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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

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Page 1: UNIVERSIDADE DO ESTADO DO AMAZONAS FUNDAÇÃO DE … · universidade do estado do amazonas . fundaÇÃo de medicina tropical dr. heitor vieira dourado . programa de pÓs-graduaÇÃo

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

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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

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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 Pós-Graduação 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

CDU: 616.928.5 Ficha Catalográfica elaborada pela Bibliotecária Maria Eliana N. Silva, lotada na

Escola Superior de Ciências da Saúde - UEA

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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

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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 essa conquista com

gratidão.

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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.

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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.

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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 5-clorometilfluoresceína 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.

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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 non-thrombocytopenic 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.

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LISTA DE FIGURAS

Figura 1: Áreas de transmissão da malária .......................................... 1 Figura 2: Mapa do Brasil com distribuição de casos de malária por

1.000 habitantes no Brasil .....................................................

3 Figura 3: Imagem da formação das plaquetas...................................... 5 Figura 4-A: Imagem de uma plaqueta quiescente, apresentando forma

discoide, com superfície rugosa (aumento 30.000 X)............

6 Figura 4-B: Aberturas na superfície da plaqueta conectadas ao sistema

canicular aberto (aumento 26.000 X).....................................

6 Figura 4-C: Grânulos α (G) e grânulos densos (D) no citoplasma de

uma plaqueta (aumento 32.000 X)........................................

6 Figura 5: Etapas do experimento de fagocitose de plaquetas.............. 27

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LISTA DE TABELAS

Tabela 1: Grânulos plaquetários e seus constituintes principais............ 7 Tabela 2: Classificação dos pacientes de acordo com a contagem de

plaquetas ................................................................................ 25

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LISTA DE QUADROS

Quadro 1: Classificação fisiopatológica da plaquetopenia......................... 9 Quadro 2: Medicamentos que podem causar plaquetopenia..................... 23

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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)

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Mg – Miligrama

mL – Mililitro

M-CSF – Fator estimulador de Colônias de Macrófagos (Macrophage colony-

stimulating 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

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SUMÁRIO

1 INTRODUÇÃO....................................................................................... 1 1.1 Aspectos epidemiológicos da malária.......................................... 1 1.2 Aspectos clínicos da malária........................................................ 4 1.3 Aspectos imunológicos da malária: produção de citocinas.......... 4 1.4 Plaquetas...................................................................................... 5 1.4.1 Formação da plaquetas................................................................ 5 1.4.2 Aspectos estruturais e funcionais das plaquetas......................... 6 1.4.3 Fagocitose de plaquetas por monócitos....................................... 7 1.4.4 Plaquetopenia............................................................................... 9 1.4.5 Plaquetopenia na malária............................................................. 10 1.4.6 Etiopatogenia da plaquetopenia na malária................................. 10 1.4.7 Relação entre plaquetopenia e citocinas produzidas durante a

infecção por malária.....................................................................

18 2 OBJETIVOS........................................................................................... 20 2.1 Objetivo geral............................................................................... 20 2.2 Objetivos específicos.................................................................... 20

3 MATERIAL E MÉTODOS...................................................................... 21

3.1 Local do estudo............................................................................ 21 3.2 Tipo e tamanho da amostra........................................................ 22 3.3 Critérios de elegibilidade.............................................................. 22 3.4 Critérios de não inclusão............................................................. 22 3.5 Critérios de exclusão................................................................... 23 3.6 Seleção dos pacientes................................................................. 24 3.7 Coleta das amostras e procedimentos laboratoriais ................ 24 3.8 Classificação de pacientes em plaquetopênicos e não-

plaquetopênicos...........................................................................

25 3.9 Cultura de células THP-1............................................................. 26 3.10 Isolamento de plaquetas.............................................................. 26 3.11 Teste in vitro de fagocitose de plaquetas.................................... 26 3.12 Avaliação da expressão de P-selectina........................................ 26 3.13 Dosagem de citocinas.................................................................. 26 3.14 Citometria de fluxo........................................................................ 27 3.15 Análise estatística......................................................................... 28 3.16 Considerações éticas................................................................... 28 3.17 Limitações do estudo................................................................... 29 4 RESULTADOS....................................................................................... 30 4.1 Artigo publicado (resultado)........................................................ 31 5 CONCLUSÃO........................................................................................ 58 6 REFERÊNCIAS BIBLIOGRÁFICAS...................................................... 59 7 ANEXOS................................................................................................ 68 ANEXO A (Termo de Consentimento Livre e Esclarecido – TCLE) ... 68 ANEXO B (Ficha clínica do participante do estudo) ........................... 71 ANEXO C (Procedimento Operacional Padrão – POP) ....................... 74 ANEXO D (Parecer do CEP da FMT-HVD.............................................. 89 ANEXO E (Artigos publicados).............................................................. 91 ANEXO F (Normas da revista PLOS ONE............................................. 113

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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 Pós-Graduação 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

CDU: 616.928.5 Ficha Catalográfica elaborada pela Bibliotecária Maria Eliana N. Silva, lotada na

Escola Superior de Ciências da Saúde - UEA

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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).

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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).

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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, transformam-

se 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).

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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).

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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).

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A B C

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).

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 P-

selectina (PSGL-1). A tabela 1 descreve os principais constituintes dos grânulos

plaquetários (36).

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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 Grânulos densos

Fator Plaquetário 4 ATP

Fibrinogênio ADP

Fator V Cálcio

Fator de vonWillebrand Serotonina

Trombospondina P-selectina Fator de crescimento derivado de plaquetas (PDGF)

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

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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).

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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;

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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.

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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).

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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

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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 α-

grânulos 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).

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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).

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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 (75-

77). 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

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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

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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

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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.

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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.

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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.

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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.

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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).

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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 Diclofenaco Ouro

Ácido aminossalicílico Dietiletilbestrol Oxipenbutazona

Ácido nalidíxico Digoxina Oxitetraciclina

Ácido valpróico Eptifibatide Paracetamol

Aminoglutetimida Fenitoína Penicilina

Amiodarona Fluconazol Piperacilina

Anfotericina B Furosemida Procainamida

Captopril Haloperidol Quinidina

Carbamazepina Heparina Quinino

Cimetidina Hidroclorotiazida Ranitidina

Clorpromazina Ibuprofeno Rifampicina

Clorotiazida Interferon-α SulfametaxozolTrimetoprima

Clorpropamida Isoniazida Sulfassalazina

Danazol Levamisol Sulfissoxazol

Deferroxamina Lítio Sulindac

Diatrizoato de meglumina Metildopa Tamoxifeno

Diazepam Minoxidil Tiotixeno

Diazóxido Nafazolina 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.

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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.

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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 anti-

HCV 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

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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.

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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/SSC-

H), 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

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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.

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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.

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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.

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Thrombocytopenia in Plasmodium vivax Malaria IsRelated to Platelets PhagocytosisHelena Cristina C. Coelho1,2, Stefanie C. P. Lopes3, Joao Paulo D. Pimentel4,6, Paulo A. Nogueira4,

Fabio T. M. Costa3, Andre 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 Fundacao de Medicina Tropical Dr. Heitor Vieira Dourado, Manaus, Amazonas, Brazil, 3 Universidade

Estadual de Campinas, Campinas, Sao Paulo, Brazil, 4 Instituto Leonidas e Maria Deane, Fiocruz, Manaus, Amazonas, Brazil, 5 Universidade Federal do Amazonas, Manaus,

Amazonas, Brazil, 6 Fundacao de Hematologia e Hemoterapia do Amazonas, Manaus, Amazonas, Brazil

Abstract

Background: Although thrombocytopenia is a hematological disorder commonly reported in malarial patients, itsmechanisms 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 plateletvolume (MPV) was higher in thrombocytopenic patients as compared to non- thrombocytopenic patients (p = 0.017) and anegative correlation was found between platelet count and MPV (r = 20.483; p = 0.003). Platelets from HV or patients werelabeled with 5-chloromethyl fluorescein diacetate (CMFDA), incubated with human monocytic cell line (THP-1) and plateletphagocytosis index was analyzed by flow cytometry. The phagocytosis index was higher in thrombocytopenic patientscompared to non-thrombocytopenic patients (p = 0.042) and HV (p = 0.048). A negative correlation was observed betweenplatelet count and phagocytosis index (r = 20.402; p = 0.016). Platelet activation was assessed measuring the expression ofP-selectin (CD62-P) in platelets’ surface by flow cytometry. No significant difference was found in the expression of P-selectin 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 andp = 0.017, respectively. In contrast, TNF-a levels were not different between the three groups, but a positive correlation wasfound between TNF-a and phagocytosis index (r = 20.305; p = 0.037).

Conclusion/Significance: Collectively, our findings indicate that platelet phagocytosis may contribute to thrombocytopeniafound in vivax malaria. Finally, we believe that this study opens new avenues to explore the mechanisms involved in plateletdysfunction, 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 PlateletsPhagocytosis. PLoS ONE 8(5): e63410. doi:10.1371/journal.pone.0063410

Editor: Luzia Helena Carvalho, Centro de Pesquisa Rene Rachou/Fundacao Oswaldo Cruz (Fiocruz-Minas), Brazil

Received February 18, 2013; Accepted April 2, 2013; Published May 24, 2013

Copyright: � 2013 Coelho et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permitsunrestricted 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 fromPrograma Estrategico de Ciencia, Tecnologia & Inovacao nas Fundacoes Estaduais de Saude (PECTI/AM-Saude) from Fundacao de Amparo a Pesquisa do Estadodo Amazonas (FAPEAM, Amazonas - Brazil). This work was supported by CNPq and FAPEAM grants. The funders had no role in study design, data collection andanalysis, decision to publish, or preparation of the manuscript.

Competing Interests: The authors have declared that no competing interests exist.

* E-mail: [email protected]

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 malaria-

associated hematological complications in P. vivax and P. falciparum

[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

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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 mecha-

nisms 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.

Materials and Methods

Ethics StatementAll protocols and consents forms were approved by the Ethics

Review Board of the Fundacao 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 SubjectsPatients 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 co-

morbidities 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 Bioea-

syH, MG, Brazil) and hepatitis B (HBsAg ELISA BioeasyH, MG,

Brazil).

Platelets IsolationPlatelets 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

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 supple-

mented 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 ExpressionP-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).

THP-1 CellsHuman 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.

In vitro Platelet PhagocytosisAfter 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 AnalysisThe 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.

Cytokine MeasurementsThe 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 manu-

facturer’s instructions. All the cytokine levels below detection limit

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were given half of the threshold value and those values above the

upper detection limit were excluded from the analysis.

Statistical AnalysisAll data were expressed as the mean 6 SD. Correlations

were analyzed using the Spearman correlation. Normal distri-

bution 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. P-

selectin 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).

Results

Patient’s Characteristics and ThrombocytopeniaFrequency

According to Table 1, thrombocytopenia (,150,000 platelets/

mL) was found in 62.9% of the patients (22/35) enrolled in this

study. Amongst thrombocytopenic patients, 18.2% (4/22) pre-

sented severe thrombocytopenia (,50,000 platelets/mL). More-

over, 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 ThrombocytopeniaMPV was significantly elevated in patients with thrombocyto-

penia (Figure 1A). Moreover, a negative correlation was observed

Table 1. Characteristics of patients with Plasmodium vivax (with and without thrombocytopenia).

Characteristics Patients P value*

Total NT T

Age, years (mean 6SD) 41.8613.6 40.1613.8 42.4613.9 0.918a

Sex (%) M 29/35 (82.9) 9/13(69.3) 19/22 (86.4) 0.383b

F 6/35 (17.1) 4/13(30.7) 3/22 (13.6)

Duration of symptoms in days (mean 6SD) 5.564.0 5.363.5 5.364.9 0.605a

Previous malaria episodes (%) Yes 27/35 (77.2) 11/13 (84.6) 16/22 (72.7) 0.680b

No 8/35 (22.8) 2/13 (15.4) 6/22 (27.3)

Nu of previous malaria episodes (mean 6SD) 3.663.6 3.162.9 3.964.0 0.876a

Last malaria (%) ,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)

SD = standard deviation.NT = non-thrombocytopenic; T = thrombocytopenic.*Non-thrombocytopenic patients6thrombocytopenic patients.aMann Whitney test.bChi-square or Fisher’s exact test.doi:10.1371/journal.pone.0063410.t001

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

Platelets Phagocytosis in Vivax Malaria

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between the MPV and the platelet count in malaria patients

(r = 20.483; p = 0.003) (Figure 1B).

Parasitemia and ThrombocytopeniaParasitemia was similar in thrombocytopenic and non-throm-

bocytopenic patients (Figure 2A) and no correlation was found

between platelet count and parasitemia (Figure 2B).

Phagocytosis AssayThe phagocytosis index was significantly higher in patients with

thrombocytopenia malaria than in patients without thrombocyto-

penia (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). Phagocy-

tosis index not corrected by MPV was also analyzed and lead to

the same results (data not shown).

P-selectin ExpressionP-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.

Cytokine Profile in Patients’ SeraOf seven cytokines analyzed in this study, IL-6, IL-10 and IFN-

c 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-thrombocytope-

nic ones (Figure 5A and 5B). Indeed, negative correlations were

found between platelet counts and IL-6 and IL-10 values

Figure 2. Parasitemia and thrombocytopenia. Comparisons of parasitemia (per mm3 of blood) between non-thrombocytopenic (NT) andthrombocytopenic (T) patients with malaria (A). Correlation between platelet count and parasitemia (mm3 of blood) (B).doi:10.1371/journal.pone.0063410.g002

Figure 3. Phagocytosis index (PI). Comparisons of phagocytosis between healthy volunteers (HV), non-thrombocytopenic (NT) andthrombocytopenic (T) patients with malaria (A). Correlation between platelet count and phagocytosis index (B).doi:10.1371/journal.pone.0063410.g003

Platelets Phagocytosis in Vivax Malaria

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(Figure 6A and 6B). A positive correlation was found only between

phagocytosis index and TNF-a values (Figure 7).

Discussion

Despite not being a criterion for severe malaria, thrombocyto-

penia 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 pathogen-

esis of thrombocytopenia in malaria. Of those, two independent

studies have shown platelets phagocytosis in malaria thrombocy-

topenic 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 anthrombocytopenic patients. The P-selectin expression in plateletswas measured in two moments, in the PRP and in isolated platelets.doi:10.1371/journal.pone.0063410.g004

Figure 5. Cytokines levels. Comparisons of IL-6 (A), IL-10 (B), and IFN-c (C) between healthy volunteers (HV), non-thrombocytopenic (NT) andthrombocytopenic (T) patients.doi:10.1371/journal.pone.0063410.g005

Platelets Phagocytosis in Vivax Malaria

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compensate the low absolute number of platelets in the periphery;

therefore preserving primary hemostasis and avoiding severe

bleeding [8].

Negative correlation between parasitemia and thrombocytope-

nia 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]. Nonethe-

less, 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 P-

selectin 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

platelet count in P. falciparum experimentally infected volunteers

[36]. In contrast, Lee and colleagues showed that circulating P-

selectin in plasma was elevated in P. falciparum severe malaria but

not in P. vivax or P. falciparum non-severe infections [20]. As P-

selectin 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 TGF-

ß in P. vivax thrombocytopenic patients compared to non-

thrombocytopenic [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-thrombocyto-

penic 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.

