malaria showed that the different clinical presentations of vivax malaria might be related to the intensity of proinflammatory responses [9,10,11,12]. Inflammatory cytokines such
as TNF-alpha and antioxidant agents have been associated with
clinical severity of P. vivax infections [13,14]. Nevertheless, data
validating their sensitivity and reliability as predictors of severe
disease are scarce. Consequently, the identification of highly
sensitive biomarkers for malaria vivax morbidity is crucial to
prevent life threatening complications.
Most of the DNA and RNA in the human body are located
within cells, but small physiologic amounts of nucleic acids can
also be found circulating freely in the blood. These DNA, RNA,
and small RNA molecules may arise from both: i) active release of
nucleic acids from living cells, or ii) break down of dying cells that
release their contents into the blood. The term Circulating Nucleic
Introduction
Plasmodium vivax malaria threatens almost 40% of the world’s
population, with an upper estimate of 300 million cases each year
[1]. Fortunately, after a long time being neglected under the
contemptible designation of benign infection, vivax malaria has
gained increasing attention in recent years.
In the last decade, a series of case reports and longitudinal
studies carried out in India [2,3], Papua in Indonesia [4,5], Papua
New Guinea [6] and Brazil [7] have demonstrated association of
P. vivax infections with severe or even fatal outcomes, with
incidence and morbidity rates similar to those for P. falciparum.
Consequently, costs due to hospitalization have significantly raised
as well as the need for intensive care, which helped vivax malaria
to be placed in a higher status of public health emergency [7].
Compared to falciparum malaria, there are remarkably large
knowledge gaps in the pathophysiology of vivax malaria, and the
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Circulating Nucleic Acids: Malaria Severity Marker
malaria, evaluated here by scoring patients’ clinical and
hematological parameters.
Acids (CNAs) refers to cell free segments of DNA or RNA found in
the bloodstream. Their existence in human plasma was first
reported more than 60 years ago [15], however, no interest was
shown in the presence of DNA in the circulatory system until high
DNA levels were demonstrated in the blood of patients with
cancer [16]. Elevated plasma CNAs levels have now been detected
during other acute illnesses and injuries. Examples include lupus
erythematosus [17,18], diabetes [19], trauma [20], stroke [21],
and myocardial infarction [22,23]. Furthermore, high usefulness of
CNAs levels in the diagnosis of infections in febrile patients and as
a prognostic marker in septic patients has been shown [24]. Their
applications in clinical diagnosis and prognosis have continuously
grown and further studies on CNAs showed that these nucleic
acids could be a powerful non-invasive approach to a wide range
of clinical disorders [25].
Aiming at finding sensitive and reliable biomarkers for P. vivax,
herein we tested the usefulness of plasma CNAs levels as markers
for the morbidity of vivax malaria. We investigated the CNAs
levels in plasma from P. vivax infected patients with different
clinical presentations and found significant higher levels of CNAs
in P. vivax infected patients, as compared to age-matched healthy
donors. We found that plasma CNAs levels were closely correlated
with variations in body temperature, platelets counts, and
increased in a linear fashion with the clinical spectrum of vivax
Results
CNAs levels were measured in plasma from P. vivax patients by
qPCR amplification of the genomic sequence of the human single
copy gene hTERT and by fluorometric quantification of the
dsDNA content with the Quant-iTTM Pico Green Reagent. The
amplification plot of hTERT shows that the mean cycle threshold
(Ct) achieved in CNAs samples from P. vivax patients (mean Ct
28.661.5) was significantly lower than the one reached in samples
from healthy donors (mean Ct 31.560.79) (p,0.0001) (Fig. 1A
and 1C). As the amount of DNA theoretically doubles every cycle
during the exponential phase of qPCR, these results suggest that
the levels of this target sequence in the CNAs preparation from P.
vivax patients are at least 8-fold higher than in healthy donors. In
fact, a difference of 11,66 between the hTERT levels in plasma
from P. vivax patients (1.278 pg/ml) and healthy donors
(0.1098 pg/ml) was confirmed when a standard curve, built from
a serial dilution of an amplified sample of hTERT sequence, was
used to interpolate the hTERT concentrations in the samples
(Figure S1). To normalize the amount of nucleic acids purified and
inputted in qPCR experiments, 5 ng of salmon sperm DNA was
Figure 1. Increased CNAs levels in plasma from P. vivax patients. CNAs levels were quantified in plasma from acute P. vivax patients or
healthy donors by measuring the amplification of the hTERT human genomic sequence (A) as compared to the amplification of the O. keta Y
chromosome marker (B) for the salmon sperm DNA spiked into plasma samples before CNAs purification. (C) Comparison of the mean cycle threshold
(Ct) from the hTERT or the O. keta Y chromosome marker in CNAs samples purified from P. vivax patients or non-exposed healthy donors. (D)
Fluorometric dsDNA quantification of CNAs levels in plasma by the Quant-iTTM Pico Green methodology. Statistical analyses were performed using
the Mann-Whitney test. A p value,0.05 was considered significant.
doi:10.1371/journal.pone.0019842.g001
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Circulating Nucleic Acids: Malaria Severity Marker
cancer [26]. Both platelets [27] and platelet derived microparticles
(PMPs) [10] have been associated with clinical manifestations of
malaria. We thus investigated if plasma CNAs levels may be
associated with thrombocytopenia and/or others hematological
parameters, such as WBC and RBC counts, hemoglobin and
hematocrit levels, mean corpuscular hemoglobin (MHC) and
mean platelet volume (MPV). Among all parameters investigated,
we found a strong negative correlation between CNAs levels,
assessed by dsDNA quantification with Pico Green, and platelet
counts (spearman r = 20.6451, p = 0.0021) (Fig. 3A). These
findings were confirmed when the mean Ct obtained after qPCR
amplification of the genomic sequence for hTERT gene was
plotted against platelet levels (Pearson r = 0.6479, p = 0.0027)
(Fig. 3B).
To further confirm the association between CNAS levels and P.
vivax morbidity, we assessed the CNAS levels in plasma from an
additional group of P. vivax patients whose selection was carriedout in a different hospital of the Amazon area, Cuiaba, MT
(,1500 miles from Manaus, AM). Once the clinical protocol used
at the hospital in Cuiaba was different from Manaus (FMT-HVD),
we were unable to build a similar clinical score. For this reason, we
compared the CNAS levels in these samples with thrombocytopenia, a common hematological disturbance seen in malaria
morbidity in the Amazon area [28]. By analyzing the amplification
of hTERT, it was possible to demonstrate a significant correlation
(Pearson r = 0.745, p = 0.0072) between CNAS levels and
thrombocytopenia in P. vivax patients from Cuiaba (Figure S2A).
As this study provides the first description of circulating nucleic
acids in malaria infection, we evaluate CNAs levels in a small group
of P. falciparum patients who sought for care at Cuiaba’s hospital
(n = 9). CNAs levels were significantly higher in samples from
falciparum malaria patients as compared to healthy donors
(p = 0.038; not shown). Importantly, CNAs levels in patient’s plasma
clearly correlated with thrombocytopenia (Figure S3A) and the
occurrence of fever during acute P. falciparum infection (Figure S3B).
spiked into plasma samples before CNAs purification (Fig. 1B). As
expected, the specific sequence of salmon sperm DNA was
similarly amplified in P. vivax patients and healthy donors plasmas
(Fig. 1B and 1C, p = 0.6925).
The increased levels of total CNAs in plasma from P. vivax
patients were confirmed by quantification of dsDNA with QuantiTTM Pico Green Reagent (Fig. 1D) (1494.761169.7 in vivax
patient vs. 689.036131.54 pg/ml in healthy donors, p,0.0001).
To investigate the potential of CNAs as biomarkers for malaria
morbidity, we compared the levels of CNAs in plasma from
patients with different clinical presentations, and scored according
to clinical and hematological parameters (Table S1). Figure 2A
illustrates the qPCR amplification of the hTERT genomic
sequence in plasma from four P. vivax patients and four
unexposed-controls. Sensitive changes in hTERT amplification
were observed according to the slightest increase in the clinical
score. Furthermore, significantly higher levels of CNAs were found
in plasma isolated from patients who presented fever at the time of
blood collection (febrile patients) compared to plasma samples
from non-febrile patients, as revealed by the two different
methodologies: amplification of hTERT genomic sequence by
qPCR (p = 0.0376) and the quantification of dsDNA content with
Quant-iTTM Pico Green (p = 0.0023) (data not shown).
To confirm whether CNAs levels reflect disease morbidity, the
sum of scores attributed to each patient (Table S1) was plotted
against the CNAs levels detected in plasma with the Quant-iTTM
Pico Green or the mean cycle threshold detected by qPCR
amplification of the hTERT genomic sequence (Fig. 2B). A clear
correlation (Spearman r = 0.4795, p = 0.0034) was found between
the CNAs levels and the intensity of clinical malaria. These data
were confirmed when the Cts from the amplification of hTERT
were analyzed (Pearson r = 20.7111, p,0.0001) (Fig. 2B).
Platelet activation exerts thrombotic and pro-inflammatory
functions and their unbalanced activation contributes to lifethreatening outcomes in diseases such as heart attack, stroke, and
Figure 2. Plasma CNAs levels reliably correlate with the P. vivax clinical spectrum. CNAs levels were quantified in plasma from P. vivax
patients with different clinical presentations. (A) Amplification of the genomic sequence of hTERT by qPCR in four healthy controls, and P. vivax
patients (Pv_01 to 04) scored according to clinical/hematological parameters. Only four patients are shown for illustration purposes. (B) Correlation
between the final clinical score of P. vivax patients (n = 21) and their plasma CNAs levels (pg/ml) (Spearman r = 0.6092, p = 0.0034), or their Ct for the
amplification of the hTERT (Pearson r = 20.7897, p,0.0001).
doi:10.1371/journal.pone.0019842.g002
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Circulating Nucleic Acids: Malaria Severity Marker
Figure 3. Correlation between plasma CNAs levels and malaria vivax thrombocytopenia. Correlation of plasma CNAs levels with platelet
counts in symptomatic vivax malaria patients. The dsDNA levels measured by Pico Green (A) and the mean cycle threshold for hTERT amplification (B)
were plotted against the platelet counts. Spearman (r = 20.6451) and Person (r = 0.6479) correlations were used respectively in A and B. A p
value,0.05 was considered significant.
doi:10.1371/journal.pone.0019842.g003
It is reasonable to speculate that parasite specific DNA is present
among the CNAs circulating in plasma. To confirm this, we
assessed the levels of P. vivax derived-CNAs in plasma in an
attempt to investigate their use as a streamline diagnostic and
prognostic tool. For this purpose, specific primers were designed to
amplify a genomic sequence unique to P. vivax. As expected,
amplification of this genomic sequence was not detected in
samples from healthy donors (Fig. 5A). Furthermore, although
parasite specific CNAs levels were weakly associated with the
presence of fever at the time of blood sampling (Ct vs. body
temperature, r = 20.5535, p = 0.0497) (Fig. 5B), they were not
In six patients attended at the FMT-HVD (Manaus, AM), the