ConclusionCollectively, 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 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

Figure 7. Correlation between TNF-a and phagocytosis index(PI).doi:10.1371/journal.pone.0063410.g007

Platelets Phagocytosis in Vivax Malaria

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brings new insights about the mechanisms involved in malaria

vivax thrombocytopenia and highlights the potential relevance of

this phenomenon.

Acknowledgments

This paper is dedicated to Prof. Maria Imaculada Muniz-Junqueira, who

introduced us all to the art of phagocytosis.

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.

References

1. World Health Organization. (2011) World malaria report 2011. Geneva: WorldHealth Organization. xii, 246 p. p.

2. Guerra CA, Gikandi PW, Tatem AJ, Noor AM, Smith DL, et al. (2008) Thelimits and intensity of Plasmodium falciparum transmission: implications for

malaria control and elimination worldwide. PLoS Med 5: e38.3. Anstey NM, Russell B, Yeo TW, Price RN (2009) The pathophysiology of vivax

malaria. Trends Parasitol 25: 220–227.

4. Lacerda MV, Mourao MP, Alexandre MA, Siqueira AM, Magalhaes BM, et al.(2012) Understanding the clinical spectrum of complicated Plasmodium vivax

malaria: a systematic review on the contributions of the Brazilian literature.Malar J 11: 12.

5. Guerra CA, Howes RE, Patil AP, Gething PW, Van Boeckel TP, et al. (2010)

The international limits and population at risk of Plasmodium vivax transmissionin 2009. PLoS Negl Trop Dis 4: e774.

6. Oliveira-Ferreira J, Lacerda MV, Brasil P, Ladislau JL, Tauil PL, et al. (2010)Malaria in Brazil: an overview. Malar J 9: 115.

7. Wickramasinghe SN, Abdalla SH (2000) Blood and bone marrow changes inmalaria. Baillieres Best Pract Res Clin Haematol 13: 277–299.

8. Lacerda MV, Mourao MP, Coelho HC, Santos JB (2011) Thrombocytopenia in

malaria: who cares? Mem Inst Oswaldo Cruz 106 Suppl 1: 52–63.9. Alecrim MGC (2000) Clinical aspects, resistance and parasitary polymorphism

of Plasmodium vivax malaria in Manaus. Brasılia: Universidade de Brasılia.176 p.

10. Kochar DK, Das A, Kochar A, Middha S, Acharya J, et al. (2010)

Thrombocytopenia in Plasmodium falciparum, Plasmodium vivax and mixedinfection malaria: a study from Bikaner (Northwestern India). Platelets 21: 623–

627.11. Tanwar GS, Khatri PC, Chahar CK, Sengar GS, Kochar A, et al. (2012)

Thrombocytopenia in childhood malaria with special reference to P. vivax

monoinfection: A study from Bikaner (Northwestern India). Platelets 23: 211–216.

12. Cox D, McConkey S (2010) The role of platelets in the pathogenesis of cerebralmalaria. Cell Mol Life Sci 67: 557–568.

13. Echeverri M, Tobon A, Alvarez G, Carmona J, Blair S (2003) Clinical andlaboratory findings of Plasmodium vivax malaria in Colombia, 2001. Rev Inst

Med Trop Sao Paulo 45: 29–34.

14. Piguet PF, Kan CD, Vesin C (2002) Thrombocytopenia in an animal model ofmalaria is associated with an increased caspase-mediated death of thrombocytes.

Apoptosis 7: 91–98.15. Erhart LM, Yingyuen K, Chuanak N, Buathong N, Laoboonchai A, et al. (2004)

Hematologic and clinical indices of malaria in a semi-immune population of

western Thailand. Am J Trop Med Hyg 70: 8–14.16. Skudowitz RB, Katz J, Lurie A, Levin J, Metz J (1973) Mechanisms of

thrombocytopenia in malignant tertian malaria. Br Med J 2: 515–518.17. Kelton JG, Keystone J, Moore J, Denomme G, Tozman E, et al. (1983)

Immune-mediated thrombocytopenia of malaria. J Clin Invest 71: 832–836.18. Looareesuwan S, Davis JG, Allen DL, Lee SH, Bunnag D, et al. (1992)

Thrombocytopenia in malaria. Southeast Asian J Trop Med Public Health 23:

44–50.19. Yamaguchi S, Kubota T, Yamagishi T, Okamoto K, Izumi T, et al. (1997)

Severe thrombocytopenia suggesting immunological mechanisms in two cases ofvivax malaria. Am J Hematol 56: 183–186.

20. Lee SH, Looareesuwan S, Chan J, Wilairatana P, Vanijanonta S, et al. (1997)

Plasma macrophage colony-stimulating factor and P-selectin levels in malaria-associated thrombocytopenia. Thromb Haemost 77: 289–293.

21. Klein E, Ronez E (2012) Peripheral hemophagocytosis in malaria infection.Blood 119: 910.

22. Jaff MS, McKenna D, McCann SR (1985) Platelet phagocytosis: a probablemechanism of thrombocytopenia in Plasmodium falciparum infection. J Clin

Pathol 38: 1318–1319.

23. Monbrison F, Angei C, Staal A, Kaiser K, Picot S (2003) Simultaneousidentification of the four human Plasmodium species and quantification of

Plasmodium DNA load in human blood by real-time polymerase chain reaction.Trans R Soc Trop Med Hyg 97: 387–390.

24. Honda S, Saito M, Dimaano EM, Morales PA, Alonzo MT, et al. (2009)

Increased phagocytosis of platelets from patients with secondary dengue virus

infection by human macrophages. Am J Trop Med Hyg 80: 841–845.

25. Gerardin P, Rogier C, Ka AS, Jouvencel P, Brousse V, et al. (2002) Prognostic

value of thrombocytopenia in African children with falciparum malaria.

Am J Trop Med Hyg 66: 686–691.

26. Rogier C, Gerardin P, Imbert P (2004) Thrombocytopenia is predictive of

lethality in severe childhood falciparum malaria. Arch Dis Child 89: 795–796.

27. Kochar DK, Saxena V, Singh N, Kochar SK, Kumar SV, et al. (2005)

Plasmodium vivax malaria. Emerg Infect Dis 11: 132–134.

28. Andrade BB, Reis-Filho A, Souza-Neto SM, Clarencio J, Camargo LM, et al.

(2010) Severe Plasmodium vivax malaria exhibits marked inflammatory

imbalance. Malar J 9: 13.

29. Maina RN, Walsh D, Gaddy C, Hongo G, Waitumbi J, et al. (2010) Impact of

Plasmodium falciparum infection on haematological parameters in children

living in Western Kenya. Malar J 9 Suppl 3: S4.

30. Gonzalez B, Rodulfo H, De Donato M, Berrizbeitia M, Gomez C, et al. (2009)

[Hematologic variations in patient with malaria caused by Plasmodium vivax

before, during and after treatment]. Invest Clin 50: 187–201.

31. Saravu K, Docherla M, Vasudev A, Shastry BA (2011) Thrombocytopenia in

vivax and falciparum malaria: an observational study of 131 patients in

Karnataka, India. Ann Trop Med Parasitol 105: 593–598.

32. Kochar DK, Tanwar GS, Agrawal R, Kochar S, Tanwar G, et al. (2012) Platelet

count and parasite density: independent variable in Plasmodium vivax malaria.

J Vector Borne Dis 49: 191–192.

33. Touze JE, Mercier P, Rogier C, Hovette P, Schmoor P, et al. (1990) [Platelet

antibody activity in malaria thrombocytopenia]. Pathol Biol (Paris) 38: 678–681.

34. Tyagi P, Biswas S (1999) Naturally occurring plasmodia-specific circulating

immune complexes in individuals of malaria endemic areas in India.

Indian J Malariol 36: 12–18.

35. Badlou BA, Wu YP, Smid WM, Akkerman JW (2006) Platelet binding and

phagocytosis by macrophages. Transfusion 46: 1432–1443.

36. Mast Q, de Groot PG, van Heerde WL, Roestenberg M, van Velzen JF, et al.

(2010) Thrombocytopenia in early malaria is associated with GP1b shedding in

absence of systemic platelet activation and consumptive coagulopathy.

Br J Haematol 151: 495–503.

37. Sosman JA, Verma A, Moss S, Sorokin P, Blend M, et al. (2000) Interleukin 10-

induced thrombocytopenia in normal healthy adult volunteers: evidence for

decreased platelet production. Br J Haematol 111: 104–111.

38. Casals-Pascual C, Kai O, Newton CR, Peshu N, Roberts DJ (2006)

Thrombocytopenia in falciparum malaria is associated with high concentrations

of IL-10. Am J Trop Med Hyg 75: 434–436.

39. Park JW, Park SH, Yeom JS, Huh AJ, Cho YK, et al. (2003) Serum cytokine

profiles in patients with Plasmodium vivax malaria: a comparison between those

who presented with and without thrombocytopenia. Ann Trop Med Parasitol

97: 339–344.

40. Tacchini-Cottier F, Vesin C, Redard M, Buurman W, Piguet PF (1998) Role of

TNFR1 and TNFR2 in TNF-induced platelet consumption in mice. J Immunol

160: 6182–6186.

41. Wroczynska A, Nahorski W, Bakowska A, Pietkiewicz H (2005) Cytokines and

clinical manifestations of malaria in adults with severe and uncomplicated

disease. Int Marit Health 56: 103–114.

42. Andrade BB, Barral-Netto M (2011) Biomarkers for susceptibility to infection

and disease severity in human malaria. Mem Inst Oswaldo Cruz 106 Suppl 1:

70–78.

43. Yeo TW, Lampah DA, Gitawati R, Tjitra E, Kenangalem E, et al. (2008)

Angiopoietin-2 is associated with decreased endothelial nitric oxide and poor

clinical outcome in severe falciparum malaria. Proc Natl Acad Sci U S A 105:

17097–17102.

44. Armah H, Wired EK, Dodoo AK, Adjei AA, Tettey Y, et al. (2005) Cytokines

and adhesion molecules expression in the brain in human cerebral malaria.

Int J Environ Res Public Health 2: 123–131.

Platelets Phagocytosis in Vivax Malaria

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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 não-

plaquetopênicos 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.

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39

6 REFERÊNCIAS BIBLIOGRÁFICAS

1. Brasil MdS. Aspectos epidemiológicos. [internet] [cited April 21, 2010]; Available from: http://portal.saude.gov.br/portal/saude/profissional/area.cfm?id_area=1526

2. WHO. World Malaria Report 2009. WHO Library Cataloguing-in-Publication 2009 [cited November 10, 2009]; Available from: http://www.who.int

3. Mendis K, Sina BJ, Marchesini P, Carter R. The neglected burden of Plasmodium vivax malaria. Am J Trop Med Hyg 2001;64(1-2 Suppl):97-106.

4. Price RN, Tjitra E, Guerra CA, Yeung S, White NJ, Anstey NM. Vivax malaria: neglected and not benign. Am J Trop Med Hyg 2007;77(6 Suppl):79-87.

5. WHO. World Malaria Report 2010/WHO- World Health Organization. WHO Library Cataloguing-in-Publication 2010 [cited January 06, 2010]; Available from: http://www.who.int

6. WHO. World Heath Organization (2010) World: Malaria Risk areas 2010. In: Global_Malaria_ITHRiskMap.JPG, editor.

7. Brasil MdS. Avaliação malaria_jan_maio_19_07_2010. 2010 [cited January 6, 2011]; Available from: http://portal.saude.gov.br/portal/arquivos/pdf/avaliacao_malaria_jan_maio_19_07_2010.pdf

8. FMT. Informe Epidemiológico Nº 12 - ANO V / 2010 da FMT. 2010 [cited November 01, 2010]; Available from: www.fmt.am.gov.br/informe/2010/MURAL%20MAL2007A2009.pdf

9. Oliveira-Ferreira J, Lacerda MV, Brasil P, Ladislau JL, Tauil PL, Daniel-Ribeiro CT. Malaria in Brazil: an overview. Malar J 2010;9:115.

10. Baird JK. Resistance to therapies for infection by Plasmodium vivax. Clin Microbiol Rev 2009;22(3):508-34.

11. de Santana Filho FS, Arcanjo AR, Chehuan YM, Costa MR, Martinez-Espinosa FE, Vieira JL, et al. Chloroquine-resistant Plasmodium vivax, Brazilian Amazon. Emerg Infect Dis 2007;13(7):1125-6.

12. WHO. malaria/publications/country-profiles/profile_bra_. In: www.who.int/malaria/publications/country-profiles/profile_bra_en.pdf, editor.

13. Alecrim MdG, Alecrim W. Malária. In: Cinerman S, Cinerman B, editors. Medicina Tropical. São Paulo: Atheneu; 2003. p. 105 -118.

Page 56: UNIVERSIDADE DO ESTADO DO AMAZONAS FUNDAÇÃO DE … · universidade do estado do amazonas . fundaÇÃo de medicina tropical dr. heitor vieira dourado . programa de pÓs-graduaÇÃo

40

14. Tan LK, Yacoub S, Scott S, Bhagani S, Jacobs M. Acute lung injury and other serious complications of Plasmodium vivax malaria. Lancet Infect Dis 2008;8(7):449-54.

15. Kochar DK, Das A, Kochar SK, Saxena V, Sirohi P, Garg S, et al. Severe Plasmodium vivax malaria: a report on serial cases from Bikaner in northwestern India. Am J Trop Med Hyg 2009;80(2):194-8.

16. Poespoprodjo JR, Fobia W, Kenangalem E, Lampah DA, Hasanuddin A, Warikar N, et al. Vivax malaria: a major cause of morbidity in early infancy. Clin Infect Dis 2009;48(12):1704-12.

17. Siqueira AM, Alexandre MA, Mourao MP, Santos VS, Nagahashi-Marie SK, Alecrim MG, et al. Severe rhabdomyolysis caused by Plasmodium vivax malaria in the Brazilian Amazon. Am J Trop Med Hyg;83(2):271-3.

18. Baird JK. Neglect of Plasmodium vivax malaria. Trends Parasitol 2007;23(11):533-9.

19. Lacerda MVG. Manifestações Clínicas e Patogênese da plaquetopenia na malária. Brasília: Universidade de Brasília; 2007.

20. Rodriguez-Morales AJ, Sanchez E, Vargas M, Piccolo C, Colina R, Arria M. Anemia and thrombocytopenia in children with Plasmodium vivax malaria. J Trop Pediatr 2006;52(1):49-51.

21. Shaikh QH, Ahmad SM, Abbasi A, Malik SA, Sahito AA, Munir SM. Thrombocytopenia in malaria. J Coll Physicians Surg Pak 2009;19(11):708-10.

22. Harish R, Gupta S. Plasmodium vivax malaria presenting with severe thrombocytopenia, cerebral complications and hydrocephalus. Indian J Pediatr 2009;76(5):551-2.

23. Makkar RP, Mukhopadhyay S, Monga A, Monga A, Gupta AK. Plasmodium vivax malaria presenting with severe thrombocytopenia. Braz J Infect Dis 2002;6(5):263-5.

24. Thapa R, Biswas B, Mallick D, Sardar S, Modak S. Childhood Plasmodium vivax malaria with severe thrombocytopenia and bleeding manifestations. J Pediatr Hematol Oncol 2009;31(10):758-9.

25. Mackintosh CL, Beeson JG, Marsh K. Clinical features and pathogenesis of severe malaria. Trends Parasitol 2004;20(12):597-603.

26. Anstey NM, Russell B, Yeo TW, Price RN. The pathophysiology of vivax malaria. Trends Parasitol 2009;25(5):220-7.

Page 57: UNIVERSIDADE DO ESTADO DO AMAZONAS FUNDAÇÃO DE … · universidade do estado do amazonas . fundaÇÃo de medicina tropical dr. heitor vieira dourado . programa de pÓs-graduaÇÃo

41

27. Karunaweera ND, Wijesekera SK, Wanasekera D, Mendis KN, Carter R. The paroxysm of Plasmodium vivax malaria. Trends Parasitol 2003;19(4):188-93.

28. Medina TS, Costa SP, Oliveira MD, Ventura AM, Souza JM, Gomes TF, et al. Increased interleukin-10 and interferon-gamma levels in Plasmodium vivax malaria suggest a reciprocal regulation which is not altered by IL-10 gene promoter polymorphism. Malar J;10:264.

29. Zeyrek FY, Kurcer MA, Zeyrek D, Simsek Z. Parasite density and serum cytokine levels in Plasmodium vivax malaria in Turkey. Parasite Immunol 2006;28(5):201-7.

30. Mirghani HA, Eltahir HG, TM AE, Mirghani YA, Elbashir MI, Adam I. Cytokine profiles in children with severe Plasmodium falciparum malaria in an area of unstable malaria transmission in central Sudan. J Trop Pediatr;57(5):392-5.

31. Andrade BB, Reis-Filho A, Souza-Neto SM, Clarencio J, Camargo LM, Barral A, et al. Severe Plasmodium vivax malaria exhibits marked inflammatory imbalance. Malar J;9:13.

32. White JG. Platelet Structure. In: Elsevier, editor. Platelets. 2 ed. San Diego; 2007. p. 45-73.

33. Alberts B, Jonson A, Lewis J, Raff M, Roberts K, Walter P. Molecular Biology of the Cell; 2002.

34. Patel SR, Hartwig JH, Italiano JE, Jr. The biogenesis of platelets from megakaryocyte proplatelets. J Clin Invest 2005;115(12):3348-54.

35. Kuter DJ, Begley CG. Recombinant human thrombopoietin: basic biology and evaluation of clinical studies. Blood 2002;100(10):3457-69.

36. Lichtman M, Beutler E, Kaushansky K, Kipps T, Seligohn U, Prchal J. Williams Hematology; 2005.

37. Gouttefangeas C, Diehl M, Keilholz W, Hornlein RF, Stevanovic S, Rammensee HG. Thrombocyte HLA molecules retain nonrenewable endogenous peptides of megakaryocyte lineage and do not stimulate direct allocytotoxicity in vitro. Blood 2000;95(10):3168-75.

38. Piguet PF, Kan CD, Vesin C, Rochat A, Donati Y, Barazzone C. Role of CD40-CVD40L in mouse severe malaria. Am J Pathol 2001;159(2):733-42.

39. Schofield L, Grau GE. Immunological processes in malaria pathogenesis. Nat Rev Immunol 2005;5(9):722-35.

Page 58: UNIVERSIDADE DO ESTADO DO AMAZONAS FUNDAÇÃO DE … · universidade do estado do amazonas . fundaÇÃo de medicina tropical dr. heitor vieira dourado . programa de pÓs-graduaÇÃo

42

40. Kaplanski G, Porat R, Aiura K, Erban JK, Gelfand JA, Dinarello CA. Activated platelets induce endothelial secretion of interleukin-8 in vitro via an interleukin-1-mediated event. Blood 1993;81(10):2492-5.

41. Bergmeier W, Wagner DD. Inflammation. In: Elsevier, editor. Platelets. 2 ed. San Diego; 2007. p. 713 -726.

42. Lee GR, Bithell TC, Foerster J, Athens JW, Lukens JN. Wintrobe Hematologia Clínica. 1 ed. São Paulo; 1998.

43. Hartwig J, Italiano J, Jr. The birth of the platelet. J Thromb Haemost 2003;1(7):1580-6.

44. Fischer TH, Merricks E, Bellinger DA, Hayes PM, Smith RS, Raymer RA, et al. Splenic clearance mechanisms of rehydrated, lyophilized platelets. Artif Cells Blood Substit Immobil Biotechnol 2001;29(6):439-51.

45. Andre P. P-selectin in haemostasis. Br J Haematol 2004;126(3):298-306.

46. Heemskerk JW, Bevers EM, Lindhout T. Platelet activation and blood coagulation. Thromb Haemost 2002;88(2):186-93.

47. Zwaal RF, Bevers EM. Platelet phospholipid asymmetry and its significance in hemostasis. Subcell Biochem 1983;9:299-334.

48. Semple JW, Aslam R, Kim M, Speck ER, Freedman J. Platelet-bound lipopolysaccharide enhances Fc receptor-mediated phagocytosis of IgG-opsonized platelets. Blood 2007;109(11):4803-5.

49. Badlou BA, Wu YP, Smid WM, Akkerman JW. Platelet binding and phagocytosis by macrophages. Transfusion 2006;46(8):1432-43.

50. Bratton DL, Henson PM. Apoptotic cell recognition: will the real phosphatidylserine receptor(s) please stand up? Curr Biol 2008;18(2):R76-9.

51. Brown SB, Clarke MC, Magowan L, Sanderson H, Savill J. Constitutive death of platelets leading to scavenger receptor-mediated phagocytosis. A caspase-independent cell clearance program. J Biol Chem 2000;275(8):5987-96.

52. Bithell TC. Trombocitopenia. In: Manole, editor. Wintrobe Hematologia Clínica. São Paulo; 1998. p. 1454-1457.

53. Lacerda MV, Mourao MP, Coelho HC, Santos JB. Thrombocytopenia in malaria: who cares? Mem Inst Oswaldo Cruz 2011;106 Suppl 1:52-63.

Page 59: UNIVERSIDADE DO ESTADO DO AMAZONAS FUNDAÇÃO DE … · universidade do estado do amazonas . fundaÇÃo de medicina tropical dr. heitor vieira dourado . programa de pÓs-graduaÇÃo

43

54. Rodriguez-Morales AJ, Sanchez E, Vargas M, Piccolo C, Colina R, Arria M, et al. Occurrence of thrombocytopenia in Plasmodium vivax malaria. Clin Infect Dis 2005;41(1):130-1.

55. Vij AS, Dandona PK, Aggarwal A. Malaria with marked thrombocytopenia: report of 2 cases. J Indian Med Assoc 2008;106(2):123, 125.

56. Erhart LM, Yingyuen K, Chuanak N, Buathong N, Laoboonchai A, Miller RS, et al. Hematologic and clinical indices of malaria in a semi-immune population of western Thailand. Am J Trop Med Hyg 2004;70(1):8-14.

57. Katira B, Shah I. Thrombocytopenia in Plasmodium vivax infected children. J Vector Borne Dis 2006;43(3):147-9.

58. Gonzalez B, Rodulfo H, De Donato M, Berrizbeitia M, Gomez C, Gonzalez L. [Hematologic variations in patient with malaria caused by Plasmodium vivax before, during and after treatment]. Invest Clin 2009;50(2):187-201.

59. Patel U, Gandhi G, Friedman S, Niranjan S. Thrombocytopenia in Malaria. Journal of the National Association 2004;96:1212-1214.

60. Skudowitz RB, Katz J, Lurie A, Levin J, Metz J. Mechanisms of thrombocytopenia in malignant tertian malaria. Br Med J 1973;2(5865):515-8.

61. Karanikas G, Zedwitz-Liebenstein K, Eidherr H, Schuetz M, Sauerman R, Dudczak R, et al. Platelet kinetics and scintigraphic imaging in thrombocytopenic malaria patients. Thromb Haemost 2004;91(3):553-7.

62. Watier H, Verwaerde C, Landau I, Werner E, Fontaine J, Capron A, et al. T-cell-dependent immunity and thrombocytopenia in rats infected with Plasmodium chabaudi. Infect Immun 1992;60(1):136-42.

63. Jaff MS, McKenna D, McCann SR. Platelet phagocytosis: a probable mechanism of thrombocytopenia in Plasmodium falciparum infection. J Clin Pathol 1985;38(11):1318-9.

64. Lee SH, Looareesuwan S, Chan J, Wilairatana P, Vanijanonta S, Chong SM, et al. Plasma macrophage colony-stimulating factor and P-selectin levels in malaria-associated thrombocytopenia. Thromb Haemost 1997;77(2):289-93.

65. Honda S, Saito M, Dimaano EM, Morales PA, Alonzo MT, Suarez LA, et al. Increased phagocytosis of platelets from patients with secondary dengue virus infection by human macrophages. Am J Trop Med Hyg 2009;80(5):841-5.

66. Saito M, Oishi K, Inoue S, Dimaano EM, Alera MT, Robles AM, et al. Association of increased platelet-associated immunoglobulins with thrombocytopenia

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and the severity of disease in secondary dengue virus infections. Clin Exp Immunol 2004;138(2):299-303.

67. Casals-Pascual C, Kai O, Newton CR, Peshu N, Roberts DJ. Thrombocytopenia in falciparum malaria is associated with high concentrations of IL-10. Am J Trop Med Hyg 2006;75(3):434-6.

68. Kelton JG, Keystone J, Moore J, Denomme G, Tozman E, Glynn M, et al. Immune-mediated thrombocytopenia of malaria. J Clin Invest 1983;71(4):832-6.

69. Yamaguchi S, Kubota T, Yamagishi T, Okamoto K, Izumi T, Takada M, et al. Severe thrombocytopenia suggesting immunological mechanisms in two cases of vivax malaria. Am J Hematol 1997;56(3):183-6.

70. Conte R, Tassi C, Belletti D, Ricci F, Tazzari PL. Autoimmune thrombocytopenia in malaria. Vox Sang 2003;85(3):221.

71. Ghosh K, Shetty S. Blood coagulation in falciparum malaria--a review. Parasitol Res 2008;102(4):571-6.

72. Cox D, McConkey S. The role of platelets in the pathogenesis of cerebral malaria. Cell Mol Life Sci 2010;67(4):557-68.

73. de Mast Q, Groot Eo, Lenting PJ, de Groot PG, McCall M, Sauerwein RW, et al. Thrombocytopenia and release of activated von Willebrand Factor during early Plasmodium falciparum malaria. J Infect Dis 2007;196(4):622-8.

74. Francischetti IM. Does activation of the blood coagulation cascade have a role in malaria pathogenesis? Trends Parasitol 2008;24(6):258-63.

75. Borochovitz D, Crosley AL, Metz J. Disseminated intravascular coagulation with fatal haemorrhage in cerebral malaria. Br Med J 1970;2(5711):710.

76. Srichaikul T, Puwasatien P, Karnjanajetanee J, Bokisch VA, Pawasatien P. Complement changes and disseminated intravascular coagulation in Plasmodium falciparum malaria. Lancet 1975;1(7910):770-2.

77. Stuart J. Intravascular coagulation in falciparum malaria. Br Med J 1978;2(6139):774.

78. Horstmann RD, Dietrich M. Haemostatic alterations in malaria correlate to parasitaemia. Blut 1985;51(5):329-35.

79. Rojanasthien S, Surakamolleart V, Boonpucknavig S, Isarangkura P. Hematological and coagulation studies in malaria. J Med Assoc Thai 1992;75 Suppl 1:190-4.

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45

80. Alonzo MT, Lacuesta TL, Dimaano EM, Kurosu T, Suarez LA, Mapua CA, et al. Platelet apoptosis and apoptotic platelet clearance by macrophages in secondary dengue virus infections. J Infect Dis 2012;205(8):1321-9.

81. Piguet PF, Kan CD, Vesin C. Thrombocytopenia in an animal model of malaria is associated with an increased caspase-mediated death of thrombocytes. Apoptosis 2002;7(2):91-8.

82. Erel O, Vural H, Aksoy N, Aslan G, Ulukanligil M. Oxidative stress of platelets and thrombocytopenia in patients with vivax malaria. Clin Biochem 2001;34(4):341-4.

83. Erel O, Kocyigit A, Bulut V, Avci S, Aktepe N. Role of lipids, lipoproteins and lipid peroxidation in thrombocytopenia in patients with vivax malaria. Haematologia (Budap) 1998;29(3):207-12.

84. Araujo CF, Lacerda MV, Abdalla DS, Lima ES. The role of platelet and plasma markers of antioxidant status and oxidative stress in thrombocytopenia among patients with vivax malaria. Mem Inst Oswaldo Cruz 2008;103(6):517-21.

85. Fajardo L, Tallent C. Malarial parasites within human platelets. J Am Med Assoc 1974;229:1205-7.

86. Abdalla SH. Hematopoiesis in human malaria. Blood Cells 1990;16(2-3):401-16; discussion 417-9.

87. Kreil A, Wenisch C, Brittenham G, Looareesuwan S, Peck-Radosavljevic M. Thrombopoietin in Plasmodium falciparum malaria. Br J Haematol 2000;109(3):534-6.

88. Sosman JA, Verma A, Moss S, Sorokin P, Blend M, Bradlow B, et al. Interleukin 10-induced thrombocytopenia in normal healthy adult volunteers: evidence for decreased platelet production. Br J Haematol 2000;111(1):104-11.

89. Silva IBA. Malária vivax: manifestações clínicas e laboratoriais relacionadas com o fator de necrose tumoral alfa. Belém: Universdiade Federal do Pará; 2004.

90. Lacerda MVG, Arcanjo ARL, Farias AF, Alecrim WD, Alecrim MGC. o papel das citocinas nas alterações hematológicas da malária por P. vivax e P. falciparum. n nais a euni o a iona e es uisa e a ria orto e o

91. Park JW, Park SH, Yeom JS, Huh AJ, Cho YK, Ahn JY, et al. Serum cytokine profiles in patients with Plasmodium vivax malaria: a comparison between those who presented with and without thrombocytopenia. Ann Trop Med Parasitol 2003;97(4):339-44.

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92. Brinker AD, Beitz J. Spontaneous reports of thrombocytopenia in association with quinine: clinical attributes and timing related to regulatory action. Am J Hematol 2002;70(4):313-7.

93. Sekhon SS, Roy V. Thrombocytopenia in adults: A practical approach to evaluation and management. South Med J 2006;99(5):491-8; quiz 499-500, 533.

94. de Monbrison F, Angei C, Staal A, Kaiser K, Picot S. Simultaneous identification of the four human Plasmodium species and quantification of Plasmodium DNA load in human blood by real-time polymerase chain reaction. Trans R Soc Trop Med Hyg 2003;97(4):387-90.