CNAs levels were further assessed 7 days after antimalarial
chemotherapy. As shown in Fig. 4A, CNAs levels decreased after
specific treatment (p = 0.0428). The comparison of the mean Ct
obtained after qPCR amplification of the hTERT in plasma
samples from acute vs. treated patients confirmed these findings
(p = 0.0243) (Fig. 4B). Seven days post-treatment, the platelet
counts returned to physiological levels (Fig. 4C). These data were
further confirmed in patients from Cuiaba area (n = 10) (Figure
S2B). In those samples, CNAs levels were significantly diminished
after 7–10 days of chemotherapy.
Figure 4. Plasma CNAs levels decrease after anti-malarial chemotherapy. For 6 patients who showed up during convalescence, the CNAs
levels in plasma were assessed by (A) fluorescence quantification of dsDNA with the Pico Green methodology or (B) comparison of the mean cycle
threshold for the qPCR amplification of hTERT genomic sequence. (C) platelet counts measured during admission and convalescence. Statistics were
performed as follow: Mann-Whitney test for panel A, and two tailed t test for panels b and C. A p value,0.05 was considered significant.
doi:10.1371/journal.pone.0019842.g004
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Circulating Nucleic Acids: Malaria Severity Marker
Figure 5. Plasmodium specific genomic sequences circulating in plasma from P. vivax patients. The presence and levels of P. vivax specific
plasma CNAs were investigated in samples from P. vivax patients with different clinical presentations by qPCR amplification of a specific P. vivax
genomic sequence. (A) For illustration purpose, qPCR amplification of a P. vivax specific genomic sequence in four healthy controls, and four P. vivax
patients (Pv_01 to 04) scored according to clinical/hematological parameters is shown. (B) Pearson correlation between the Ct of parasite specific
genomic sequence and the body temperature measured at the time of blood collection (r = 20.5535, p = 0.0497) or the clinical score of the patients
(r = 20.3604, p = 0.2056). A p value,0.05 was considered significant.
doi:10.1371/journal.pone.0019842.g005
associated with the clinical spectrum of the disease (r = 20.3604,
p = 0.2056) (Fig. 5C). Also, parasite specific CNAs genomic
sequences did not reflect peripheral parasitemia (r = 20.3735,
p = 0.1884).
malaria patients. The kinetics by which CNAs levels rise and fall
during acute malaria requires further investigation.
The source of CNAs levels during malaria remains unknown.
Apoptosis and necrosis have been pointed as the main source of
cell-free DNA circulating in blood [41,42]. Usually apoptosisinduced cleavage of DNA results in DNA fragments of
approximately 180 bp; thus, quantification of a small and a long
PCR product allows indirect inferences about the underlying celldeath entity. Although apoptosis has not been directly addressed in
this study, our results do not rule out this possibility, as most of the
fragments amplified were in the range of 90 bp to be suitable for
qPCR analysis. In malaria, apoptosis is a process highly
represented in the annotation of gene expression profile of acute
infection as revealed by several microarray studies involving both
human and mouse models [43,44]. Nevertheless, it was recently
shown that apoptosis and or necrosis might not be the main
sources of CNAs in plasma of patients with a variety of other
conditions, and active release of free circulating DNA by living
cells was pointed as a plausible mechanism [45]. At this time, it is
unknown whether apoptosis and/or DNA release contribute to the
higher levels of cell-free DNA observed here in P. vivax patients.
Thrombocytopenia (platelet counts ,150,000/mm3) is a
common hematological finding in patients with Plasmodium
infection particularly in vivax malaria [28,46]. Recent studies
carried out in northwest India highlighted the higher occurrence
of severe thrombocytopenia in P. vivax in comparison to either P.
falciparum monoinfection or mixed infections [47,48]. We show
here that CNAs levels in vivax malaria strongly correlate with a
drop in platelet counts, a data confirmed in two different hospitals
of the Amazon area. Although it is not possible, at this point, to
speculate on the role of platelets in the increase of CNAs levels in
plasma, our results indicate that CNAs might contribute to cell
activation and inflammation that are associated with malaria
infection.
Although P. falciparum infection was not the main scope of the
present study, by having access to a small group of patients, it was
possible to demonstrate that CNAS levels are increased during
acute P. falciparum infection. In this malaria model, increased
CNAs levels in plasma were associated with thrombocytopenia
and the occurrence of fever at the time of blood collection (Figure
Discussion
This study is the first to investigate the use of plasma levels of
cell-free circulating nucleic acids (CNAs) as a marker of P. vivax
malaria morbidity. We show here that CNAs levels in plasma from
P. vivax patients increase linearly with the clinical spectrum of the
disease. This confirms that this powerful marker can also be used
in malaria as a sensitive indicator of inflammation and injury. In
fact, plasma CNAs levels have been regarded as a noninvasive
universal cancer biomarker [29] as their levels have been shown to
be distinctly increased in most patients with solid tumors (E.g. lung
[30], colon [31], cervical [32], ovarian [33], breast [34], testis [35],
bladder [36], and prostate [37]), allowing their discrimination
from patients with nonmalignant disease or healthy individuals.
Plasma CNAs levels have also been associated with the severity of
several other inflammatory disorders [17,18,19,20,21,22,23].
Other molecules circulating in plasma, such as adhesion
molecules [38], pro-inflammatory cytokines [39], the superoxide
dismutase-1 [14], and, more recently, microparticles [10], have
been suggested as biomarkers for human P. vivax malaria as their
levels are often associated with malaria clinical manifestations.
Nevertheless, we believe that CNAs offer a more sensitive tool
since qPCR amplification of hTERT, a specific single copy human
genomic sequence, revealed that levels as low as 100 fentogram of
CNAs could be detected circulating in plasma, and were able to
discriminate different degrees of disease morbidity (Figure S1).
We show here that plasma CNAs reach physiologic levels after
7–10 days of antimalarial chemotherapy and patient’s recovery. It
has been shown that clearance of cell-free DNA from the
bloodstream occurs rapidly; the half-life time of fetal DNA in
the blood of mothers after delivery was approximately 16 minutes
[40]. Cell-free DNA seems to be eliminated by different manners
including renal and hepatic mechanisms as well as degradation by
plasma nucleases [29]. It is unknown whether a different clearance
time is also contributing to the higher levels of cell-free DNA in
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Circulating Nucleic Acids: Malaria Severity Marker
number of P. vivax malaria cases in Latin America [54]. In 2009, a
total of 19,698 malaria cases were reported in Manaus with a large
dominance of vivax (92.6%) over falciparum malaria [55].
Individuals who sought care at FTM-HVD and whose thick
blood smear was positive for P. vivax were invited to participate in
the study. Exclusion criteria included: (i) refuse or inability to sign
the informed consent; (ii) age ,18 years; (ii) pregnant women; (ii)
mixed infection with P. falciparum or P. malariae; (iv) any other comorbidity that could be traced. Twenty-one patients, aging 21 to
72 years, were enrolled in the study. Selected volunteers were all
negative for P. falciparum and/or Plasmodium malariae infection by
both microscopic examination and a nested-PCR, carried out
latter in our laboratory. Clinical and demographical data were
acquired through a standardized questionnaire, and the hematological profiles were assessed by automated complete blood count
carried out at FMTA hematology facility. Table 1 summarizes
demographic, epidemiological, parasitological and hematological
data of P. vivax infected-volunteers.
The study was approved by the Ethical Review Board of the
René Rachou Research Center, FIOCRUZ, Brazilian Ministry of
Health (Reporter CEPSH/CPqRR 05/2008). All participants
were instructed about the objectives of the study and signed an
S3). While these results support the association between CNAS
and malaria, the size of our sample precludes any definitive
comparison between P. falciparum and P. vivax infection. Further
studies will be required to proper address this question.
In uncomplicated P. vivax malaria, we have recently shown that
the levels of circulating platelet-derived microparticles (PMPs) are
associated with the clinical spectrum of disease, including fever
and prolonged time with malaria symptoms [10]. The fact that
CNAs levels as well as PMPs were higher in febrile and
symptomatic vivax patients suggests a possible association with
PMP and CNAs. MPs are important carriers of membrane
components or bioactive molecules and their association with
nucleic acids has been proposed [49]. The presence of host and/or
parasite DNA associated with MPs circulating in plasma and their
role in inflammation is currently being addressed in your
laboratory.
To investigate if parasite derived-sequences are part of the pool
of nucleic acids circulating in blood during vivax malaria, and if
these sequences correlate with disease morbidity, we assessed the
levels of a parasite specific single copy genomic sequence in CNAs
purified from P. vivax patients. Although our results revealed that
host and parasite sequences are part of the total plasma CNAs
levels in acute P. vivax infected patients, the levels of a host specific
(hTERT) but not parasite specific sequence correlated with vivax
clinical disease. These results are in agreement with a recently
study carried out in the Amazon area in which high parasitemia
was not the rule among patients with severe disease according to
the WHO criteria [50].
Whether CNAs are merely inert debris of cellular injury, or if
they possess pro-inflammatory properties and are, therefore,
players in the immunopathogenic basis of malaria requires further
investigation. Although at this point is not possible to draw
conclusions, their role in the inflammatory response during
malaria cannot be rule out. In fact, it is well known that dying
cells spill their content and release a myriad of endogenous proinflammatory danger signals, including proteins, nucleic acids,
extracellular matrix components, lipid mediators and adenosine
triphosphate (ATP) [51]. These endogenous danger signals have
been shown to play important roles in inflammation [51,52,53]. As
human and parasite derived nucleic acid sequences have been
shown to posses immune-stimulatory properties, the implication of
CNAs in cellular activation and in innate immunity is likely.
Likewise, the frequency of immune stimulatory vs. non-stimulatory
circulating nucleic acids in plasma from patients with different
clinical outcomes would provide important insights into the role of
CNAS in malaria pathogenesis.
In conclusion, we show that host circulating nucleic acids in
plasma constitute a reliable and non-invasive biomarker to
evaluate vivax malaria morbidity. CNAs levels were closely
associated with P. vivax malaria clinical spectrum, and may have
a role in malaria-induced inflammation. Given the enormous
economic scourge of P. vivax in endemic areas, plasma CNAs levels
provide a welcome prognostic tool to rapidly identify potentially
severe cases and improve clinical management.
Table 1. Characteristics of the Plasmodium vivax patients
enrolled in the study.
CHARACTERISTICS
Demographical and epidemiological
Sex, male/female, proportion
13/8
Age, median, range
49 (21–72)
Nu of previous malaria episodes
3 (0–30)
Parasitological and hematological, median (range)
Parasitemia, parasites/ml of blood
305 (25–2255)
Hematocrit %,
42.6 (30.5–48.9)
Hemoglobin levels g/dL
13.2 (9.5–14.9)
WBC counts6106/mm3
4.9 (2–8.6)
RBC counts6106/mm3
4.8 (3.57–5.42)
Platelet counts6106/mm3
125.5 (39–225)
MCV (fL)
89.3 (82.9–96)
MPV (fL)
9.8 (8.1–13.2)
MCH (pg)
27.1 (25–30.1)
MCHC (g/dL)
30.6 (29.8–33.2)
Clinical parameters
Duration of symptoms in days, median, (range)
3 (.1–20)
Fever at the time of blood sampling, n (%)
7 (33.3%)
Symptoms in the last 3 days, n (%)
Fever
21 (100%)
Myalgia
21 (100%)
Chills
19 (90.5%)
Headache
18 (85.7%)
Study area and subjects
Nausea
15 (71.4%)
This study was conducted in May 2010, at Fundação de
Medicina Tropical Dr. Heitor Vieira Dourado (FMT-HVD), a
tertiary care center for infectious diseases in Manaus (3u89S,
60u19W), the capital of the state of Amazonas, Brazil. Manaus is
clearly part of a new frontier in the economic development of the
Amazon and is considered as one of the leading cities in terms of
Anorexia
12 (57.1%)
Vomiting
6 (28.6%)
Dyspnea
6 (28.6%)
Diarrhea
3 (14.3%)
Materials and Methods
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doi:10.1371/journal.pone.0019842.t001
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Circulating Nucleic Acids: Malaria Severity Marker
informed consent in accordance with guidelines for human
research, as specified by the Brazilian National Council of Health
(Resolution 196/96). Patients diagnosed with vivax malaria were
treated according to the standard protocols recommended by the
National Malaria Control Program (chloroquine plus primaquine).
Peripheral blood samples (10 mL in EDTA) were obtained from
patients on admission and, in those who attended follow-up,
during convalescence 7 days later. Plasma samples from 14 agematched malaria-unexposed donors from Belo Horizonte, a
malaria free area, were used as baseline control. Aiming to avoid
bias of selection, we further include an additional group of P. vivax
patients (n = 14; age range, 18–41 years) from a second hospital of
the Amazon area, Julio Muller Hospital, Cuiaba, MT, which was
located about 1500 miles from Manaus city. CNAS levels were
also evaluated in plasma samples from a small group of P.
falciparum patients (n = 9; age range, 18–52 yrs.). Plasma samples
were isolated immediately after blood sampling and stored at
280uC until use.
specific marker was amplified in parallel with hTERT using the
specific primers: Fw: 59 AGG CAA CCC TTG CTC GAA TT 39;
Rev 59 TGG GCA CAT GGC TTA CCG 39; (ii) total dsDNA
levels in plasmas were also quantified fluorometrically using the
Quant-iTTM Pico Green Reagent (Molecular Probes, Netherlands) according to the manufacturer’s instructions.
To identify parasite derived sequences in plasma samples from
infected patients the following primer pair Fw: 59 CAA CAG GTC
CTT CAC GCT TAG TG 39; Rev: 59 CGA CAG CAC CAT
TGG CG 39 was designed based on the P. vivax genomic sequence
[59] retrieved from PlasmoDB version 6.4 (http://plasmodb.org/
plasmo/). The Primer Express software (PE Applied Biosystems)
was used for primer design. Quantitative PCR reactions were
carried out in an ABI Prism 7000 Sequence Detection System
SDS (PE Applied Biosystems, CA, USA). The temperature profile
was 95uC for 10 min followed by 40 cycles of denaturation at
95uC for 15 s and annealing/extension at 60uC for 1 min. The
cycle threshold for DNA quantification was set to 0.2 for all
experiments in this study.
Malaria vivax clinical score
Statistical analysis
Since at present no clear criteria define vivax malaria severity,
the present study used the World Health Organization standard
criteria built for P. falciparum malaria [50]. One patient (Pv_04,
Table 1) presented clinical signs of severe malaria according to
the WHO criteria. This patient presented with hyperbilirrubinemia (total bilirubin = 4.3 mg/dL) and acute renal failure
(creatinin = 2.3 mg/dL), and other common infectious diseases
were ruled out during his hospitalization. To define different
degrees of morbidity for the remaining P. vivax malaria patients,
we adapted the criteria originally described by Karunaweera et
al [56], and previously validated in the Amazon area [57].
Briefly, the occurrence of fever at the time of blood collection
and other 8 signs and/or symptoms that commonly accompany
a malarial infection - headache, chills, myalgia, nausea,
vomiting and diarrhea - were addressed into the questionnaire
applied to each patient. Additionally, hematological parameters
were also included in the score calculation: white blood cells
(WBC), red blood cells (RBC) and platelets counts, hemoglobin
and hematocrit levels (Table 1). Numerical scores of 0 or 1 were
assigned to clinical and hematological parameters reported as
absent (or within normal range) or present (or outside normal
range), respectively. For those 15 parameters analyzed, the sum
of scores provides the patient’s final clinical score, as shown in
Table S1 (supporting information). This semi quantitative
clinical assessment enabled numerical comparisons between
the plasma CNAs levels and the clinical spectrum of vivax
malaria.
Data were analyzed using GraphPad Prism version 5.00 for
Windows (GraphPad Software, CA, US). Differences in the means
were analyzed using two-tailed student’s t test or Mann-Whitney
test when data did not fit a Gaussian distribution. Spearman
nonparametric correlation coefficient was used to analyze the
association between the variables.
Supporting Information
Figure S1 Absolute quantification of hTERT levels in
plasma from P. vivax patients. The human genomic
sequence of hTERT was amplified by PCR using the primers
described in M&M. The concentration of the PCR product was
determined spectrophotometrically using Nanodrop. (A) A standard curve was built by re-amplifying known amounts of the
hTERT PCR product in 10-fold serial dilutions. (B) Amplification
of hTERT in CNAs samples purified from healthy donors or
malaria patients. (C) Results of interpolated hTERT concentrations in CNAs samples purified from plasma of healthy donors or
malaria patients. Levels are expressed as pg/ml. Differences were
calculated by the Mann-Whitney test. A p value,0.05 was
considered significant.
(TIFF)
Figure S2 Plasma CNAs levels correlates with vivax
thrombocytopenia in a different Brazilian endemic area,
Cuiaba, Mato Grosso. Correlation of plasma CNAs levels with
platelet counts in 14 symptomatic vivax malaria patients attended
at the hospital Julio Muller, Cuiaba, MT. (A) The mean cycle
threshold for hTERT amplification was plotted against the platelet
counts (Pearson r = 0.745, p = 0.0072). (B) Assessment of CNAs
levels and mean cycle threshold for hTERT amplification in
samples from 10 out of 14 patients who returned after 7–10 days
post treatment.
(TIFF)
Purification and quantification of CNAs from plasma
Cell-free circulating nucleic acids (CNAs) were isolated from
plasma from P. vivax patients or healthy donors with QIAamp
Circulating Nucleic Acid Kit (Qiagen, CA, US) according to the
manufacturer’s instructions. Two different methodologies were
used to quantify CNAs levels in plasma: (i) amplification of the
genomic sequence of the human telomerase reverse transcriptase
(hTERT), an ubiquitous single copy gene mapped on 5p 15.33,
used here as a marker of the total amount of DNA present in
plasma samples. For that, we used the following specific primers
Fw: 59GGC ACA CGT GGC TTT TCG 39; Rev: 59 GGT GAA
CCT GCT AAG TTT ATG CAA 39, previously described [58].
To normalize the amount of DNA in plasma samples, 5 ng of
Salmon Sperm DNA solution (Invitrogen, CA, USA) were spiked
into plasma samples before purification of CNAs. The genomic
sequence of the chum salmon (Oncorhynchus keta) Y-chromosome
PLoS ONE | www.plosone.org
Plasma CNAs levels correlates with thrombocytopenia in P. falciparum patients. CNAs levels were
assessed in plasma from 9 samples from P. falciparum patients and
correlated with (A) their platelet counts and (B) body temperature
measured at the time of blood collection. Fluorometric dsDNA
measurement by PicoGreen and qPCR amplification of hTERT
genomic sequence were used for comparisons.
(TIFF)
Figure S3
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Circulating Nucleic Acids: Malaria Severity Marker
Real Time PCR Instrumet awarded to BSF as the 2009 winner of the
Helixis Young Investigator Award.
Table S1 Patient final clinical score and plasma CNAs
levels.
(DOC)
Author Contributions
Acknowledgments
Conceived and designed the experiments: BSF LHC MVL. Performed the
experiments: BSF BLFV HCC MLSS FMFC AM-N. Analyzed the data:
BSF LHC MVL. Contributed reagents/materials/analysis tools: CFB
MVL. Wrote the paper: BSF LHC. Coordinated the study in the endemic
areas: MVL CJF.
The authors would like to thank the Program for Technological
Development in Tools for Health - PDTIS - FIOCRUZ for use of its
facilities; Belisa Maria Lopes Magalhães and Raimunda Ericilda da Silva
for their help with patients in the endemic area. The authors would also
like to thank the Helixis Incorporation (currently Illumina) for the PIXO
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PLoS ONE | www.plosone.org
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May 2011 | Volume 6 | Issue 5 | e19842
52
Mem Inst Oswaldo Cruz, Rio de Janeiro, Vol. 106(Suppl. I): 52-63, 2011
Thrombocytopenia in malaria: who cares?
Marcus Vinícius Guimarães Lacerda1,2,3/+, Maria Paula Gomes Mourão1,2,3,
Helena Cristina Cardoso Coelho2, João Barberino Santos4
Fundação de Medicina Tropical Dr. Heitor Vieira Dourado, Av. Pedro Teixeira 25, 69040-000 Manaus, AM, Brasil
2
Universidade do Estado do Amazonas, Manaus, AM, Brasil 3Universidade Nilton Lins, Manaus, AM, Brasil
4
Universidade de Brasília, Brasília, DF, Brasil
1
Despite not being a criterion for severe malaria, thrombocytopenia is one of the most common complications
of both Plasmodium vivax and Plasmodium falciparum malaria. In a systematic review of the literature, platelet
counts under 150,000/mm3 ranged from 24-94% in patients with acute malaria and this frequency was not different
between the two major species that affected humans. Minor bleeding is mentioned in case reports of patients with P.
vivax infection and may be explained by medullary compensation with the release of mega platelets in the peripheral
circulation by megakaryocytes, thus maintaining a good primary haemostasis. The speculated mechanisms leading
to thrombocytopenia are: coagulation disturbances, splenomegaly, bone marrow alterations, antibody-mediated
platelet destruction, oxidative stress and the role of platelets as cofactors in triggering severe malaria. Data from
experimental models are presented and, despite not being rare, there is no clear recommendation on the adequate
management of this haematological complication. In most cases, a conservative approach is adopted and platelet
counts usually revert to normal ranges a few days after efficacious antimalarial treatment. More studies are needed
to specifically clarify if thrombocytopenia is the cause or consequence of the clinical disease spectrum.
Key words: Plasmodium falciparum - Plasmodium vivax - malaria - thrombocytopenia - platelets
Malaria affects almost all blood components and is
a true haematological infectious disease. Anaemia and
thrombocytopenia are the most frequent malaria-associated haematological complications (Wickramasinghe &
Abdalla 2000) and have received more attention in the scientific literature due to their associated mortality. On the
other hand, thrombocytopenia is less studied, causes negligible mortality and is an isolated phenomenon; there is
no report of a single patient in the literature who has died
only because of malaria-associated thrombocytopenia.
In the current field of Travel Medicine, the rapid increase in the number of people travelling to tropical areas
has added a great challenge for malaria diagnosis because
the thick blood smear (the standard diagnosis in endemic