95. Brasil MdS. Manual de terapêutica da malária. 2001 [cited December 7, 2009]; Available from: http://portal aquivos pdf manu_terapeutica_malaria.pdf

96. World Health Organization. World malaria report 2011. Geneva: World Health Organization; 2011.

97. Oliveira-Ferreira J, Lacerda MV, Brasil P, Ladislau JL, Tauil PL, Daniel-Ribeiro CT. Malaria in Brazil: an overview. Malar J;9:115.

98. Wickramasinghe SN, Abdalla SH. Blood and bone marrow changes in malaria. Baillieres Best Pract Res Clin Haematol 2000;13(2):277-99.

99. Alecrim MGC. Clinical aspects, resistance and parasitary polymorphism of Plasmodium vivax malaria in Manaus. Brasília: Universidade de Brasília; 2000.

100. Echeverri M, Tobon A, Alvarez G, Carmona J, Blair S. Clinical and laboratory findings of Plasmodium vivax malaria in Colombia, 2001. Rev Inst Med Trop Sao Paulo 2003;45(1):29-34.

101. Klein E, Ronez E. Peripheral hemophagocytosis in malaria infection. Blood 2012;119(4):910.

102. Maina RN, Walsh D, Gaddy C, Hongo G, Waitumbi J, Otieno L, et al. Impact of Plasmodium falciparum infection on haematological parameters in children living in Western Kenya. Malar J 2010;9 Suppl 3:S4.

103. Kochar DK, Das A, Kochar A, Middha S, Acharya J, Tanwar GS, et al. Thrombocytopenia in Plasmodium falciparum, Plasmodium vivax and mixed infection malaria: a study from Bikaner (Northwestern India). Platelets 2010;21(8):623-7.

104. World Health Organization. Guidelines for the treatment of malaria. 2nd ed. Geneva: World Health Organization; 2010.

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105. Tacchini-Cottier F, Vesin C, Redard M, Buurman W, Piguet PF. Role of TNFR1 and TNFR2 in TNF-induced platelet consumption in mice. J Immunol 1998;160(12):6182-6.

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7. ANEXOS

ANEXO A: Termo de Consentimento Livre e Esclarecido – TCLE

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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.

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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 Data

..............................................................................................

Assinatura do pesquisador que conversou com o paciente

....................................................

Local e Data

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ANEXO B: Ficha clínica do participante do estudo

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Mestrado do Programa de Pós-Grauduação UEA/FMT-HVD

Aluna responsável: Helena Cristina Cardoso Coelho

Data de inclusão: ........ /........ /........

1 IDENTIFICAÇÃO

Nome:............................................................................................................... Registro:..........................

Data de nascimento: ........ /........ / ........ Idade:.................... Sexo: 1-M 2- F

Endereço:................................................................................................................................................

Bairro:............................................................... Município:.............................. Estado:........................

Telefone residencial:......................................................... 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

2.6 Tipo da última malária 1-V 2- F 3-F+V 4- não sabe

2.7 Usou alguma medicação antimalária nos últimos 60 dias? 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? 1- sim 2-não

2.16 Usa alguma medicação regular? 1- sim 2-não

Qual?.......................................................................................................................................................

FICHA CLÍNICA DO PARTICIPANTE DO ESTUDO

AVALIAÇÃO IN VITRO DA FAGOCITOSE DE PLAQUETAS EM PACIENTES COM

MALÁRIA VIVAX

Última malária foi há quanto tempo ?......................................................................................................

Número:

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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 3 - V + F 4 - Negativo

4,2 Parasitemia da malária vivax 1- < 1/2+ 2- 1/2+ 3- + 4- ++ 5- +++ 6- ++++

4,3 Esquizontes ? 1- sim 2-não Gametócitos 1- sim 2-não

4,4 Hemácias.......................x106/µL MCV................../µm3MCH...................pg MCHCM...................g/dL

4,5 Hematrócrito..................................% Hb .....................................................

4,6 Leucócitos .......................................x103/µL Leucócitos .....................................%

4,7 Linfócitos ........................................x103/µL Linfócitos.....................................%

4,8 Monócitos........................................x103/µL Monócitos.....................................%

4,9 Neutrófilos........................................x103/µL Neutrófilos.....................................%

4,10 Eosinófilos........................................x103/µL Eosinófilos.....................................%

4,11 Plaquetas........................................x103/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 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.

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ANEXO C: Procedimento Operacional Padrão - POP

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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

Data de aplicação:

Escrito ou traduzido por: Helena C. C. Coelho Data & assinatura

Revisado por: Paulo Nogueira , João Paulo Pimentel e Stefanie Lopes

Data & assinatura

Aprovado por: Marcus V. G. Lacerda Data & assinatura Data da próxima

revisão:

Emenda Razão da emenda

1 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

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POP_MAL_LB_009_v01_PT

6 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 1mL de meio de congelamento para cada 1x106células);

6.5.7 Transferir o meio de congelamento para o tubo com células e homogeneizar bem com o auxílio da

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POP_MAL_LB_009_v01_PT

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.

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Figura 1: Câmara de Neubauer. Em vermelho: os 4 quadrantes que devem ser contados para determinar o

número de células por mL.

7 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.

8 REGISTROS DOS ANEXOS

Não se aplica.

A versão atual deste POP foi traduzida a ____________________ e a versão traduzida entra em efeito em ___/___/_____.

Assinatura do responsável: ______________________

dd mm aa

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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

Data de aplicação:

Escrito ou traduzido por:

Helena C. C. Coelho Data & assinatura

Revisado por: Paulo Nogueira, João Paulo Pimentel e Stefanie Lopes

Data & assinatura

Aprovado por:

Marcus V. G. Lacerda Data & assinatura Data da próxima

revisão:

Emenda Razão da emenda

1 OBJETIVOS

Este procedimento visa isolar, marcar e opsonizar plaquetas do sangue periférico.

2 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.

3 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

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6 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).

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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;

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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.

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• 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.

8 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 ___/___/_____.

Assinatura do responsável: ______________________

dd mm aa

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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:

Helena C. C. Coelho

Data & assinatura

Revisado por:

Paulo Nogueira, João Paulo Pimentel e Stefanie Lopes

Data & assinatura

Aprovado por:

Marcus V. G. Lacerda

Data & assinatura

Data de aplicação:

Data da próxima revisão:

Emenda Razão da emenda

1 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.

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4 RESPONSABILIDADES

Gerente da unidade, coordenador da subunidade e pessoal técnico.

5 POP’S RELACIONADOS

POP_MAL_LB_010_v01_PT

6 PROCEDIMENTOS

6.1 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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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.

6.7.3 Adicionar 100 µL da solução de plaquetas marcadas com CMFDA (50x106/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.

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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 THP-1 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 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.

8 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 ___/___/_____.

Assinatura do responsável: ______________________

dd mm aa

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69

ANEXO D: Parecer do CEP da FMT-HVD

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91

ANEXO E: Artigos publicados

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Plasma Circulating Nucleic Acids Levels IncreaseAccording to the Morbidity of Plasmodium vivax MalariaBernardo 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 Laboratorio de Malaria, Centro de Pesquisa Rene, Fundacao Oswaldo Cruz, Belo Horizonte, Minas Gerais, Brazil, 2 Gerencia de Malaria, Fundacao de Medicina Tropical Dr.

Heitor Vieira Dourado, Manaus, Amazonas, Brazil, 3 Departamento de Clınica Medica, 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, theidentification 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 diseasesand 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 Amazonand 14 malaria non-exposed healthy donors were quantified by two different methodologies: amplification of the humantelomerase reverse transcriptase (hTERT) genomic sequence by quantitative real time PCR (qPCR), and the fluorometricdsDNA quantification by Pico Green. CNAs levels were significantly increased in plasma from P. vivax patients as comparedto 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 inplasma samples from 14 additional P. vivax patients of a different endemic area in Brazil, in which CNAS levels stronglycorrelated with thrombocytopenia (p = 0.0072). We further show that plasma CNAs levels decrease and reach physiologicallevels after antimalarial treatment. Although we found both host and parasite specific genomic sequences circulating inplasma, 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 theirpotential 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 theMorbidity 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: � 2011 Franklin et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permitsunrestricted 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 OswaldoCruz 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)

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

true spectrum of clinical disease in endemic areas remains

unknown [8]. The few studies that have addressed the pathogen-

esis of vivax malaria showed that the different clinical presenta-

tions of vivax malaria might be related to the intensity of pro-

inflammatory 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

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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

malaria, evaluated here by scoring patients’ clinical and

hematological parameters.

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 orhealthy donors by measuring the amplification of the hTERT human genomic sequence (A) as compared to the amplification of the O. keta Ychromosome 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 usingthe Mann-Whitney test. A p value,0.05 was considered significant.doi:10.1371/journal.pone.0019842.g001

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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 Quant-

iTTM 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 life-

threatening outcomes in diseases such as heart attack, stroke, and

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 carried-

out 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 thrombocyto-

penia, 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).

Figure 2. Plasma CNAs levels reliably correlate with the P. vivax clinical spectrum. CNAs levels were quantified in plasma from P. vivaxpatients with different clinical presentations. (A) Amplification of the genomic sequence of hTERT by qPCR in four healthy controls, and P. vivaxpatients (Pv_01 to 04) scored according to clinical/hematological parameters. Only four patients are shown for illustration purposes. (B) Correlationbetween 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 theamplification of the hTERT (Pearson r = 20.7897, p,0.0001).doi:10.1371/journal.pone.0019842.g002

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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.

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

Figure 3. Correlation between plasma CNAs levels and malaria vivax thrombocytopenia. Correlation of plasma CNAs levels with plateletcounts 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 pvalue,0.05 was considered significant.doi:10.1371/journal.pone.0019842.g003

Figure 4. Plasma CNAs levels decrease after anti-malarial chemotherapy. For 6 patients who showed up during convalescence, the CNAslevels in plasma were assessed by (A) fluorescence quantification of dsDNA with the Pico Green methodology or (B) comparison of the mean cyclethreshold for the qPCR amplification of hTERT genomic sequence. (C) platelet counts measured during admission and convalescence. Statistics wereperformed 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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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).

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

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 apoptosis-

induced 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 cell-

death 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

Figure 5. Plasmodium specific genomic sequences circulating in plasma from P. vivax patients. The presence and levels of P. vivax specificplasma CNAs were investigated in samples from P. vivax patients with different clinical presentations by qPCR amplification of a specific P. vivaxgenomic sequence. (A) For illustration purpose, qPCR amplification of a P. vivax specific genomic sequence in four healthy controls, and four P. vivaxpatients (Pv_01 to 04) scored according to clinical/hematological parameters is shown. (B) Pearson correlation between the Ct of parasite specificgenomic 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

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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 pro-

inflammatory 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.

Materials and Methods

Study area and subjectsThis study was conducted in May 2010, at Fundacao 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

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 co-

morbidity 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 hemato-

logical 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

Rene 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

Table 1. Characteristics of the Plasmodium vivax patientsenrolled 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%)

Nausea 15 (71.4%)

Anorexia 12 (57.1%)

Vomiting 6 (28.6%)

Dyspnea 6 (28.6%)

Diarrhea 3 (14.3%)

doi:10.1371/journal.pone.0019842.t001

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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 age-

matched 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.

Malaria vivax clinical scoreSince 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 hyperbilirrubi-

nemia (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.

Purification and quantification of CNAs from plasmaCell-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

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, Nether-

lands) 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.

Statistical analysisData 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 inplasma 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 stan-

dard 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 concentra-

tions 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 vivaxthrombocytopenia 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)

Figure S3 Plasma CNAs levels correlates with throm-bocytopenia 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)

Circulating Nucleic Acids: Malaria Severity Marker

PLoS ONE | www.plosone.org 7 May 2011 | Volume 6 | Issue 5 | e19842

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Table S1 Patient final clinical score and plasma CNAslevels.(DOC)

Acknowledgments

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 Magalhaes 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

Real Time PCR Instrumet awarded to BSF as the 2009 winner of the

Helixis Young Investigator Award.

Author Contributions

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.

References

1. Guerra CA, Howes RE, Patil AP, Gething PW, Van Boeckel TP, et al. (2010)

The international limits and population at risk of Plasmodium vivax transmission

in 2009. PLoS Negl Trop Dis 4: e774.

2. Kochar D, Saxena V, Singh N, Kochar S, Kumar S, et al. (2005) Plasmodium

vivax malaria. Emerg Infect Dis 11: 132–134.

3. Kochar D, Das A, Kochar S, Saxena V, Sirohi P, et al. (2009) Severe

Plasmodium vivax malaria: a report on serial cases from Bikaner in northwesternIndia. Am J Trop Med Hyg 80: 194–198.

4. Poespoprodjo J, Fobia W, Kenangalem E, Lampah D, Hasanuddin A, et al.(2009) Vivax malaria: a major cause of morbidity in early infancy. Clin Infect

Dis 48: 1704–1712.

5. Tjitra E, Anstey N, Sugiarto P, Warikar N, Kenangalem E, et al. (2008)

Multidrug-resistant Plasmodium vivax associated with severe and fatal malaria: a

prospective study in Papua, Indonesia. PLoS Med 5: e128.

6. Genton B, D’Acremont V, Rare L, Baea K, Reeder J, et al. (2008) Plasmodium

vivax and mixed infections are associated with severe malaria in children: aprospective cohort study from Papua New Guinea. PLoS Med 5: e127.

7. Alexandre M, Ferreira C, Siqueira A, Magalhaes B, Mourao M, et al. (2010)Severe Plasmodium vivax malaria, Brazilian Amazon. Emerg Infect Dis 16:

1611–1614.

8. Anstey N, Russell B, Yeo T, Price R (2009) The pathophysiology of vivax

malaria. Trends Parasitol 25: 220–227.

9. Hemmer C, Holst F, Kern P, Chiwakata C, Dietrich M, et al. (2006) Stronger

host response per parasitized erythrocyte in Plasmodium vivax or ovale than in

Plasmodium falciparum malaria. Trop Med Int Health 11: 817–823.

10. Campos F, Franklin B, Teixeira-Carvalho A, Filho A, de Paula S, et al. (2010)

Augmented plasma microparticles during acute Plasmodium vivax infection.Malar J 9: 327.

11. Karunaweera N, Grau G, Gamage P, Carter R, Mendis K (1992) Dynamics offever and serum levels of tumor necrosis factor are closely associated during

clinical paroxysms in Plasmodium vivax malaria. Proc Natl Acad Sci U S A 89:3200–3203.

12. Andrade B, Reis-Filho A, Souza-Neto S, Clarencio J, Camargo L, et al. (2010)Severe Plasmodium vivax malaria exhibits marked inflammatory imbalance.

Malar J 9: 13.

13. Karunaweera N, Wijesekera S, Wanasekera D, Mendis K, Carter R (2003) Theparoxysm of Plasmodium vivax malaria. Trends Parasitol 19: 188–193.

14. Andrade BB, Reis-Filho A, Souza-Neto SM, Raffaele-Netto I, Camargo LM,et al. (2010) Plasma superoxide dismutase-1 as a surrogate marker of vivax

malaria severity. PLoS Negl Trop Dis 4: e650.

15. Mandel P, Metais P (1948) Les acides nucleiques du plasma sanguine chez

I’homme. C R Acad Sci Paris 142.