areas) has high specificity but only when performed by
experienced microscopists. The presence of thrombocytopenia in acute febrile travellers returning from tropical areas has become a highly sensitive clinical marker
for malaria diagnosis (D’Acremont et al. 2002). Another
study has reported 60% sensitivity and 88% specificity
of thrombocytopenia for malaria diagnosis in acute febrile patients (Lathia & Joshi 2004). The sensitivity of
thrombocytopenia together with the acute febrile syndrome was 100% for malaria diagnosis, with a specificity
of 70%, a positive predictive value of 86% and a negative
predictive value of 100% (Patel et al. 2004).
Financial support: CAPES (scholarship for HCCC), CNPq (MVGL is
a level 2 research productivity fellow), ASH
+ Corresponding author: [email protected]
Received 1 April 2011
Accepted 26 May 2011
online | memorias.ioc.fiocruz.br
Thrombocytopenia is a well-documented and frequent complication in Plasmodium vivax malaria. In
one study, platelet count normalised after treatment and
only one patient, concomitant with the lowest platelet
count, exhibited “purpuric lesions” on the lower extremities (Hill et al. 1964).
Since the beginning of the 1970s, there have been
reports proposing that malaria-associated thrombocytopenia is quite similar in P. vivax and Plasmodium falciparum infections (Beale et al. 1972). However, more
recent data in India has shown how thrombocytopenia
exhibited a heightened frequency and severity among
patients with P. vivax infection (Kochar et al. 2010).
In 1903, the young physician Carlos Chagas (who
become more famous afterwards for the discovery of
American trypanosomiasis, which is named after him),
published his MD thesis on the Hematological Studies on Paludism (Chagas 1903). Within it, he described
anaemia and leukocyte abnormalities, but also normal
megakaryocytes in the bone marrow were referred to in
patients with acute and chronic malaria from Rio de Janeiro. He also drew our attention to evidence of bleeding
in the 46 patients he followed.
In the city of Manaus, state of Amazonas, located in
the Western Brazilian Amazon, Djalma Batista authored
Paludism in the Amazon, a book in which he described
observations about patients with malaria seen at his private clinics (Batista 1946). Similar to Carlos Chagas,
there is no mention of platelet count in his study because
it was not routinely performed. However, there is a vivid
description of haemostasis disorders in some patients.
Particularly noteworthy is the presence of huge spleen
enlargement and prolonged bleeding time accompanied
by recurrent gingival bleeding.
Malaria and thrombocytopenia • Marcus Vinícius Guimarães Lacerda et al.
Data on the real burden of thrombocytopenia associated with malaria is contradictory in the literature and
it is not usually considered when conducting patient selection. Table I shows the major publications estimating
the frequency of thrombocytopenia. Most of these data
were published in the late 1990s, probably in time with
the surge in the availability of affordable automated machines capable of performing full blood counts (FBC).
Manual platelet counting is time-consuming and usually
needs to be requested by the physician with the routine
blood count in most of the endemic areas for malaria. In
only three publications is there an adequate randomised
enrollment of patients with appropriate sample size calculation to estimate the frequency of bleeding and its
association with the respective platelet count (Lacerda
2007, Silva 2009, Kochar et al. 2010). Only one study
has ruled out other common causes of thrombocytopenia
that are also endemic in the studied area (Lacerda 2007).
There is a wide range of thrombocytopenia occurrence
in these reports, which may be explained by distinct selection criteria of the enrolled patients. There are also
differences in the selection of outpatients or inpatients
from tertiary care centres that tend to present with severe
thrombocytopenia. Furthermore, clinical manifestations
of thrombocytopenia are usually described as case reports and most of these are due to P. vivax (Table II).
In 2005, 138 of 684 (20.1%) malarial cases hospitalised in a tertiary care centre in Manaus had thrombocytopenia as the cause of admission, which corresponded
to 6.8% of hospitalisations due to all causes in this reference institution (unpublished observations). Hospitalisation, however, does not add any benefit to the patient and
because there is no evidence for any intervention, this
simply increases public health costs in underdeveloped
and under-resourced areas.
Pathogenesis of malarial thrombocytopenia - Coagulation disturbances - A study based on 31 American
soldiers in Vietnam with chloroquine-resistant falciparum malaria noted the following changes in the acute
phase of the disease using the same patients as their own
controls during convalescence: decrease in the platelet
count and prothrombim activation time, increase in the
activated thromboplastin time, and reduction in factors
V, VII and VIII with normal fibrinogen (Dennis et al.
1967). This report suggested that thrombocytopenia was
simply a consequence of the coagulation disorders presented by these patients, an idea that persisted for many
decades in the literature. In another series of 21 patients
with falciparum malaria, six had developed disseminated
intravascular coagulation (DIC). The authors noted that
the patients with more severe thrombocytopenia also
had DIC and that there was correlation between platelet
count and C3 protein levels. However, the reduction in
C3 was proportional to that in parasitaemia, suggesting
that thrombocytopenia was not independently associated with C3 (Srichaikul et al. 1975). In Manaus, 2004, a
study with falciparum and vivax patients demonstrated a
negative correlation between platelet counts, thrombinanti-thrombin complex and D-dimers, suggesting that
the activation of coagulation could be partially responsible for thrombocytopenia (Marques et al. 2005).
53
Splenomegaly - The spleen in malaria has played a
crucial role in the immune response against the parasite,
as well as controlling parasitaemia due to the phagocytosis of parasitised red blood cells (RBCs) (Engwerda et al.
2005). Some data suggested that platelets were sequestered in the spleen during the acute infection (Skudowitz
et al. 1973). In the experimental model with Plasmodium
chabaudi, thrombocytopenia was absent in splenectomised mice, showing that the spleen was essential for
thrombocytopenia (Watier et al. 1992). The term hypersplenism was proposed to describe the clinical picture of
the enlarged spleen followed by the decrease in one or
more peripheral blood lineages (usually reverted after
splenectomy), probably due to sequestration or destruction of cells inside the spleen, in liver diseases, which
lead to increased portal system pressure. However, it is
recently believed that not only mechanical alterations take
place, but also compromise of haematopoietic growth
factors produced in the liver (Peck-Radosavljevic 2001).
On the other hand, the isolated spleen enlargement does
not explain per se the destruction of cells as formerly believed. This organ represents outstanding architectural
organisation and controls, with great sophistication, the
exposure of cells screened by it. In patients with malaria,
the increase in the macrophage-colony stimulating factor
is associated to thrombocytopenia, suggesting that macrophages play a role in the destruction of these particles
(Lee et al. 1997). In the comparison of spleens from patients with severe falciparum malaria vs. those of control
and septic patients, it was shown that splenic dendritic
cells are increased in malaria and there is a reduction in B
lymphocytes and macrophages in the splenic cords (Urban et al. 2005). The mechanisms related to the formation
of splenic hematomas are mostly associated with P. vivax
infection and the interface with thrombocytopenia is
noted to be imprecise (Lacerda et al. 2007). In vivax malaria, the role of the spleen in the expression of vir genes
is still unrecognised. P. vivax passing through the spleen
would activate the transcription of polymorphic Vir proteins to escape from macrophage destruction in this organ. On the other hand, these same proteins would permit
the binding of parasitised RBCs to barrier cells, creating
an isolated microenvironment in the spleen that would be
rich in reticulocytes (del Portillo et al. 2004). More recent
studies with the murine model of Plasmodium yoelii evidenced that there was higher parasite accumulation, reduced motility, loss of directionality, increased residence
time and altered magnetic resonance only in the spleens
of mice infected with the non-lethal 17X strain (MartinJaular et al. 2011). This same model has never been used
to study the role of the spleen in thrombocytopenia, but
opens new avenues for functional and structural studies
of this lymphoid organ.
Bone marrow alterations - The finding of a P. vivax
trophozoite inside a human platelet suggested that thrombocytopenia could be the result of invasion of these particles by the parasites themselves, similar to what was
classically proposed for RBCs. As these same authors did
not find parasites inside megakaryocytes, they proposed
that the penetration took place in the peripheral circulation (Fajardo & Tallent 1974). However, this observa-
54
Mem Inst Oswaldo Cruz, Rio de Janeiro, Vol. 106(Suppl. I), 2011
TABLE I
Systematic review of studies, estimating thrombocytopenia in malarial patients (1997-2011)
References
Study site
Type of patients
Age range
Species
n
Thrombocytopenia
% [criterion (mm3)]
Mohanty et al. (1997)
India
Inpatients and outpatients All ages
P.v.
P.f.
24
76
29 (< 150,000)
39 (< 150,000)
Noronha (1998)
Kortepeter and Brown (1998)
Murthy et al. (2000)
Brazil
USA
India
Inpatients and outpatients < 14 y
Inpatients and outpatients > 18 y
Inpatients
10-80 y
P.f.
P.f./P.v.
P.f.
54
79
158
51.8 (< 150,000)
74 (< 150,000)
40.5 (< 150,000)
Gonzalez et al. (2000)
Alecrim (2000)
Silva et al. (2000)
Oh et al. (2001)
Robinson et al. (2001)
Mourão et al. (2001)
Lacerda et al. (2001)
Ladhani et al. (2002)
Park et al. (2002)
Mohapatra et al. (2002)
Bashawri et al. (2002)
Araújo Filho et al. (2003)
Echeverri et al. (2003)
Colombia
Inpatients
All ages
P.f.
P.v.
113
128
33.6 (< 150,000)
39 (< 150.000)
Brazil
Inpatients
Outpatients
> 12 y
P.v.
73
319
91.8 (< 150,000)
60.8 (< 150,000)
Brazil
South Korea
Australia
Brazil
Brazil
Kenya
Brazil
India
Saudi Arabia
Brazil
Colombia
Inpatients
Inpatients and outpatients
Inpatients
Inpatients
Inpatients
Inpatients
Inpatients
Inpatients and outpatients
Inpatients and outpatients
Inpatients and outpatients
Outpatients
All ages
> 17 y
NA
< 12 y
> 12 y
Children
All ages
15-60 y
2 m-74 y
4-64 y
All ages
P.v.
P.v.
P.f./P.v./P.o.
P.f.
P.f.
P.f.
P.v.
P.v.
P.v./P.f.
P.v.
P.v.
429
101
246
255
218
1,369
237
110
727
68
104
46.6 (< 140,000)
85.1 (< 150,000)
71 (< 150,000)
73.7 (< 150,000)
87.6 (< 150,000)
56.7 (< 150,000)
61.6 (NA)
3.6 (< 100,000)
55.6 (< 150,000)
20.6 (< 50,000)
8 (< 130,000)
Jadhav et al. (2004)
India
Inpatients and outpatients All ages
P.v.
P.f.
973 65 (50,000-150,000)
590 50 (50,000-150,000)
Marques (2004)
Brazil
Inpatients and outpatients
P.f.
P.v.
44
106
79 (< 150,000)
94 (< 150,000)
Rodriguez-Morales et al. (2005) Venezuela
NA
NA
Rodriguez-Morales et al. (2006) Venezuela
Inpatients
< 12 y
Casals-Pascual et al. (2006)
Kenya
Inpatients and outpatients 6 m-10 y
Kumar and Shashirekha (2006)
India
Inpatients and outpatients All ages
P.v.
P.v.
P.f.
P.v.
116
78
120
27
87.6 (< 150,000)
58.9 (< 150,000)
34.4 (< 150,000)
88.8 (< 150,000)
Lacerda (2007)
> 15 y
Brazil
Outpatients
> 18 y
P.v.
P.f.
142
26
71.8 (< 150,000)
65.4 (< 150,000)
Koltas et al. (2007)
Taylor et al. (2008)
Turkey
Indonesia
Outpatients
Outpatients
All ages
All ages
P.v.
P.v./P.f.
90
151
NA
78.8 (< 150,000)
Tan et al. (2008)
Thailand
P.v.
P.f.
523
694