16. Leon S, Shapiro B, Sklaroff D, Yaros M (1977) Free DNA in the serum of cancer

patients and the effect of therapy. Cancer Res 37: 646–650.

17. Chen J, Meister S, Urbonaviciute V, Rodel F, Wilhelm S, et al. (2007) Sensitive

detection of plasma/serum DNA in patients with systemic lupus erythematosus.

Autoimmunity 40: 307–310.

18. Koffler D, Agnello V, Winchester R, Kunkel H (1973) The occurrence of single-

stranded DNA in the serum of patients with systemic lupus erythematosus andother diseases. J Clin Invest 52: 198–204.

19. Rani S, Clynes M, O’Driscoll L (2007) Detection of amplifiable mRNAextracellular to insulin-producing cells: potential for predicting beta cell mass

and function. Clin Chem 53: 1936–1944.

20. Lam N, Rainer T, Chan L, Joynt G, Lo Y (2003) Time course of early and late

changes in plasma DNA in trauma patients. Clin Chem 49: 1286–1291.

21. Lam NY, Rainer TH, Wong LK, Lam W, Lo YM (2006) Plasma DNA as a

prognostic marker for stroke patients with negative neuroimaging within the first

24 h of symptom onset. Resuscitation 68: 71–78.

22. Antonatos D, Patsilinakos S, Spanodimos S, Korkonikitas P, Tsigas D (2006)

Cell-free DNA levels as a prognostic marker in acute myocardial infarction.Ann N Y Acad Sci 1075: 278–281.

23. Chang CP, Chia RH, Wu TL, Tsao KC, Sun CF, et al. (2003) Elevated cell-freeserum DNA detected in patients with myocardial infarction. Clin Chim Acta

327: 95–101.

24. Moreira VG, Prieto B, Rodrıguez JS, Alvarez FV (2010) Usefulness of cell-free

plasma DNA, procalcitonin and C-reactive protein as markers of infection infebrile patients. Ann Clin Biochem 47: 253–258.

25. Butt A, Swaminathan R (2008) Overview of circulating nucleic acids in plasma/

serum. Ann N Y Acad Sci 1137: 236–242.

26. Ombrello C, Block RC, Morrell CN (2010) Our expanding view of platelet

functions and its clinical implications. J Cardiovasc Transl Res 3: 538–546.

27. Cox D, McConkey S (2010) The role of platelets in the pathogenesis of cerebral

malaria. Cell Mol Life Sci 67: 557–568.

28. Araujo C, Lacerda M, Abdalla D, Lima E (2008) The role of platelet and plasma

markers of antioxidant status and oxidative stress in thrombocytopenia among

patients with vivax malaria. Mem Inst Oswaldo Cruz 103: 517–521.

29. Ellinger J, Muller S, Stadler T, Jung A, von Ruecker A, et al. (2009) The role of

cell-free circulating DNA in the diagnosis and prognosis of prostate cancer. Urol

Oncol.

30. Sozzi G, Conte D, Mariani L, Lo Vullo S, Roz L, et al. (2001) Analysis of

circulating tumor DNA in plasma at diagnosis and during follow-up of lung

cancer patients. Cancer Res 61: 4675–4678.

31. Umetani N, Kim J, Hiramatsu S, Reber H, Hines O, et al. (2006) Increased

integrity of free circulating DNA in sera of patients with colorectal or

periampullary cancer: direct quantitative PCR for ALU repeats. Clin Chem

52: 1062–1069.

32. Trejo-Becerril C, Perez-Cardenas E, Trevino-Cuevas H, Taja-Chayeb L,

Garcıa-Lopez P, et al. (2003) Circulating nucleosomes and response to

chemotherapy: an in vitro, in vivo and clinical study on cervical cancer patients.

Int J Cancer 104: 663–668.

33. Chang HW, Lee SM, Goodman SN, Singer G, Cho SK, et al. (2002) Assessment

of plasma DNA levels, allelic imbalance, and CA 125 as diagnostic tests for

cancer. J Natl Cancer Inst 94: 1697–1703.

34. Huang ZH, Li LH, Hua D (2006) Quantitative analysis of plasma circulating

DNA at diagnosis and during follow-up of breast cancer patients. Cancer Lett

243: 64–70.

35. Ellinger J, Wittkamp V, Albers P, Perabo F, Mueller S, et al. (2009) Cell-free

circulating DNA: diagnostic value in patients with testicular germ cell cancer.

J Urol 181: 363–371.

36. Ellinger J, Bastian P, Ellinger N, Kahl P, Perabo F, et al. (2008) Apoptotic DNA

fragments in serum of patients with muscle invasive bladder cancer: a prognostic

entity. Cancer Lett 264: 274–280.

37. Jung K, Stephan C, Lewandowski M, Klotzek S, Jung M, et al. (2004) Increased

cell-free DNA in plasma of patients with metastatic spread in prostate cancer.

Cancer Lett 205: 173–180.

38. Jakobsen P, Morris-Jones S, Rønn A, Hviid L, Theander T, et al. (1994)

Increased plasma concentrations of sICAM-1, sVCAM-1 and sELAM-1 in

patients with Plasmodium falciparum or P. vivax malaria and association with

disease severity. Immunology 83: 665–669.

39. Kern P, Hemmer CJ, Van Damme J, Gruss HJ, Dietrich M (1989) Elevated

tumor necrosis factor alpha and interleukin-6 serum levels as markers for

complicated Plasmodium falciparum malaria. Am J Med 87: 139–143.

40. Lo YM, Zhang J, Leung TN, Lau TK, Chang AM, et al. (1999) Rapid clearance

of fetal DNA from maternal plasma. Am J Hum Genet 64: 218–224.

41. Jahr S, Hentze H, Englisch S, Hardt D, Fackelmayer FO, et al. (2001) DNA

fragments in the blood plasma of cancer patients: quantitations and evidence for

their origin from apoptotic and necrotic cells. Cancer Res 61: 1659–1665.

42. Atamaniuk J, Ruzicka K, Stuhlmeier KM, Karimi A, Eigner M, et al. (2006)

Cell-free plasma DNA: a marker for apoptosis during hemodialysis. Clin Chem

52: 523–526.

43. Lovegrove F, Gharib S, Patel S, Hawkes C, Kain K, et al. (2007) Expression

microarray analysis implicates apoptosis and interferon-responsive mechanisms

in susceptibility to experimental cerebral malaria. Am J Pathol 171: 1894–1903.

44. Miu J, Hunt N, Ball H (2008) Predominance of interferon-related responses in

the brain during murine malaria, as identified by microarray analysis. Infect

Immun 76: 1812–1824.

45. van der Vaart M, Pretorius P (2008) Circulating DNA. Its origin and fluctuation.

Ann N Y Acad Sci 1137: 18–26.

46. Shaikh Q, Ahmad S, Abbasi A, Malik S, Sahito A, et al. (2009)

Thrombocytopenia in malaria. J Coll Physicians Surg Pak 19: 708–710.

47. Kochar DK, Tanwar GS, Khatri PC, Kochar SK, Sengar GS, et al. (2010)

Clinical features of children hospitalized with malaria–a study from Bikaner,

northwest India. Am J Trop Med Hyg 83: 981–989.

Circulating Nucleic Acids: Malaria Severity Marker

PLoS ONE | www.plosone.org 8 May 2011 | Volume 6 | Issue 5 | e19842

Page 96: UNIVERSIDADE DO ESTADO DO AMAZONAS FUNDAÇÃO DE … · universidade do estado do amazonas . fundaÇÃo de medicina tropical dr. heitor vieira dourado . programa de pÓs-graduaÇÃo

48. Kochar DK, Das A, Kochar A, Middha S, Acharya J, et al. (2010)

Thrombocytopenia in Plasmodium falciparum, Plasmodium vivax and mixed

infection malaria: a study from Bikaner (Northwestern India). Platelets 21:

623–627.

49. Anker P, Stroun M (2002) Progress in the knowledge of circulating nucleic acids:

plasma RNA is particle-associated. Can it become a general detection marker for

a cancer blood test? Clin Chem 48: 1210–1211.

50. WHO (2000) Severe falciparum malaria. 90 p.

51. Kono H, Rock KL (2008) How dying cells alert the immune system to danger.

Nat Rev Immunol 8: 279–289.

52. McDonald B, Pittman K, Menezes GB, Hirota SA, Slaba I, et al. (2010)

Intravascular danger signals guide neutrophils to sites of sterile inflammation.

Science 330: 362–366.

53. Hornung V, Latz E (2010) Intracellular DNA recognition. Nat Rev Immunol 10:

123–130.

54. Saraiva M, Amorim R, Moura M, Martinez-Espinosa F, Barbosa M (2009)

[Urban expansion and spatial distribution of malaria in the municipality ofManaus, State of Amazonas]. Rev Soc Bras Med Trop 42: 515–522.

55. Oliveira-Ferreira J, Lacerda M, Brasil P, Ladislau J, Tauil P, et al. (2010)

Malaria in Brazil: an overview. Malar J 9: 115.56. Karunaweera N, Carter R, Grau G, Mendis K (1998) Demonstration of anti-

disease immunity to Plasmodium vivax malaria in Sri Lanka using a quantitativemethod to assess clinical disease. Am J Trop Med Hyg 58: 204–210.

57. Souza-Silva FA, da Silva-Nunes M, Sanchez BA, Ceravolo IP, Malafronte RS,

et al. (2010) Naturally acquired antibodies to Plasmodium vivax Duffy bindingprotein (DBP) in Brazilian Amazon. Am J Trop Med Hyg 82: 185–193.

58. Sozzi G, Conte D, Leon M, Ciricione R, Roz L, et al. (2003) Quantification offree circulating DNA as a diagnostic marker in lung cancer. J Clin Oncol 21:

3902–3908.59. Carlton J, Adams J, Silva J, Bidwell S, Lorenzi H, et al. (2008) Comparative

genomics of the neglected human malaria parasite Plasmodium vivax. Nature

455: 757–763.

Circulating Nucleic Acids: Malaria Severity Marker

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52

online | memorias.ioc.fiocruz.br

Mem Inst Oswaldo Cruz, Rio de Janeiro, Vol. 106(Suppl. I): 52-63, 2011

Malaria affects almost all blood components and is a true haematological infectious disease. Anaemia and thrombocytopenia are the most frequent malaria-associ-ated haematological complications (Wickramasinghe & Abdalla 2000) and have received more attention in the sci-entific literature due to their associated mortality. On the other hand, thrombocytopenia is less studied, causes neg-ligible 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 in-crease 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 thrombocy-topenia in acute febrile travellers returning from tropi-cal 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 fe-brile patients (Lathia & Joshi 2004). The sensitivity of thrombocytopenia together with the acute febrile syn-drome 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).

Thrombocytopenia is a well-documented and fre-quent 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 ex-tremities (Hill et al. 1964).

Since the beginning of the 1970s, there have been reports proposing that malaria-associated thrombocy-topenia is quite similar in P. vivax and Plasmodium fal-ciparum 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 Stud-ies 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 Ja-neiro. 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 pri-vate 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.

Financial support: CAPES (scholarship for HCCC), CNPq (MVGL is a level 2 research productivity fellow), ASH+ Corresponding author: [email protected] 1 April 2011Accepted 26 May 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

1Fundação de Medicina Tropical Dr. Heitor Vieira Dourado, Av. Pedro Teixeira 25, 69040-000 Manaus, AM, Brasil 2Universidade do Estado do Amazonas, Manaus, AM, Brasil 3Universidade Nilton Lins, Manaus, AM, Brasil

4Universidade de Brasília, Brasília, DF, Brasil

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

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Malaria and thrombocytopenia • Marcus Vinícius Guimarães Lacerda et al. 53

Data on the real burden of thrombocytopenia associ-ated with malaria is contradictory in the literature and it is not usually considered when conducting patient se-lection. 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 ma-chines 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 cal-culation 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 se-lection 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 re-ports and most of these are due to P. vivax (Table II).

In 2005, 138 of 684 (20.1%) malarial cases hospital-ised in a tertiary care centre in Manaus had thrombocy-topenia as the cause of admission, which corresponded to 6.8% of hospitalisations due to all causes in this refer-ence institution (unpublished observations). Hospitalisa-tion, 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 - Co-agulation disturbances - A study based on 31 American soldiers in Vietnam with chloroquine-resistant falci-parum 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 pre-sented 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 associ-ated with C3 (Srichaikul et al. 1975). In Manaus, 2004, a study with falciparum and vivax patients demonstrated a negative correlation between platelet counts, thrombin-anti-thrombin complex and D-dimers, suggesting that the activation of coagulation could be partially respon-sible for thrombocytopenia (Marques et al. 2005).

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 phagocyto-sis of parasitised red blood cells (RBCs) (Engwerda et al. 2005). Some data suggested that platelets were seques-tered in the spleen during the acute infection (Skudowitz et al. 1973). In the experimental model with Plasmodium chabaudi, thrombocytopenia was absent in splenec-tomised mice, showing that the spleen was essential for thrombocytopenia (Watier et al. 1992). The term hyper-splenism 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 destruc-tion 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 be-lieved. 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 mac-rophages play a role in the destruction of these particles (Lee et al. 1997). In the comparison of spleens from pa-tients 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 (Ur-ban 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 ma-laria, 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 pro-teins to escape from macrophage destruction in this or-gan. 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 evi-denced that there was higher parasite accumulation, re-duced motility, loss of directionality, increased residence time and altered magnetic resonance only in the spleens of mice infected with the non-lethal 17X strain (Martin-Jaular 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 throm-bocytopenia could be the result of invasion of these par-ticles 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 circu-lation (Fajardo & Tallent 1974). However, this observa-

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TABLE ISystematic review of studies, estimating thrombocytopenia in malarial patients (1997-2011)

References Study site Type of patients Age range Species nThrombocytopenia % [criterion (mm3)]

Mohanty et al. (1997) India Inpatients and outpatients All ages P.v. 24 29 (< 150,000)P.f. 76 39 (< 150,000)

Noronha (1998) Brazil Inpatients and outpatients < 14 y P.f. 54 51.8 (< 150,000)Kortepeter and Brown (1998) USA Inpatients and outpatients > 18 y P.f./P.v. 79 74 (< 150,000)Murthy et al. (2000) India Inpatients 10-80 y P.f. 158 40.5 (< 150,000)Gonzalez et al. (2000) Colombia Inpatients All ages P.f. 113 33.6 (< 150,000)

P.v. 128 39 (< 150.000)Alecrim (2000) Brazil Inpatients > 12 y P.v. 73 91.8 (< 150,000)

Outpatients 319 60.8 (< 150,000)