22 (< 75,000)
34 (< 75,000)
Silva (2009)
Rasheed et al. (2009)
Shaikh et al. (2009)
Prasad et al. (2009)
Gonzalez et al. (2009)
Poespoprodjo et al. (2009)
Khan et al. (2009)
Maina et al. (2010)
Kochar et al. (2010)
George and Alexander (2010)
Srivastava et al. (2011)
Brazil
Outpatients
Pakistan
Inpatients
Pakistan
Outpatients
India
Inpatients
Venezuela
Outpatients
Indonesia
Inpatients
Qatar
Outpatients
Kenya
Outpatients
India
Inpatients and outpatients
India
Inpatients
India
Inpatients
Inpatients and outpatients Pregnant
women
All ages
397
P.v.
All ages
502
P.v./P.f
All ages
124
P.v./P.f.
<5y
40
P.f.
3-67
59
P.v.
0-3 m P.v./P.f. and mixed 179
All ages
81
P.v.
<5y
523
P.f.
All ages P.v./P.f. and mixed 1,064
18-66 y
30
P.v.
All ages
50
P.v.
77.1 (< 150,000)
80 (< 150,000)
82.5 (< 150,000)
85 (< 150,000)
55.9 (< 150,000)
61.3 (< 100,000)
63 (< 150,000)
49 (< 150,000)
24.6 (< 150,000)
93.3 (< 150,000)
82 (< 150,000)
m: months; NA: non-available; P.f.: Plasmodium falciparum; P.o.: Plasmodium ovale; P.v.: Plasmodium vivax; y: years.
Malaria and thrombocytopenia • Marcus Vinícius Guimarães Lacerda et al.
55
TABLE II
Collated case reports of Plasmodium vivax-associated thrombocytopenia (1964-2011)
Study site
Platelet count
(x 1,000/mm3)
Bleeding
Platelet
transfusion
Observation
United States of America
Solomon Islands
Bali
NA
Thailand and Sri Lanka
Brazil
India
India
Mexico
Brazil
India
India
South America
India
20-49
NA
22
NA
22-53
1
5
8
19
1
6
14-92
15
30
Petecchiae
NA
No
NA
No
Several
No
Gingival bleeding
Epistaxis
Gingival bleeding
Petecchiae
No
NA
No
No
NA
No
NA
No
Yes
No
Yes
Yes
Yes
Yes
Yes
NA
No
Experimental infection
DIC
PAIgG increase
PAIgG increase
ITP
ITP
Acute renal failure
South Korea
25-20
No
No
DIC, lung edema, acute
renal failure and shock
Kaur et al. (2007)
Lacerda et al. (2008)
Vij et al. (2008)
Rifakis et al. (2008)
India
Brazil
India
Venezuela
30
6
NA
57
Petecchiae
No
Gingival bleeding
No
No
No
No
No
Acute renal failure
Chronic splenomegaly
NA
Hydronephrosis and shock
Parakh et al. (2009)
India
5-42
Petecchiae
No
Cerebral malaria, shock
and acute renal failure
Thapa et al. (2009)
India
11
Petecchiae and
mucosal bleeding
Yes
Hepatitis and shock
Harish and Gupta (2009)
Bhatia and Bhatia (2010)
India
India
1
NA
Intracranial bleed
Yes
No
NA
Seizures
NA
References
Hill et al. (1964)
Takaki et al. (1991)
Anstey et al. (1992)
Ohtaka et al. (1993)
Yamaguchi et al. (1997)
Victoria et al. (1998)
Kakar et al. (1999)
Makkar et al. (2002)
Holland et al. (2004)
Lacerda et al. (2004)
Aggarwal et al. (2005)
Katira and Shah (2006)
Komoda et al. (2006)
Kaur et al. (2007)
Song et al. (2007)
DIC: disseminated intravascular coagulation; ITP: immune thrombocytopenic purpura; NA: non-available; PAIgG: plateletassociated IgG.
tion was never seen again in the literature. Likewise, a
“dysmegakaryopoiesis” was proposed, similar to what
happened in the human malarial anaemia model, where
dyserythropoiesis was triggered by cytokines (Menendez et al. 2000). In the few studies that examined the
bone marrow for this purpose, megakaryocytic lineage
was apparently preserved (Naveira 1970, Beale et al.
1972). Thrombopoietin indeed seems to rise during the
acute disease even in the presence of liver compromise,
suggesting that no bone marrow inhibition is seen (Kreil
et al. 2000). Additional data from FBC samples in vivax
patients showed that there is a significant negative correlation between platelet count and mean platelet volume
(Lacerda 2007), suggesting that megakaryocytes are able
to release mega platelets in the circulation to compensate
for the low absolute number of platelets in the periphery. Similar results were shown in children with falciparum malaria (Maina et al. 2010). These mega platelets
are probably able to sustain a good primary haemostasis
that could explain the low frequency of severe bleeding
in malarial patients, as shown in Table II. Non-human
primates, on the other hand, are an unexplored model to
study megakaryopoiesis alterations and its implication
on thrombocytopenia (Llanos et al. 2006).
Antibody-mediated platelet destruction - There is evidence that platelet-associated IgG (PAIgG) is increased
in malaria and is associated with thrombocytopenia.
However, this is a generic definition for all types of
IgGs that may be found on the platelet surface, including
antibodies stored inside platelet α-granules. Therefore,
increased PAIgG could also be interpreted as platelet activation and exposition of IgGs on the surface, and not
necessarily auto-immunity, as suggested in anecdotal
case reports where antibodies against glycoproteins
were detected in malaria (Panasiuk 2001, Conte et al.
2003). The detection of auto-antibodies against platelets
by flow cytometry (Rios-Orrego et al. 2005) should not
be seen as specific for malaria, as natural auto-antibody
formation is a common defence of the infected organism
56
Mem Inst Oswaldo Cruz, Rio de Janeiro, Vol. 106(Suppl. I), 2011
and is frequently seen in most viral, bacterial and parasitic diseases without any repercussion (Daniel-Ribeiro
& Zanini 2000). Molecular mimicry, however, provides
evolutionary advantage for microorganisms that escape
immune aggression (Daniel-Ribeiro 2000). The relationship between malaria and auto-immunity has been
discussed in the literature and the first epidemiological
association was made based on the presence of fewer auto-immune diseases in malarigenous areas (Greenwood
1968). The formation of circulating immune complexes
(CIC) in vivo in malaria, as well as in other infectious
diseases, is a continuous process from antigens and
antibodies and/or complement elements. CIC seems to
modulate the immune response to several antigens that
remain sequestered in B lymphocyte or dendritic cellrich follicles for a longer time, which contributes to the
formation of B-cell immunological memory, as seen in
vaccine studies (Davidson 1985). During acute malaria,
thrombocytopenia is most probably associated with the
binding of parasite antigens to the surface of platelets to
which antimalarial antibodies also bind, leading to the
in situ formation of immune complexes (ICs) (Kelton et
al. 1983). In an experimental model with Plasmodium
berghei, the same correlation between platelet count and
IC’s was evidenced (Grau et al. 1988). No association
was found with IgM (Beale et al. 1972). It is clear that
CICs are elevated in vivax and falciparum malaria, but
their role in the development of thrombocytopenia is still
obscure (Touze et al. 1990, Tyagi & Biswas 1999) as well
as its immune suppressing effect (Brown & Kreier 1982,
Shear 1984). Because the generation of IC’s is proportional to the amount of available antigen, the negative
correlation between platelet count and peripheral parasitaemia reported in many studies (Lacerda 2007, Silva 2009) corroborates ICs as a potential mechanism of
platelet destruction. The presence of amino acid residues
tyrosine 193 [9Y(193)] and serine 210 [S(210)] on apical
membrane antigen-1 (AMA-1) was significantly associated with normal platelet counts in P. vivax malaria independent of the level of parasitaemia that also provides
supporting evidence for this (Grynberg et al. 2007). In
only one study, circulating monocytes were found to
phagocytose platelets, but this mechanism still needs to
be associated to thrombocytopenia more closely (Jaff
et al. 1985). The finding of immune thrombocytopenic
purpura (ITP) secondary to malarial infection is rare and
may be due to idiosyncratic auto-immune mechanisms
not well understood (Lacerda et al. 2004).
Oxidative stress - Free radicals may play an important role in the platelet destruction in malarial infection.
There is evidence that the decrease in total cholesterol in
vivax malaria is due to lipidic peroxidation (Erel et al.
1998). Also, in vivax malaria, there is a negative correlation between platelet count and platelet lipid peroxidation in addition to the positive correlation between platelet count and the activity of gluthatione peroxidase and
superoxide dismutase, which are considered anti-oxidant
enzymes (Erel et al. 2001). In a study of 103 patients with
acute falciparum malaria, there was a negative correlation between platelet count and nitrogen reactive intermediates (Santos 2000). There is also a strong associa-
tion between platelet count and intra-platelet gluthatione
peroxidase, suggesting that a compensatory mechanism
is presented by platelets to face the oxidative burst found
in malaria (Araujo et al. 2008).
Platelet aggregation - Platelets from patients with
acute malaria are highly sensitive to adenosine diphosphate (ADP) addition in vitro (Essien & Ebhota 1981),
and it is believed that ADP release following haemolysis
could contribute to higher platelet aggregation. Actually,
the incubation of platelets with P. falciparum-parasitised
RBCs also increases platelet aggregation per se in vitro,
especially after ADP and thromboxane A2 addition (Inyang et al. 1987). Even electron microscopic examination
of non-stimulated, fresh platelets from malarial patients
show centralisation of dense granules, glycogen depletion and microaggregates and phylopoids as a sign of in
vivo activation, which could be responsible for a pseudothrombocytopenia due to sequestration of these activated
particles in the interior of the vessels (Mohanty et al.
1988). Contradictory data were presented showing aggregation impairment in severe falciparum patients after
ADP addition in vitro (Srichaikul et al. 1988). P. falciparum induces systemic acute endothelial cell activation
and the release of activated von Willerbrand factor (vWF)
immediately after the onset of the blood-stage infection
(Mast et al. 2007). Even without consumptive coagulopathy, the increase in soluble glycoprotein-1b (GP1b) concentrations results from vWF-mediated GP1b shedding,
a process that may prevent excessive adhesion of platelets
and parasitised erythrocytes (Mast et al. 2010). Antimalarial drugs have also been shown as potential inhibitors
of platelet aggregation in vivo and in vitro, what precludes
careful inclusion and exclusion criteria of patients to be
used in clinical research (Cummins et al. 1990).
The relationship between thrombocytopenia and severe malaria - Severe thrombocytopenia (platelet count
under 50,000/mm3), despite not being considered severe
malaria according to World Health Organization criteria
(WHO 2010) due to the inability to cause death per se,
has been occasionally associated with severity (Gerardin
et al. 2002, Rogier et al. 2004) or not (Moulin et al. 2003).
But thrombocytopenia has also been described in severe
vivax patients (Kochar et al. 2005, Andrade et al. 2010).
In 17 patients from Manaus affected by any of the WHO
malaria severity criteria with confirmed P. vivax monoinfection, 14 presented with thrombocytopenia, suggesting
that this haematological complication can be explored
as a marker of the severity for this species (Alexandre
et al. 2010). From the case reports described in Table
II, the association between severe cases with thrombocytopenia is evident. However, that can be due to bias
publication, where prospective studies would be needed
to validate this association. On the other hand, considering that many studies point to a clear negative correlation
between platelet count and parasitaemia (Grynberg et
al. 2007, Silva 2009), it should be investigated if thrombocytopenia could be used in the surveillance of drug
resistance, where higher parasitaemias for prolonged
periods are usually found. Interestingly, in areas where
thrombocytopenia and other types of clinical severity are
Malaria and thrombocytopenia • Marcus Vinícius Guimarães Lacerda et al.
frequently reported, resistant parasites are also being simultaneously detected (Santana Filho et al. 2007, Tjitra
et al. 2008), possibly explaining why the prevalence of
thrombocytopenia worldwide is not homogeneous.