Silva et al. (2000) Brazil Inpatients All ages P.v. 429 46.6 (< 140,000)Oh et al. (2001) South Korea Inpatients and outpatients > 17 y P.v. 101 85.1 (< 150,000)Robinson et al. (2001) Australia Inpatients NA P.f./P.v./P.o. 246 71 (< 150,000)Mourão et al. (2001) Brazil Inpatients < 12 y P.f. 255 73.7 (< 150,000)Lacerda et al. (2001) Brazil Inpatients > 12 y P.f. 218 87.6 (< 150,000)Ladhani et al. (2002) Kenya Inpatients Children P.f. 1,369 56.7 (< 150,000)Park et al. (2002) Brazil Inpatients All ages P.v. 237 61.6 (NA)Mohapatra et al. (2002) India Inpatients and outpatients 15-60 y P.v. 110 3.6 (< 100,000)Bashawri et al. (2002) Saudi Arabia Inpatients and outpatients 2 m-74 y P.v./P.f. 727 55.6 (< 150,000)Araújo Filho et al. (2003) Brazil Inpatients and outpatients 4-64 y P.v. 68 20.6 (< 50,000)Echeverri et al. (2003) Colombia Outpatients All ages P.v. 104 8 (< 130,000)Jadhav et al. (2004) India Inpatients and outpatients All ages P.v. 973 65 (50,000-150,000)

P.f. 590 50 (50,000-150,000)Marques (2004) Brazil Inpatients and outpatients > 15 y P.f. 44 79 (< 150,000)

P.v. 106 94 (< 150,000)Rodriguez-Morales et al. (2005) Venezuela NA NA P.v. 116 87.6 (< 150,000)Rodriguez-Morales et al. (2006) Venezuela Inpatients < 12 y P.v. 78 58.9 (< 150,000)Casals-Pascual et al. (2006) Kenya Inpatients and outpatients 6 m-10 y P.f. 120 34.4 (< 150,000)Kumar and Shashirekha (2006) India Inpatients and outpatients All ages P.v. 27 88.8 (< 150,000)Lacerda (2007) Brazil Outpatients > 18 y P.v. 142 71.8 (< 150,000)

P.f. 26 65.4 (< 150,000)Koltas et al. (2007) Turkey Outpatients All ages P.v. 90 NATaylor et al. (2008) Indonesia Outpatients All ages P.v./P.f. 151 78.8 (< 150,000)Tan et al. (2008) Thailand Inpatients and outpatients Pregnant

womenP.v. 523 22 (< 75,000)P.f. 694 34 (< 75,000)

Silva (2009) Brazil Outpatients All ages P.v. 397 77.1 (< 150,000)Rasheed et al. (2009) Pakistan Inpatients All ages P.v./P.f 502 80 (< 150,000)Shaikh et al. (2009) Pakistan Outpatients All ages P.v./P.f. 124 82.5 (< 150,000)Prasad et al. (2009) India Inpatients < 5 y P.f. 40 85 (< 150,000)Gonzalez et al. (2009) Venezuela Outpatients 3-67 P.v. 59 55.9 (< 150,000)Poespoprodjo et al. (2009) Indonesia Inpatients 0-3 m P.v./P.f. and mixed 179 61.3 (< 100,000)Khan et al. (2009) Qatar Outpatients All ages P.v. 81 63 (< 150,000)Maina et al. (2010) Kenya Outpatients < 5 y P.f. 523 49 (< 150,000)Kochar et al. (2010) India Inpatients and outpatients All ages P.v./P.f. and mixed 1,064 24.6 (< 150,000)George and Alexander (2010) India Inpatients 18-66 y P.v. 30 93.3 (< 150,000)Srivastava et al. (2011) India Inpatients All ages P.v. 50 82 (< 150,000)

m: months; NA: non-available; P.f.: Plasmodium falciparum; P.o.: Plasmodium ovale; P.v.: Plasmodium vivax; y: years.

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Malaria and thrombocytopenia • Marcus Vinícius Guimarães Lacerda et al. 55

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 (Menen-dez 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 cor-relation 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 periph-ery. Similar results were shown in children with falci-parum 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 ev-idence 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 ac-tivation 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

TABLE IICollated case reports of Plasmodium vivax-associated thrombocytopenia (1964-2011)

References Study sitePlatelet count(x 1,000/mm3) Bleeding

Platelettransfusion Observation

Hill et al. (1964) United States of America 20-49 Petecchiae No Experimental infectionTakaki et al. (1991) Solomon Islands NA NA NA DICAnstey et al. (1992) Bali 22 No No -Ohtaka et al. (1993) NA NA NA NA PAIgG increaseYamaguchi et al. (1997) Thailand and Sri Lanka 22-53 No No PAIgG increaseVictoria et al. (1998) Brazil 1 Several Yes ITPKakar et al. (1999) India 5 No No -Makkar et al. (2002) India 8 Gingival bleeding Yes -Holland et al. (2004) Mexico 19 Epistaxis Yes -Lacerda et al. (2004) Brazil 1 Gingival bleeding Yes ITPAggarwal et al. (2005) India 6 Petecchiae Yes -Katira and Shah (2006) India 14-92 No Yes -Komoda et al. (2006) South America 15 NA NA -Kaur et al. (2007) India 30 No No Acute renal failureSong et al. (2007) South Korea 25-20 No No DIC, lung edema, acute

renal failure and shockKaur et al. (2007) India 30 Petecchiae No Acute renal failureLacerda et al. (2008) Brazil 6 No No Chronic splenomegalyVij et al. (2008) India NA Gingival bleeding No NARifakis et al. (2008) Venezuela 57 No No Hydronephrosis and shockParakh et al. (2009) India 5-42 Petecchiae No Cerebral malaria, shock

and acute renal failureThapa et al. (2009) India 11 Petecchiae and

mucosal bleedingYes Hepatitis and shock

Harish and Gupta (2009) India 1 Intracranial bleed No SeizuresBhatia and Bhatia (2010) India NA Yes NA NA

DIC: disseminated intravascular coagulation; ITP: immune thrombocytopenic purpura; NA: non-available; PAIgG: platelet-associated IgG.

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56 Mem Inst Oswaldo Cruz, Rio de Janeiro, Vol. 106(Suppl. I), 2011

and is frequently seen in most viral, bacterial and para-sitic diseases without any repercussion (Daniel-Ribeiro & Zanini 2000). Molecular mimicry, however, provides evolutionary advantage for microorganisms that escape immune aggression (Daniel-Ribeiro 2000). The rela-tionship between malaria and auto-immunity has been discussed in the literature and the first epidemiological association was made based on the presence of fewer au-to-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 cell-rich 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 propor-tional to the amount of available antigen, the negative correlation between platelet count and peripheral para-sitaemia reported in many studies (Lacerda 2007, Sil-va 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 associ-ated with normal platelet counts in P. vivax malaria in-dependent 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 impor-tant 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 correla-tion between platelet count and platelet lipid peroxida-tion in addition to the positive correlation between plate-let 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 correla-tion between platelet count and nitrogen reactive inter-mediates (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 diphos-phate (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 (In-yang et al. 1987). Even electron microscopic examination of non-stimulated, fresh platelets from malarial patients show centralisation of dense granules, glycogen deple-tion and microaggregates and phylopoids as a sign of in vivo activation, which could be responsible for a pseudo-thrombocytopenia due to sequestration of these activated particles in the interior of the vessels (Mohanty et al. 1988). Contradictory data were presented showing ag-gregation impairment in severe falciparum patients after ADP addition in vitro (Srichaikul et al. 1988). P. falci-parum 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 coagulopa-thy, the increase in soluble glycoprotein-1b (GP1b) con-centrations results from vWF-mediated GP1b shedding, a process that may prevent excessive adhesion of platelets and parasitised erythrocytes (Mast et al. 2010). Antima-larial 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 se-vere 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 monoin-fection, 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 thrombo-cytopenia is evident. However, that can be due to bias publication, where prospective studies would be needed to validate this association. On the other hand, consider-ing 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 throm-bocytopenia 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

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Malaria and thrombocytopenia • Marcus Vinícius Guimarães Lacerda et al. 57

frequently reported, resistant parasites are also being si-multaneously 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 plasmo-dial infection, platelet counts were never performed in as-ymptomatic 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 hae-mostasis, 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 non-stimulated 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). Plate-lets 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 pro-duction 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 show-ing 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 fal-ciparum 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 ma-laria, an inverse correlation between platelet count and plasmatic IL-10 was seen (Casals-Pascual et al. 2006). This interpretation is not straightforward, because IL-10 is generally associated with protection against severe disease. The authors hypothesise, though, that IL-10 could reduce platelet counts to avoid infected-RBC ad-hesion to the endothelium, as if thrombocytopenia was a mechanism of defence against severe disease and not

the cause. Studies of vivax infection have shown throm-bocytopenia 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 ac-tivation or apoptosis) has yet to be determined in vivo. There is evidence that these MPs participate in the en-dothelial activation responsible for severe cerebral ma-laria in murine models (Combes et al. 2006). MPs were also associated with coma and thrombocytopenia in se-vere 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 (Cam-pos 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 con-firmed efficacy. The indication of prophylactic platelet transfusion when platelet counts are under 10,000/mm3 probably applies only when the bone marrow is compro-mised and is not able to release efficacious platelets (Re-bulla 2000). This does not seem to be the case in malar-ia. 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 mi-nor bleeding despite having non-severe thrombocytope-nia; 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 diag-nosed, the formal clinical indication persists, as recom-mended elsewhere (Franchini 2005). Due to the impossi-bility of using frozen platelets in routine clinical practice, other platelet substitutes and preparations are being in-vestigated (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, prob-ably due to the fact that the recovery of thrombocytope-nia 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-

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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 exacer-bated the neurological symptoms and increased the fre-quency 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 adju-vant antimalarial therapy (Muniz-Junqueira et al. 2005, Mohanty et al. 2006), based on the drug-induced inhibi-tion of adhesion molecules in RBCs and platelets (Mu-niz-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 ac-quired immunodeficiency syndrome (Scaradavou 2002). The first cases of homosexuals with profound thrombo-cytopenia 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 chron-ic 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, anti-platelet 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). Further-more, during infection with any of the four dengue vi-ruses, 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 dis-ease (Honda et al. 2009). Thrombocytopenia is also ob-served 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 infec-tious diseases, as a rule, changes the paradigm that plate-lets are essential only to haemostasis, supporting their role as important contributors to modulate the immune response. In any case, studies focusing on the pathogen-esis of thrombocytopenia in malarial patients should

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 pathogen-esis are highlighted, but further studies are still needed to clarify the impact of each mechanism and its clinical rel-evance. The clinical management of malarial thrombocy-topenia 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 re-sponse to the parasite present or whether platelets are ac-tually involved in the generation of severe disease.

ACKNOWLEDGEMENTS

To Alex Kumar, for critical and linguistic review of the man-uscript, and to Mary Galinski, for inspiring the title. This review is dedicated to Simon Karpatkin and Vanize Oliveira Macêdo.

REFERENCES

Aggarwal A, Rath S, Shashiraj 2005. Plasmodium vivax malaria pre-senting with severe thrombocytopenia. J Trop Pediatr 51: 120-121.

Alecrim MGC 2000. Clinical aspects, resistance and parasitary poly-morphism of Plasmodium vivax malaria in Manaus, PhD Thesis, Universidade de Brasília, Brasília, 176 pp.

Alexandre MA, Ferreira CO, Siqueira AM, Magalhaes BL, Mourao MPG, Lacerda MVG, Alecrim MGC 2010. Severe Plasmodium vivax malaria, Brazilian Amazon. Emerg Infect Dis 16: 1611-1614.

Almeida AJ 2003. Trombocitopenia associada ao HCV: aspectos clíni-co-laboratoriais e virológicos, MD Thesis, Fundação Oswaldo Cruz, Rio de Janeiro, 110 pp.

Andrade BB, Reis-Filho A, Souza-Neto SM, Clarencio J, Camargo LM, Barral A, Barral-Netto M 2010. Severe Plasmodium vivax malaria exhibits marked inflammatory imbalance. Malar J 9: 13.

Major mechanisms associated to malaria-triggered thrombocytope-nia and the possible relationship with severe disease.

Page 104: UNIVERSIDADE DO ESTADO DO AMAZONAS FUNDAÇÃO DE … · universidade do estado do amazonas . fundaÇÃo de medicina tropical dr. heitor vieira dourado . programa de pÓs-graduaÇÃo

Malaria and thrombocytopenia • Marcus Vinícius Guimarães Lacerda et al. 59

Anstey NM, Currie BJ, Dyer ME 1992. Profound thrombocytopenia due to Plasmodium vivax malaria. Aust N Z J Med 22: 169-170.

Araujo CF, Lacerda MV, Abdalla DS, Lima ES 2008. The role of platelet and plasma markers of antioxidant status and oxidative stress in thrombocytopenia among patients with vivax malaria. Mem Inst Oswaldo Cruz 103: 517-521.

Araújo Filho JA, Bressan FR, Tourinho TM, Souza MVL, Pereira LI 2003. Plaquetopenia acentuada associada à malária pelo P. vivax. Anais do XIII Congresso Brasileiro de Infectologia, Goiânia. Braz J Infect Dis 7 (Suppl. 1): 25.

Bashawri LA, Mandil AA, Bahnassy AA, Ahmed MA 2002. Malaria: hematological aspects. Ann Saudi Med 22: 372-376.

Batista D 1946. O paludismo na Amazônia: contribuição à epidemiolo-gia, à protozoologia e à clínica; estudo sôbre a febre biliosa-hemo-globinúrica; síntese, Imprensa Nacional, Rio de Janeiro, 212 pp.

Beale PJ, Cormack JD, Oldrey TB 1972. Thrombocytopenia in ma-laria with immunoglobulin (IgM) changes. BMJ 1: 345-349.

Becchi C, Al Malyan M, Fabbri LP, Marsili M, Boddi V, Boncinelli S 2006. Mean platelet volume trend in sepsis: is it a useful param-eter? Minerva Anestesiol 72: 749-756.

Bhatia V, Bhatia J 2010. Severe thrombocytopenia with bleeding manifestations in two children secondary to Plasmodium vivax. Platelets 21: 307-309.

Blajchman MA 2003. Substitutes and alternatives to platelet transfusions in thrombocytopenic patients. J Thromb Haemost 1: 1637-1641.

Brown KM, Kreier JP 1982. Plasmodium berghei malaria: blockage by immune complexes of macrophage receptors for opsonized plasmodia. Infect Immun 37: 1227-1233.

Campos FM, Franklin BS, Teixeira-Carvalho A, Filho AL, de Paula SC, Fontes CJ, Brito CF, Carvalho LH 2010. Augmented plasma microparticles during acute Plasmodium vivax infection. Malar J 9: 327.

Carvalho BO, Lopes SC, Nogueira PA, Orlandi PP, Bargieri DY, Blanco YC, Mamoni R, Leite JA, Rodrigues MM, Soares IS, O- liveira TR, Wunderlich G, Lacerda MV, Del Portillo HA, Araujo MO, Russell B, Suwanarusk R, Snounou G, Renia L, Costa FT 2010. On the cytoadhesion of Plasmodium vivax-infected eryth-rocytes. J Infect Dis 202: 638-647.

Casals-Pascual C, Kai O, Newton CR, Peshu N, Roberts DJ 2006. Thrombocytopenia in falciparum malaria is associated with high concentrations of IL-10. Am J Trop Med Hyg 75: 434-436.

Chagas C 1903. Hematological studies of impaludism, MD Thesis, Manguinhos Institute, Rio de Janeiro, 143 pp.