On the other side of the clinical presentation of plasmodial infection, platelet counts were never performed in asymptomatic parasite carriers. However, due to the very low
parasitaemia (sometimes submicroscopic) presented by
these patients, it is possible that platelet counts are normal
and parallel clinical symptoms (Suarez-Mutis et al. 2007).
Avoiding the consensual understanding that platelets
are particles devoted to the maintenance of primary haemostasis, it has been shown that platelets participate in the
pathogenesis of microvascular malaria, adhering to the
endothelium when it is previously stimulated with tumor
necrosis factor (TNF) (Lou et al. 1997). Even in the nonstimulated cerebral endothelium, platelets may adhere
and facilitate the adhesion of P. falciparum-parasitised
RBCs, through CD36 is ubiquitous in endothelial cells
and, coincidentally, platelets (Wassmer et al. 2004). Platelets therefore act by stabilising and strengthening bridges
between RBCs and endothelial cells, which is considered
the cornerstone of severe falciparum malaria. Rosetting
of parasitised RBCs is also mediated through CD36 in
platelets in severe malaria (Pain et al. 2001, Chotivanich et
al. 2004). In mice infected with P. berghei ANKA, mice
deficient of tissue and uroquinase plasminogen activators
demonstrated less capillary sequestration of platelets and
less severe malaria (Piguet et al. 2000). Blocking GPIIb
with anti-CD41 monoclonal antibodies in the first day of
murine infection with P. berghei also led to higher production of interleukin (IL)-10, IL-1α, IL-6, interferon-α
and TNF and less mortality among mice, suggesting that
platelets may act as cofactors of severe malaria (Sun et
al. 2003, van der Heyde et al. 2005). There was also an
inverse correlation between platelet count and TNF in
patients with vivax infection and no association between
specific mutation G→A in the position 308 in the TNF
gene (a polymorphism whose functional effect upon
severe disease is hypothesised) and platelet count was
observed. More severe patients presented more severe
thrombocytopenia and higher TNF levels (Silva 2004).
Platelets stimulated by parasitised RBCs may also trigger
apoptosis in endothelial cells pre-treated with TNF in a
pathway mediated by tumor growth factor (TGF)-β1 from
platelets (Wassmer et al. 2006a, b). Recent evidence showing P. vivax-infected RBCs adhering to lung endothelial
cells and to the placental tissue ex vivo indicates that in
vivax, mechanisms similar to those associated with falciparum severity may be involved (Carvalho et al. 2010).
The contribution of platelets to this adhesion, however,
requires further investigation.
In children in Kenya suffering from falciparum malaria, an inverse correlation between platelet count and
plasmatic IL-10 was seen (Casals-Pascual et al. 2006).
This interpretation is not straightforward, because IL10 is generally associated with protection against severe
disease. The authors hypothesise, though, that IL-10
could reduce platelet counts to avoid infected-RBC adhesion to the endothelium, as if thrombocytopenia was
a mechanism of defence against severe disease and not
57
the cause. Studies of vivax infection have shown thrombocytopenia to be associated with an increase in IL-1,
IL-6, IL-10 and TGF-β (Park et al. 2003).
The role of platelet-derived microparticles (MPs)
(submicron-sized vesicles released from cells upon activation or apoptosis) has yet to be determined in vivo.
There is evidence that these MPs participate in the endothelial activation responsible for severe cerebral malaria in murine models (Combes et al. 2006). MPs were
also associated with coma and thrombocytopenia in severe falciparum malaria patients (Pankoui Mfonkeu et al.
2010). Apparently, there is an increase in the amount of
MPs in vivax malaria patients, which may play a role in
the acute inflammatory symptoms of this disease (Campos et al. 2010); this role requires further investigation.
Clinical management of malarial thrombocytopenia
- To date, there is no robust evidence on how to manage
patients with malaria and thrombocytopenia. Platelet
transfusion has been widely followed, but with no confirmed efficacy. The indication of prophylactic platelet
transfusion when platelet counts are under 10,000/mm3
probably applies only when the bone marrow is compromised and is not able to release efficacious platelets (Rebulla 2000). This does not seem to be the case in malaria. Keeping platelet counts between 50,000 and 100,000/
mm3 is a formal indication only in patients undergoing
surgical procedures (Rebulla 2001). In a tertiary care
centre in the Western Brazilian Amazon over a 12-month
period, 10.4% (20/191) of patients who received platelet
transfusion were diagnosed with vivax or falciparum
malaria (Lacerda et al. 2006). The dosage was usually
below that recommended in the literature (Schlossberg &
Herman 2003). In 40% of patients, the only justifications
for transfusion were maintaining a platelet count below
10,000/mm3 and discrete bleeding. In a further 6% of
patients, only a very low platelet count was described. In
this group of 40% of patients, the alleged reason was minor bleeding despite having non-severe thrombocytopenia; in 33%, no indication was verified. These data point
to the little existing evidence of the recommendations
for platelet transfusion in these patients. The corrected
count increment to evaluate transfusion efficacy was not
calculated for any patient. The low efficacy of platelet
transfusion in general is well described for several acute
infectious diseases (de Paula et al. 1993), probably due to
peripheral immune mechanisms of destruction that do
not spare the transfused platelets. Indications for platelet
transfusion in cases when DIC is suspected and diagnosed, the formal clinical indication persists, as recommended elsewhere (Franchini 2005). Due to the impossibility of using frozen platelets in routine clinical practice,
other platelet substitutes and preparations are being investigated (Blajchman 2003). Except in atypical cases of
ITP with severe bleeding, there is no evidence for the use
of human intravenous immunoglobulin, even in cases of
severe thrombocytopenia (Lacerda et al. 2004).
The use of corticoids has never been followed, probably due to the fact that the recovery of thrombocytopenia following antimalarial treatment is seen in almost
all cases, with good prognosis for all species that infect
humans (Lacerda 2007) and with the lack of robust evi-
58
Mem Inst Oswaldo Cruz, Rio de Janeiro, Vol. 106(Suppl. I), 2011
dence of immune-mediated destruction of platelets as a
major mechanism. It was also found that in patients with
cerebral falciparum malaria, dexamethasone exacerbated the neurological symptoms and increased the frequency of gastrointestinal bleeding (Warrell et al. 1982,
Hoffman et al. 1988). However, in none of these studies
was platelet recovery analysed as a secondary endpoint.
Immune modulators are also candidates in the adjuvant antimalarial therapy (Muniz-Junqueira et al. 2005,
Mohanty et al. 2006), based on the drug-induced inhibition of adhesion molecules in RBCs and platelets (Muniz-Junqueira 2007). The exploration of drugs known by
their anti-inflammatory effect, modulating TNF, e.g.,
pentoxyfylline and thalidomide, upon severe malaria,
could not only contribute to the understanding of the
mechanisms of severity but also clarify the association
between platelets and severe disease.
Thrombocytopenia in other infectious diseases Many other acute and chronic infectious diseases share
similar thrombocytopenia as part of the clinical picture
and these mechanisms may be used by proxy to explain
malarial disease.
Chronic thrombocytopenia is found in approximately
10% of patients with human immunodeficiency virus
(HIV)-1 infection and in one-third of those with acquired immunodeficiency syndrome (Scaradavou 2002).
The first cases of homosexuals with profound thrombocytopenia in New York were classified as ITP (Karpatkin
2002), involving the presence of serum IgG anti-GPIIIa
(Karpatkin et al. 1995). Later on, this IgG was found to
be directed against GPIIIa49-66 (Nardi et al. 1997). More
recently, molecular mimicry was proposed between nef
HIV-1 protein and GPIIIa49-66 (Li et al. 2005). Other chronic infectious diseases known to cause thrombocytopenia
include chronic viral hepatitis, where CIC (Samuel et al.
1999) and PAIgG (Doi et al. 2002) are also implicated.
In the case of hepatitis C virus infection, the blockage
in the maturation of megakaryocytes is mediated by the
viral RNA itself (Almeida 2003). Despite an associated
medullary compromise in visceral leishmaniasis in the
canine model of Leishmania infantum infection, antiplatelet IgG and IgM were also observed (Terrazzano
et al. 2006). In acute infection with Trypanosoma cruzi,
frequent thrombocytopenia is related to the presence of
parasite trans-sialidase (Tribulatti et al. 2005). Furthermore, during infection with any of the four dengue viruses, thrombocytopenia is frequent and is supposed to
be a criterion of dengue hemorrhagic fever (Mourão et
al. 2007). Platelet phagocytosis ex vivo has already been
shown as a potential mechanism in this acute viral disease (Honda et al. 2009). Thrombocytopenia is also observed in leptospirosis (Nicodemo 1993), typhoid fever
(Huang & DuPont 2005), hantavirus infection (Santos et
al. 2006), yellow fever (Monath 2001) and sepsis (Becchi
et al. 2006), whose mechanisms are poorly understood.
The high frequency of thrombocytopenia in other infectious diseases, as a rule, changes the paradigm that platelets are essential only to haemostasis, supporting their
role as important contributors to modulate the immune
response. In any case, studies focusing on the pathogenesis of thrombocytopenia in malarial patients should
Major mechanisms associated to malaria-triggered thrombocytopenia and the possible relationship with severe disease.
always rule out other concomitant infectious diseases,
which is difficult in socio-economically deprived study
populations suffering large burdens of multiple diseases.
The frequency of thrombocytopenia (i.e., platelet
count below 150,000/mm3) in malarial infection ranges
from 24-94% in the literature, despite the low occurrence
of severe bleeding, even in the case of severe malaria. It
is still unclear whether this haematological complication
is more frequent in P. vivax or P. falciparum malaria. In
Figure, the major mechanisms involved in the pathogenesis are highlighted, but further studies are still needed to
clarify the impact of each mechanism and its clinical relevance. The clinical management of malarial thrombocytopenia is expectant and the level of evidence for platelet
transfusion is insufficient to recommend this practice. It
is not clear whether platelets are diminished during acute
malarial infection as a consequence of the immune response to the parasite present or whether platelets are actually involved in the generation of severe disease.
ACKNOWLEDGEMENTS
To Alex Kumar, for critical and linguistic review of the manuscript, and to Mary Galinski, for inspiring the title. This review
is dedicated to Simon Karpatkin and Vanize Oliveira Macêdo.
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Tables
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concise title. Footnotes can be used to explain abbreviations. Citations should be
indicated using the same style as outlined above. Tables occupying more than one
printed page should be avoided, if possible. Larger tables can be published as
Supporting Information. Please ensure that table formatting conforms to our Guidelines
for table preparation.
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3. Specific Reporting Guidelines
Additional information about reporting guidelines for specific article types can be found
at the PLOS Editorial and Publishing Policies.
Human Research
Methods sections of papers on research using human subject or samples must include
ethics statements that specify:

The name of the approving institutional review board or equivalent committee(s).
If approval was not obtained, the authors must provide a detailed statement
explaining why it was not needed

Whether informed consent was written or oral. If informed consent was oral, it
must be stated in the paper:
o
Why written consent could not be obtained
o
That the Institutional Review Board (IRB) approved use of oral consent
o
How oral consent was documented
For studies involving humans categorized by race/ethnicity, age, disease/disabilities,
religion, sex/gender, sexual orientation, or other socially constructed groupings, authors
should:

Explicitly describe their methods of categorizing human populations

Define categories in as much detail as the study protocol allows

Justify their choices of definitions and categories, including for example whether
any rules of human categorization were required by their funding agency

Explain whether (and if so, how) they controlled for confounding variables such
as socioeconomic status, nutrition, environmental exposures, or similar factors in
their analysis
In addition, outmoded terms and potentially stigmatizing labels should be changed to
more current, acceptable terminology. Examples: "Caucasian" should be changed to
"white" or "of [Western] European descent" (as appropriate); "cancer victims" should be
changed to "patients with cancer."
For papers that include identifying, or potentially identifying, information, authors must
download the Consent Form for Publication in a PLOS Journal(PDF), which the
individual, parent, or guardian must sign once they have read the paper and been
informed about the terms of PLOS open-access license. The signed consent form
should not be submitted with the manuscript, but authors should securely file it in the
individual's case notes and the methods section of the manuscript should explicitly state
that consent authorization for publication is on file, using wording like:
"The individual in this manuscript has given written informed consent (as
outlined in PLOS consent form) to publish these case details."
For more information about PLOS ONE policies regarding human subject research, see
the Publication Criteria and Editorial Policies.
Clinical Trials
Authors of manuscripts describing the results of clinical trials must adhere to
the CONSORT reporting guidelines appropriate to their trial design, available on
the CONSORT Statement website. Before the paper can enter peer review, authors
must:
1. Provide the registry name and number in the methods section of the manuscript
2. Provide a copy of the trial protocol as approved by the ethics committee and a
completed CONSORT checklist as Supporting Information (which will be
published alongside the paper, if accepted)
3. Include the CONSORT flow diagram as the manuscript's "Figure 1"
Any deviation from the trial protocol must be explained in the paper. Authors must
explicitly discuss informed consent in their paper, and we reserve the right to ask for a
copy of the patient consent form.
The methods section must include the name of the registry, the registry number, and
the URL of your trial in the registry database for each location in which the trial is
registered.
For more information about PLOS ONE policies regarding clinical trials, see the Editorial
Policies.
Animal research
Methods sections of papers reporting results of animal research must include required
ethics statements that specify:

The full name of the relevant ethics committee that approved the work, and the
associated permit numbers; where ethical approval is not required, the article
should include a clear statement of this and the reason why

Relevant details for efforts taken to ameliorate animal suffering
Example: "This study was carried out in strict accordance with the
recommendations in the Guide for the Care and Use of Laboratory Animals
of the National Institutes of Health. The protocol was approved by the
Committee on the Ethics of Animal Experiments of the University of
Minnesota (Permit Number: 27-2956). All surgery was performed under
sodium pentobarbital anesthesia, and all efforts were made to minimize
suffering."
The organism(s) studied should always be stated in the abstract. Where research may
be confused as pertaining to clinical research, the animal model should also be stated in
the title.
We encourage authors to use the ARRIVE (Animal Research: Reporting of In
Vivo Experiments) guidelines as a reference.
For more information about PLOS ONE policies regarding animal research, see
the Publication Criteria and Editorial Policies.
Observational and field studies
Methods sections for submissions reporting on any type of field study must include
ethics statements that specify:

Permits and approvals obtained for the work, including the full name of the
authority that approved the study; if none were required, authors should explain
why

Whether the land accessed is privately owned or protected

Whether any protected species were sampled

Full details of animal husbandry, experimentation, and care/welfare, where
relevant
For more information about PLOS ONE policies regarding observational and field
studies, see the Publication Criteria and Editorial Policies.
Work using cell lines
Methods sections for submissions reporting on research with cell lines should state the
origin of any cell lines. For established cell lines it should be stated from where/who the
cell line was obtained, and references must also be given to either a published paper or
to a commercial source. If previously unpublished de novo cell lines were used,
including those gifted from another laboratory, details of institutional review board or
ethics committee approval must be given, and confirmation of written informed consent
must be provided if the line is of human origin.
For more information about PLOS ONE policies regarding observational and field
studies, see the Publication Criteria.
Systematic Review/Meta-Analysis
A systematic review paper, as defined by The Cochrane Collaboration, is a review of a
clearly formulated question that uses explicit, systematic methods to identify, select, and
critically appraise relevant research, and to collect and analyze data from the studies
that are included in the review. These reviews differ substantially from narrative-based
reviews or synthesis articles. Statistical methods (meta-analysis) may or may not be
used to analyze and summarize the results of the included studies.
Reports of systematic reviews and meta-analyses should use the PRISMA (Preferred
Reporting Items for Systematic Reviews and Meta-Analyses) statement as a guide, and
must include a completed PRISMA checklist and flow diagram to accompany the main
text. Blank templates of the checklist and flow diagram can be downloaded from the
PRISMA website. Authors must also state in their "Methods" section whether a protocol
exists for their systematic review, and if so, provide a copy of the protocol as Supporting
Information and provide the registry number in the abstract.
If your article is a Systematic Review or a Meta-Analysis you should:

State this in your cover letter

Select 'Research Article' as your article type when submitting

Upload your PRISMA flowchart as Figure 1 (required where applicable)

Include the PRISMA checklist as Supporting Information.
Software Papers
Manuscripts describing software should provide full details of the algorithms designed.
Describe any dependencies on commercial products or operating system. Include
details of the supplied test data and explain how to install and run the software. A brief
description of enhancements made in the major releases of the software may also be
given. Authors should provide a direct link to the deposited software from within the
paper.
See the PLOS ONE Editorial Policies and the PLOS Editorial and Publishing Policies for
more information about submitting papers describing software.
Database Papers
For descriptions of databases, provide details about how the data were curated, as well
as plans for long-term database maintenance, growth, and stability. Authors should
provide a direct link to the database hosting site from within the paper.
See the PLOS ONE Editorial Policies for more information about submitting papers
describing databases.
New Zoological Taxon
PLOS ONE aims to comply with the requirements of the International Commission on
Zoological Nomenclature (ICZN) when publishing papers that describe a new zoological
taxon name. However, the ICZN does not yet recognize online-only journals. There is
a proposal to amend the Code to accommodate such publications.
Until acceptance of this amendment, the ICZN has proposed an interim solution for
authors publishing in PLOS ONE to ensure that the scientific animal name is be
considered "available" (legally published) under the rules of the Code. PLOS
ONE provides a limited hardcopy print-run of the article and makes it publicly
obtainable. Therefore, for all papers that include the naming of a new zoological taxon,
PLOS will make a printed version available for outside parties (at a cost of $10, to cover
postage and printing) at the same time as the publication of the online-only article
(which remains freely available).
The printed version of the article contains the text below in the footer of the first page.
This text will be added by PLOS staff, and does not need to be included by the authors:
Footer text: "This printed document was produced by a method that assures
numerous identical and durable copies, and those copies were simultaneously
obtainable for the purpose of providing a public and permanent scientific record,
in accordance with Article 8.1 of the International Code of Zoological
Nomenclature. Date of publication: XXXXXXXX. This document is otherwise
identical to DOI: XXXXX
For proper registration of the new taxon, we also require two specific statements to be
included in your manuscript.
In the Results section, the globally unique identifier (GUID), currently in the form of a
Life Science Identifier (LSID), should be listed under the new species name, for
example:
Anochetus boltoni Fisher sp. nov. urn:lsid:zoobank.org:act:B6C072CF-1CA6-40C78396-534E91EF7FBB
You will need to contact Zoobank to obtain a GUID (LSID). Please do this as early as
possible to avoid delay of publication upon acceptance of your manuscript. It is your
responsibility to provide us with this information so we can include it in the final
published paper.
Please also insert the following text into the Methods section, in a sub-section to be
called "Nomenclatural Acts":
The electronic version of this document does not represent a published work
according to the International Code of Zoological Nomenclature (ICZN), and
hence the nomenclatural acts contained in the electronic version are not available
under that Code from the electronic edition. Therefore, a separate edition of this
document was produced by a method that assures numerous identical and
durable copies, and those copies were simultaneously obtainable (from the
publication date noted on the first page of this article) for the purpose of
providing a public and permanent scientific record, in accordance with Article 8.1
of the Code. The separate print-only edition is available on request from PLOS by
sending a request to PLOS ONE, PLOS, 1160 Battery Street, Suite 100, San
Francisco, CA 94111, USA along with a check for $10 (to cover printing and
postage) payable to "PLOS".
In addition, this published work and the nomenclatural acts it contains have been
registered in ZooBank, the proposed online registration system for the ICZN. The
ZooBank LSIDs (Life Science Identifiers) can be resolved and the associated
information viewed through any standard web browser by appending the LSID to
the prefix "http://zoobank.org/". The LSID for this publication is: (insert here). The
online version of this work is archived and available from the following digital
repositories: [INSERT NAMES OF DIGITAL REPOSITORIES WHERE ACCEPTED
MANUSCRIPT WILL BE SUBMITTED (PubMed Central, LOCKSS etc)].
All PLOS ONE articles are deposited in PubMed Central and LOCKSS. If your institute,
or those of your co-authors, has its own repository, we recommend that you also
deposit the published online article there and include the name in your article.
New Botanical Taxon
When publishing papers that describe a new botanical taxon name, PLOS aims to
comply with the requirements of the International Code of Nomenclature for algae,
fungi, and plants (ICN). In association with the International Plant Names Index (IPNI),
the following guidelines for publication in an online-only journal have been agreed such
that any scientific botanical name published by us is considered effectively published
under the rules of the Code. Please note that these guidelines differ from those for
zoological nomenclature.
Effective January 2012, "the description or diagnosis required for valid publication of the
name of a new taxon" can be in either Latin or English. This does not affect the
requirements for scientific names, which are still to be Latin.
Also effective January 2012, the electronic PDF represents a published work according
to the ICN for algae, fungi, and plants. Therefore the new names contained in the
electronic publication of a PLOS ONE article are effectively published under that Code
from the electronic edition alone, so there is no longer any need to provide printed
copies.
Additional information describing recent changes to the Code can be found here.
For proper registration of the new taxon, we require two specific statements to be
included in your manuscript.
In the Results section, the globally unique identifier (GUID), currently in the form of a
Life Science Identifier (LSID), should be listed under the new species name, for
example:
Solanum aspersum S.Knapp, sp. nov. [urn:lsid:ipni.org:names:77103633-1]
Type: Colombia. Putumayo: vertiente oriental de la Cordillera, entre Sachamates
y San Francisco de Sibundoy, 1600-1750 m, 30 Dec 1940, J. Cuatrecasas 11471
(holotype, COL; isotypes, F [F-1335119], US [US-1799731]).
You will need to contact IPNI to obtain the GUID (LSID) after your manuscript is
accepted for publication. You must then make sure to provide us with this information so
we can include it in the final published paper.
In the Methods section, include a sub-section called "Nomenclature" using the following
wording:
The electronic version of this article in Portable Document Format (PDF) will
represent a published work according to the International Code of Nomenclature
for algae, fungi, and plants, and hence the new names contained in the electronic
version are effectively published under that Code from the electronic edition
alone.
In addition, new names contained in this work have been submitted to IPNI, from
where they will be made available to the Global Names Index. The IPNI LSIDs can
be resolved and the associated information viewed through any standard web
browser by appending the LSID contained in this publication to the prefix
http://ipni.org/. The online version of this work is archived and available from the
following digital repositories: [INSERT NAMES OF DIGITAL REPOSITORIES
WHERE ACCEPTED MANUSCRIPT WILL BE SUBMITTED (PubMed Central,
LOCKSS etc)].
All PLOS ONE articles are deposited in PubMed Central and LOCKSS. If your institute,
or those of your co-authors, has its own repository, we recommend that you also
deposit the published online article there and include the name in your article.
New Fungal Taxon
When publishing papers that describe a new fungal taxon name, PLOS aims to comply
with the requirements of the International Code of Nomenclature for algae, fungi, and
plants (ICN). The following guidelines for publication in an online-only journal have been
agreed such that any scientific fungal name published by us is considered effectively
published under the rules of the Code. Please note that these guidelines differ from
those for zoological nomenclature.
Effective January 2012, "the description or diagnosis required for valid publication of the
name of a new taxon" can be in either Latin or English. This does not affect the
requirements for scientific names, which are still to be Latin.
Also effective January 2012, the electronic PDF represents a published work according
to the ICN for algae, fungi, and plants. Therefore the new names contained in the
electronic publication of a PLOS ONE article are effectively published under that Code
from the electronic edition alone, so there is no longer any need to provide printed
copies.
Additional information describing recent changes to the Code can be found here.
For proper registration of the new taxon, we require two specific statements to be
included in your manuscript.
In the Results section, the globally unique identifier (GUID), currently in the form of a
Life Science Identifier (LSID), should be listed under the new species name, for
example:
Hymenogaster huthii. Stielow et al. 2010, sp. nov.
[urn:lsid:indexfungorum.org:names:518624]
You will need to contact either Mycobank or Index Fungorum to obtain the GUID (LSID).
Please do this as early as possible to avoid delay of publication upon acceptance of
your manuscript. It is your responsibility to provide us with this information so we can
include it in the final published paper. Effective January 2013, all papers describing new
fungal species must reference the identifier issued by a recognized repository in the
protologue in order to be considered effectively published.
In the Methods section, include a sub-section called "Nomenclature" using the following
wording (this example is for taxon names submitted to MycoBank; please substitute
appropriately if you have submitted to Index Fungorum):
The electronic version of this article in Portable Document Format (PDF) will
represent a published work according to the International Code of Nomenclature
for algae, fungi, and plants, and hence the new names contained in the electronic
version are effectively published under that Code from the electronic edition
alone.
In addition, new names contained in this work have been submitted to MycoBank
from where they will be made available to the Global Names Index. The unique
MycoBank number can be resolved and the associated information viewed
through any standard web browser by appending the MycoBank number
contained in this publication to the prefix
http://www.mycobank.org/MycoTaxo.aspx?Link=T&Rec=. The online version of
this work is archived and available from the following digital repositories:
[INSERT NAMES OF DIGITAL REPOSITORIES WHERE ACCEPTED MANUSCRIPT
WILL BE SUBMITTED (PubMed Central, LOCKSS etc)].
All PLOS ONE articles are deposited in PubMed Central and LOCKSS. If your institute,
or those of your co-authors, has its own repository, we recommend that you also
deposit the published online article there and include the name in your article.
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4. Language polishing services
Prior to submission, authors who believe their manuscripts would benefit from
professional editing are encouraged to use language-editing and copyediting services.
Obtaining this service is the responsibility of the author, and should be done before
initial submission. Submissions are not copyedited before publication. Please
contact plosone [at] plos.org for assistance if needed. Submissions that do not meet
the PLOS ONE Publication Criteria for language standards may be rejected.