Chotivanich K, Sritabal J, Udomsangpetch R, Newton P, Stepniewska KA, Ruangveerayuth R, Looareesuwan S, Roberts DJ, White NJ 2004. Platelet-induced autoagglutination of Plasmodium falci-parum-infected red blood cells and disease severity in Thailand. J Infect Dis 189: 1052-1055.

Combes V, Coltel N, Faille D, Wassmer SC, Grau GE 2006. Cerebral malaria: role of microparticles and platelets in alterations of the blood-brain barrier. Int J Parasitol 36: 541-546.

Conte R, Tassi C, Belletti D, Ricci F, Tazzari PL 2003. Autoimmune thrombocytopenia in malaria. Vox Sang 85: 221.

Cummins D, Faint R, Yardumian DA, Dawling S, Mackie I, Machin SJ 1990. The in vitro and ex vivo effects of chloroquine sulphate on platelet function: implications for malaria prophylaxis in pa-tients with impaired haemostasis. J Trop Med Hyg 93: 112-115.

D’Acremont V, Landry P, Mueller I, Pecoud A, Genton B 2002. Clini-cal and laboratory predictors of imported malaria in an outpatient setting: an aid to medical decision making in returning travelers with fever. Am J Trop Med Hyg 66: 481-486.

Daniel-Ribeiro CT 2000. Is there a role for autoimmunity in immune protection against malaria? Mem Inst Oswaldo Cruz 95: 199-207.

Daniel-Ribeiro CT, Zanini G 2000. Autoimmunity and malaria: what are they doing together? Acta Trop 76: 205-221.

Davidson RA 1985. Immunology of parasitic infections. Med Clin North Am 69: 751-758.

de Paula LV, Klafke A, Bordin R, Pereira JPM, Job FM 1993. Avalia-ção da eficácia das transfusões de concentrados de plaquetas nos Serviços de Hematologia e Hemoterapia do Hospital de Clínicas de Porto Alegre. Bol Soc Bras Hematol Hemoter 15: 9-13.

del Portillo HA, Lanzer M, Rodriguez-Malaga S, Zavala F, Fernan-dez-Becerra C 2004. Variant genes and the spleen in Plasmodium vivax malaria. Int J Parasitol 34: 1547-1554.

Dennis LH, Eichelberger JW, Inman MM, Conrad ME 1967. Deple-tion of coagulation factors in drug-resistant Plasmodium falci-parum malaria. Blood 29: 713-721.

Doi T, Homma H, Mezawa S, Kato J, Kogawa K, Sakamaki S, Niitsu Y 2002. Mechanisms for increment of platelet associated IgG and platelet surface IgG and their implications in immune thrombocytopenia associated with chronic viral liver disease. Hepatol Res 24: 23.

Echeverri M, Tobon A, Alvarez G, Carmona J, Blair S 2003. Clinical and laboratory findings of Plasmodium vivax malaria in Colom-bia 2001. Rev Inst Med Trop Sao Paulo 45: 29-34.

Engwerda CR, Beattie L, Amante FH 2005. The importance of the spleen in malaria. Trends Parasitol 21: 75-80.

Erel O, Kocyigit A, Bulut V, Avci S, Aktepe N 1998. Role of lipids, lipoproteins and lipid peroxidation in thrombocytopenia in pa-tients with vivax malaria. Haematologia (Budap) 29: 207-212.

Erel O, Vural H, Aksoy N, Aslan G, Ulukanligil M 2001. Oxidative stress of platelets and thrombocytopenia in patients with vivax malaria. Clin Biochem 34: 341-344.

Essien EM, Ebhota MI 1981. Platelet hypersensitivity in acute ma-laria (Plasmodium falciparum) infection in man. Thromb Hae-most 46: 547-549.

Fajardo LF, Tallent C 1974. Malarial parasites within human platelets. J Am Med Assoc 229: 1205-1207.

Franchini M 2005. Pathophysiology, diagnosis and treatment of dissem-inated intravascular coagulation: an update. Clin Lab 51: 633-639.

George P, Alexander LM 2010. A study on the clinical profile of com-plicated Plasmodium vivax mono-infections. Asian Pac J Trop Med 3: 560-562.

Gerardin P, Rogier C, Ka AS, Jouvencel P, Brousse V, Imbert P 2002. Prognostic value of thrombocytopenia in African children with falciparum malaria. Am J Trop Med Hyg 66: 686-691.

Gonzalez B, Rodulfo H, De Donato M, Berrizbeitia M, Gomez C, Gonzalez L 2009. Hematologic variations in patient with malaria caused by Plasmodium vivax before, during and after treatment. Invest Clin 50: 187-201.

Gonzalez LM, Guzman M, Carmona J, Lopera T, Blair S 2000. Clinical and epidemiologic characteristics of 291 hospitalized patients for malaria in Medellin (Colombia). Acta Med Colomb 25: 163-170.

Grau GE, Piguet PF, Gretener D, Vesin C, Lambert PH 1988. Immu-nopathology of thrombocytopenia in experimental malaria. Im-munology 65: 501-506.

Greenwood BM 1968. Autoimmune disease and parasitic infections in Nigerians. Lancet 2: 380-382.

Grynberg P, Fernandes Fontes CJ, Braga EM 2007. Association be-tween particular polymorphic residues on apical membrane anti-

Page 105: UNIVERSIDADE DO ESTADO DO AMAZONAS FUNDAÇÃO DE … · universidade do estado do amazonas . fundaÇÃo de medicina tropical dr. heitor vieira dourado . programa de pÓs-graduaÇÃo

60 Mem Inst Oswaldo Cruz, Rio de Janeiro, Vol. 106(Suppl. I), 2011

gen 1 (AMA-1) and platelet levels in patients with vivax malaria. Clin Microbiol Infect 13: 1089-1094.

Harish R, Gupta S 2009. Plasmodium vivax malaria presenting with severe thrombocytopenia, cerebral complications and hydro-cephalus. Indian J Pediatr 76: 551-552.

Hill GJ, Knight V, Jeffery GM 1964. Thrombocytopenia in vivax ma-laria. Lancet 39: 240-241.

Hoffman SL, Rustama D, Punjabi NH, Surampaet B, Sanjaya B, Dim-pudus AJ, McKee KT, Jr., Paleologo FP, Campbell JR, Marwoto H 1988. High-dose dexamethasone in quinine-treated patients with cerebral malaria: a double-blind, placebo-controlled trial. J Infect Dis 158: 325-331.

Holland BH, Walker AN, Collier L, Stephens JL 2004. Severe throm-bocytopenia and epistaxis secondary to Plasmodium vivax in-fection. Internet J Infect Dis 3. Available from: ispub.com/ostia/index.php?xmlFilePath=journals/ijid/vol3n2/vivax.xml.

Honda S, Saito M, Dimaano EM, Morales PA, Alonzo MT, Suarez LA, Koike N, Inoue S, Kumatori A, Matias RR, Natividad FF, Oishi K 2009. Increased phagocytosis of platelets from patients with secondary dengue virus infection by human macrophages. Am J Trop Med Hyg 80: 841-845.

Huang DB, DuPont HL 2005. Problem pathogens: extra-intestinal complications of Salmonella enterica serotype typhi infection. Lancet Infect Dis 5: 341-348.

Inyang AL, Sodeinde O, Okpako DT, Essien EM 1987. Platelet re-actions after interaction with cultured Plasmodium falciparum infected erythrocytes. Br J Haematol 66: 375-378.

Jadhav UM, Patkar VS, Kadam NN 2004. Thrombocytopenia in ma-laria - correlation with type and severity of malaria. J Assoc Phy-sicians India 52: 615-618.

Jaff MS, McKenna D, McCann SR 1985. Platelet phagocytosis: a probable mechanism of thrombocytopenia in Plasmodium falci-parum infection. J Clin Pathol 38: 1318-1319.

Kakar A, Bhoi S, Prakash V, Kakar S 1999. Profound thrombocy-topenia in Plasmodium vivax malaria. Diagn Microbiol Infect Dis 35: 243-244.

Karpatkin S 2002. HIV-1 related thrombocytopenia. In AD Michel-son, Platelets, Elsevier Academic Press, California, p. 559-570.

Karpatkin S, Nardi MA, Hymes KB 1995. Sequestration of anti-platelet GPIIIa antibody in rheumatoid factor immune complexes of human immunodeficiency virus 1 thrombocytopenic patients. Proc Natl Acad Sci USA 92: 2263-2267.

Katira B, Shah I 2006. Thrombocytopenia in Plasmodium vivax in-fected children. J Vector Borne Dis 43: 147-149.

Kaur D, Wasir V, Gulati S, Bagga A 2007. Unusual presentation of Plasmodium vivax malaria with severe thrombocytopenia and acute renal failure. J Trop Pediatr 53: 210-212.

Kelton JG, Keystone J, Moore J, Denomme G, Tozman E, Glynn M, Neame PB, Gauldie J, Jensen J 1983. Immune-mediated thrombo-cytopenia of malaria. J Clin Invest 71: 832-836.

Khan FY, Lutof AK, Yassin MA, Khattab MA, Saleh M, Rezeq HY, Almaslamani M 2009. Imported malaria in Qatar: a one year hos-pital-based study in 2005. Travel Med Infect Dis 7: 111-117.

Kochar DK, Das A, Kochar A, Middha S, Acharya J, Tanwar GS, Gupta A, Pakalapati D, Garg S, Saxena V, Subudhi AK, Boopathi PA, Sirohi P, Kochar SK 2010. Thrombocytopenia in Plasmodi-um falciparum, Plasmodium vivax and mixed infection malaria: a study from Bikaner (Northwestern India). Platelets 21: 623-627.

Kochar DK, Saxena V, Singh N, Kochar SK, Kumar SV, Das A 2005. Plasmodium vivax malaria. Emerg Infect Dis 11: 132-134.

Koltas IS, Demirhindi H, Hazar S, Ozcan K 2007. Supportive pre-sumptive diagnosis of Plasmodium vivax malaria. Thrombocy-topenia and red cell distribution width. Saudi Med J 28: 535-539.

Komoda M, Fujimoto T, Kawaguchi Y, Tsushima H, Fukushima T, Hata T, Miyazaki Y, Tsukasaki K, Tomonaga M 2006. Plasmo-dium vivax malaria with clinical presentation mimicking acute type idiopathic thrombocytopenic purpura (abstract). Rinsho Ketsueki 47: 1453-1456.

Kortepeter M, Brown JD 1998. A review of 79 patients with malaria seen at a military hospital in Hawaii from 1979 to 1995. Mil Med 163: 84-89.

Kreil A, Wenisch C, Brittenham G, Looareesuwan S, Peck-Radosav-ljevic M 2000. Thrombopoietin in Plasmodium falciparum ma-laria. Br J Haematol 109: 534-536.

Kumar A, Shashirekha 2006. Thrombocytopenia: an indicator of acute vivax malaria. Indian J Pathol Microbiol 49: 505-508.

Lacerda MV, Alexandre MA, Santos PD, Arcanjo AR, Alecrim WD, Alecrim MGC 2004. Idiopathic thrombocytopenic purpura due to vivax malaria in the Brazilian Amazon. Acta Trop 90: 187-190.

Lacerda MV, Hipolito JR, Passos LN 2008. Chronic Plasmodium vivax infection in a patient with splenomegaly and severe throm-bocytopenia. Rev Soc Bras Med Trop 41: 522-523.

Lacerda MVG 2007. Manifestações clínicas e patogênese da plaque-topenia na malária, PhD Thesis, Universidade de Brasília, 439 pp.

Lacerda MVG, Cavalcante TB, Silva AC, Macêdo VO 2006. Avalia-ção das indicações de transfusão de concentrado de plaquetas. Anais do XLII Congresso da Sociedade Brasileira de Medicina Tropical, Teresina. Rev Soc Bras Med Trop 39 (Suppl. I): 104.

Lacerda MVG, Mourão MPG, Alecrim WD, Alecrim MGC 2001. Clinical study of patients with falciparum malaria admitted to the Tropical Medicine Foundation of Amazonas - Brazil. Annals of the 50th Annual Meeting of the American Society of Tropical Medi-cine and Hygiene, Atlanta (USA). Am J Trop Med Hyg 65: 336.

Lacerda MVG, Oliveira SL, Alecrim MGC 2007. Splenic hematoma in a patient with Plasmodium vivax malaria. Rev Soc Bras Med Trop 40: 96-97.

Ladhani S, Lowe B, Cole AO, Kowuondo K, Newton CR 2002. Changes in white blood cells and platelets in children with falciparum ma-laria: relationship to disease outcome. Br J Haematol 119: 839-847.

Lathia TB, Joshi R 2004. Can hematological parameters discriminate malaria from nonmalarious acute febrile illness in the tropics? Indian J Med Sci 58: 239-244.

Lee SH, Looareesuwan S, Chan J, Wilairatana P, Vanijanonta S, Chong SM, Chong BH 1997. Plasma macrophage colony-stimu-lating factor and P-selectin levels in malaria-associated thrombo-cytopenia. Thromb Haemost 77: 289-293.

Li Z, Nardi MA, Karpatkin S 2005. Role of molecular mimicry to HIV-1 peptides in HIV-1-related immunologic thrombocytope-nia. Blood 106: 572-576.

Llanos C, Quintero G, Castellanos A, Arévalo-Herrera M, Herrera S 2006. Surgical bone marrow aspiration in Aotus lemurinus gri-seimembra. J Med Primatol 35: 131-135.

Lou J, Donati YR, Juillard P, Giroud C, Vesin C, Mili N, Grau GE 1997. Platelets play an important role in TNF-induced microvas-cular endothelial cell pathology. Am J Pathol 151: 1397-1405.

Maina RN, Walsh D, Gaddy C, Hongo G, Waitumbi J, Otieno L, Jones D, Ogutu BR 2010. Impact of Plasmodium falciparum infection on haematological parameters in children living in Western Ke-nya. Malar J 9 (Suppl. 3): S4.

Page 106: UNIVERSIDADE DO ESTADO DO AMAZONAS FUNDAÇÃO DE … · universidade do estado do amazonas . fundaÇÃo de medicina tropical dr. heitor vieira dourado . programa de pÓs-graduaÇÃo

Malaria and thrombocytopenia • Marcus Vinícius Guimarães Lacerda et al. 61

Makkar RP, Mukhopadhyay S, Monga A, Gupta AK 2002. Plasmo-dium vivax malaria presenting with severe thrombocytopenia. Braz J Infect Dis 6: 263-265.

Marques HO 2004. Alterações da hemostasia em pacientes com malária, MSc Thesis, Universidade Federal de São Paulo, São Paulo, 140 pp.

Marques HO, Alexandre MAA, Oliveira VM, Marreira L, Lacerda MVG, Alecrim MGC, Morelli VM, Lourenço DM 2005. Hemo-static changes in patients with malaria. Annals of the XX Con-gress of the International Society on Thrombosis and Hemosta-sis, Sydney (Australia). J Thromb Haemost 3 (Suppl. I): 1452.

Martin-Jaular L, Ferrer M, Calvo M, Rosanas-Urgell A, Kalko S, Graewe S, Soria G, Cortadellas N, Ordi J, Planas A, Burns J, Heussler V, Del Portillo HA 2011. Strain-specific spleen remodelling in Plasmodi-um yoelii infections in Balb/c mice facilitates adherence and spleen macrophage-clearance escape. Cell Microbiol 13: 109-122.

Mast Q, de Groot PG, van Heerde WL, Roestenberg M, van Velzen JF, Verbruggen B, Roest M, McCall M, Nieman AE, Westendorp J, Syafruddin D, Fijnheer R, van Dongen-Lases EC, Sauerwein RW, van der Ven AJ 2010. Thrombocytopenia in early malaria is asso-ciated with GP1b shedding in absence of systemic platelet activa-tion and consumptive coagulopathy. Br J Haematol 151: 495-503.

Mast Q, Groot E, Lenting PJ, de Groot PG, McCall M, Sauerwein RW, Fijnheer R, van der Ven A 2007. Thrombocytopenia and release of activated von Willebrand Factor during early Plasmodium fal-ciparum malaria. J Infect Dis 196: 622-628.

Menendez C, Fleming AF, Alonso PL 2000. Malaria-related anaemia. Parasitol Today 16: 469-476.

Mohanty D, Ghosh K, Nandwani SK, Shetty S, Phillips C, Rizvi S, Parmar BD 1997. Fibrinolysis, inhibitors of blood coagulation, and monocyte derived coagulant activity in acute malaria. Am J Hematol 54: 23-29.

Mohanty D, Marwaha N, Ghosh K, Sharma S, Garewal G, Shah S, Devi S, Das KC 1988. Functional and ultrastructural changes of platelets in malarial infection. Trans R Soc Trop Med Hyg 82: 369-375.

Mohanty S, Patel DK, Pati SS, Mishra SK 2006. Adjuvant therapy in cerebral malaria. Indian J Med Res 124: 245-260.

Mohapatra MK, Padhiary KN, Mishra DP, Sethy G 2002. Atypical manifestations of Plasmodium vivax malaria. Indian J Malariol 39: 18-25.

Monath TP 2001. Yellow fever: an update. Lancet Infect Dis 1: 11-20.

Moulin F, Lesage F, Legros AH, Maroga C, Moussavou A, Guyon P, Marc E, Gendrel D 2003. Thrombocytopenia and Plasmodium falciparum malaria in children with different exposures. Arch Dis Child 88: 540-541.

Mourão MP, Lacerda MV, Macedo VO, Santos JB 2007. Thrombocy-topenia in patients with dengue virus infection in the Brazilian Amazon. Platelets 18: 605-612.

Mourão MPG, Lacerda MVG, Magalhães L, Alecrim WD, Alecrim MGC 2001. Estudo clínico em crianças internadas com malária (P. falciparum) na FMT-AM. Anais do XXXVII Congresso da Sociedade Brasileira de Medicina Tropical, Salvador. Rev Soc Bras Med Trop 34 (Suppl. 1): 71.

Muniz-Junqueira MI 2007. Immunomodulatory therapy associated to anti-parasite drugs as a way to prevent severe forms of malaria. Curr Clin Pharmacol 2: 59-73.

Muniz-Junqueira MI, Silva FO, de Paula Júnior MR, Tosta CE 2005. Thalidomide influences the function of macrophages and in-creases the survival of Plasmodium berghei-infected CBA mice. Acta Trop 94: 128-138.

Murthy GL, Sahay RK, Srinivasan VR, Upadhaya AC, Shantaram V, Gayatri K 2000. Clinical profile of falciparum malaria in a tertiary care hospital. J Indian Med Assoc 98: 160-162.

Nardi MA, Liu LX, Karpatkin S 1997. GPIIIa(49-66) is a major pathophysiologically relevant antigenic determinant for anti-platelet GPIIIa of HIV-1-related immunologic thrombocytopenia. Proc Natl Acad Sci USA 94: 7589-7594.

Naveira JB 1970. Malária - aspectos hematológicos, Associate pro-fessorship Thesis, Universidade Federal do Rio de Janeiro, Rio de Janeiro, 104 pp.

Nicodemo AC 1993. Análise de aspectos microscópicos, imuno-his-toquímicos e ultra-estruturais do pulmão na leptospirose para a compreensão da patogenia da plaquetopenia, PhD Thesis, Uni-versidade de São Paulo, São Paulo, 153 pp.

Noronha EF 1998. Estudo clínico-epidemiológico da malária fal-ciparum em crianças de 0 a 14 anos, atendidas no Instituto de Medicina Tropical do Amazonas em Manaus - AM - Brasil, MSc Thesis, Universidade de Brasília, Brasília, 126 pp.

Oh MD, Shin H, Shin D, Kim U, Lee S, Kim N, Choi MH, Chai JY, Choe K 2001. Clinical features of vivax malaria. Am J Trop Med Hyg 65: 143-146.

Ohtaka M, Ohyashiki K, Iwabuchi H, Iwabuchi A, Lin KY, Toyama K 1993. A case of vivax malaria with thrombocytopenia suggesting immunological mechanisms. Rinsho Ketsueki 34: 490-492.

Pain A, Ferguson DJ, Kai O, Urban BC, Lowe B, Marsh K, Roberts DJ 2001. Platelet-mediated clumping of Plasmodium falciparum-in-fected erythrocytes is a common adhesive phenotype and is associ-ated with severe malaria. Proc Natl Acad Sci USA 98: 1805-1810.

Panasiuk A 2001. Autoimmune thrombocytopenia in recurrent poli-etiological malaria (Plasmodium falciparum, Plasmodium vivax). Wiad Parazytol 47: 85-89.

Pankoui Mfonkeu JB, Gouado I, Fotso Kuate H, Zambou O, Amvam Zollo PH, Grau GE, Combes V 2010. Elevated cell-specific mi-croparticles are a biological marker for cerebral dysfunctions in human severe malaria. PLoS ONE 5: e13415.

Parakh A, Agarwal N, Aggarwal A, Aneja A 2009. Plasmodium vivax malaria in children: uncommon manifestations. Ann Trop Pae-diatr 29: 253-256.

Park CHL, Ferreira CB, Bianchi CP, Fazio FS, Costa JC, Padilha A, Fonseca MO, Boulos M 2002. Plaquetopenia em pacientes com malária por Plasmodium vivax. Anais do XXXVIII Congresso da Sociedade Brasileira de Medicina Tropical, Foz do Iguaçu. Rev Soc Bras Med Trop 35 (Suppl. 1): 370.

Park JW, Park SH, Yeom JS, Huh AJ, Cho YK, Ahn JY, Min GS, Song GY, Kim YA, Ahn SY, Woo SY, Lee BE, Ha EH, Han HS, Yoo K, Seoh JY 2003. Serum cytokine profiles in patients with Plasmodium vivax malaria: a comparison between those who presented with and without thrombocytopenia. Ann Trop Med Parasitol 97: 339-344.

Patel U, Gandhi G, Friedman S, Niranjan S 2004. Thrombocytopenia in malaria. J Natl Med Assoc 96: 1212-1214.

Peck-Radosavljevic M 2001. Hypersplenism. Eur J Gastroenterol He-patol 13: 317-323.

Piguet PF, Da Laperrousaz C, Vesin C, Tacchini-Cottier F, Senaldi G, Grau GE 2000. Delayed mortality and attenuated thrombocy-topenia associated with severe malaria in urokinase- and uroki-nase receptor-deficient mice. Infect Immun 68: 3822-3829.

Poespoprodjo JR, Fobia W, Kenangalem E, Lampah DA, Hasanuddin A, Warikar N, Sugiarto P, Tjitra E, Anstey NM, Price RN 2009. Vivax malaria: a major cause of morbidity in early infancy. Clin Infect Dis 48: 1704-1712.

Prasad R, Das BK, Pengoria R, Mishra OP, Shukla J, Singh TB 2009. Coagulation status and platelet functions in children with severe falciparum malaria and their correlation of outcome. J Trop Pediatr 55: 374-378.

Page 107: UNIVERSIDADE DO ESTADO DO AMAZONAS FUNDAÇÃO DE … · universidade do estado do amazonas . fundaÇÃo de medicina tropical dr. heitor vieira dourado . programa de pÓs-graduaÇÃo

62 Mem Inst Oswaldo Cruz, Rio de Janeiro, Vol. 106(Suppl. I), 2011

Rasheed A, Saeed S, Khan SA 2009. Clinical and laboratory findings in acute malaria caused by various plasmodium species. J Pak Med Assoc 59: 220-223.

Rebulla P 2000. Trigger for platelet transfusion. Vox Sang 78 (Suppl. 2): 179-182.

Rebulla P 2001. Revisitation of the clinical indications for the transfu-sion of platelet concentrates. Rev Clin Exp Hematol 5: 288-310.

Rifakis PM, Hernandez O, Fernandez CT, Rodriguez-Morales AJ, Von A, Franco-Paredes C 2008. Atypical Plasmodium vivax ma-laria in a traveler: bilateral hydronephrosis, severe thrombocy-topenia, and hypotension. J Travel Med 15: 119-121.

Rios-Orrego A, Alvarez-Castillo T, Carmona-Fonseca J, Blair-Trujil-lo S 2005. Temporal evolution of platelets and anti-platelet anti-bodies in patients of endemic area with non complicated malaria. An Med Interna 22: 561-568.

Robinson P, Jenney AW, Tachado M, Yung A, Manitta J, Taylor K, Biggs BA 2001. Imported malaria treated in Melbourne, Austra-lia: epidemiology and clinical features in 246 patients. J Travel Med 8: 76-81.

Rodriguez-Morales AJ, Sanchez E, Vargas M, Piccolo C, Colina R, Arria M 2006. Anemia and thrombocytopenia in children with Plasmodium vivax malaria. J Trop Pediatr 52: 49-51.

Rodriguez-Morales AJ, Sanchez E, Vargas M, Piccolo C, Colina R, Arria M, Franco-Paredes C 2005. Occurrence of thrombocytope-nia in Plasmodium vivax malaria. Clin Infect Dis 41: 130-131.

Rogier C, Gerardin P, Imbert P 2004. Thrombocytopenia is predic-tive of lethality in severe childhood falciparum malaria. Arch Dis Child 89: 795-796.

Samuel H, Nardi M, Karpatkin M, Hart D, Belmont M, Karpatkin S 1999. Differentiation of autoimmune thrombocytopenia from thrombocytopenia associated with immune complex disease: systemic lupus erythematosus, hepatitis-cirrhosis, and HIV-1 in-fection by platelet and serum immunological measurements. Br J Haematol 105: 1086-1091.

Santana Filho FS, Arcanjo AR, Chehuan YM, Costa MR, Martinez-Espinosa FE, Vieira JL, Barbosa MG, Alecrim WD, Alecrim MG 2007. Chloroquine-resistant Plasmodium vivax, Brazilian Ama-zon. Emerg Infect Dis 13: 1125-1126.

Santos MC, Lacerda MVG, Benedetti SM, Albuquerque BC, Aguiar Filho AA, Elkhoury MR, Rosa ES, Vasconcelos PF, Medeiros DB, Mourao MPG 2006. Human hantavirus infection, Brazilian Amazon. Emerg Infect Dis 12: 1165-1167.

Santos PD 2000. Correlação entre níveis séricos de Intermediários Reativos de Nitrogênio (IRN) e malária em pacientes da Funda-ção de Medicina Tropical do Amazonas (FMT/IMT-AM), MSc Thesis, Universidade Federal do Amazonas, Manaus, 133 pp.

Scaradavou A 2002. HIV-related thrombocytopenia. Blood Rev 16: 73-76.

Schlossberg HR, Herman JH 2003. Platelet dosing. Transfus Apher Sci 28: 221-226.

Shaikh QH, Ahmad SM, Abbasi A, Malik SA, Sahito AA, Munir SM 2009. Thrombocytopenia in malaria. J Coll Physicians Surg Pak 19: 708-710.

Shear HL 1984. Murine malaria: immune complexes inhibit Fc recep-tor-mediated phagocytosis. Infect Immun 44: 130-136.

Silva IBA 2004. Malária vivax: manifestações clínicas e laboratori-ais relacionadas com o fator de necrose tumoral alfa, PhD The-sis, Universidade Federal do Pará, Belém, 128 pp.

Silva SBR 2009. Avaliação da frequência e dos fatores associados à plaquetopenia causada pelo Plasmodium vivax, MSc Thesis, Universidade Federal do Mato Grosso, 64 pp.

Silva SL, Santana Filho FS, Arcanjo ARL, Alecrim WD, Alecrim MGC 2000. Perfil clínico e hematológico dos pacientes inter-nados com malária por Plasmodium vivax e plaquetopenia, na Fundação de Medicina Tropical do Amazonas, no período de ja-neiro de 1997 a setembro de 1999. Anais do XXXVI Congresso da Sociedade Brasileira de Medicina Tropical, São Luís do Mara-nhão , Rev Soc Bras Med Trop 33 (Suppl. 1): 348.

Skudowitz RB, Katz J, Lurie A, Levin J, Metz J 1973. Mechanisms of thrombocytopenia in malignant tertian malaria. BMJ 2: 515-518.

Song JY, Park CW, Jo YM, Kim JY, Kim JH, Yoon HJ, Kim CH, Lim CS, Cheong HJ, Kim WJ 2007. Two cases of Plasmodium vivax malaria with the clinical picture resembling toxic shock. Am J Trop Med Hyg 77: 609-611.

Srichaikul T, Pulket C, Sirisatepisarn T, Prayoonwiwat W 1988. Platelet dysfunction in malaria. Southeast Asian J Trop Med Pub-lic Health 19: 225-233.

Srichaikul T, Puwasatien P, Karnjanajetanee J, Bokisch VA, Pawasatien P 1975. Complement changes and disseminated intravascular co-agulation in Plasmodium falciparum malaria. Lancet 1: 770-772.

Srivastava S, Ahmad S, Shirazi N, Kumar Verma S, Puri P 2011. Ret-rospective analysis of vivax malaria patients presenting to ter-tiary referral centre of Uttarakhand. Acta Trop 117: 82-85.

Suarez-Mutis MC, Cuervo P, Leoratti FM, Moraes-Avila SL, Fer-reira AW, Fernandes O, Coura JR 2007. Cross sectional study reveals a high percentage of asymptomatic Plasmodium vivax infection in the Amazon Rio Negro area, Brazil. Rev Inst Med Trop Sao Paulo 49: 159-164.

Sun G, Chang WL, Li J, Berney SM, Kimpel D, van der Heyde HC 2003. Inhibition of platelet adherence to brain microvasculature protects against severe Plasmodium berghei malaria. Infect Im-mun 71: 6553-6561.

Takaki K, Aoki T, Akeda H, Kajiwara T, Honda S, Maeda Y, Okada K, Sawae Y 1991. A case of Plasmodium vivax malaria with find-ings of DIC. Kansenshogaku Zasshi 65: 488-492.

Tan SO, McGready R, Zwang J, Pimanpanarak M, Sriprawat K, Thwai KL, Moo Y, Ashley EA, Edwards B, Singhasivanon P, White NJ, Nosten F 2008. Thrombocytopaenia in pregnant women with ma-laria on the Thai-Burmese border. Malar J 7: 209.

Taylor WR, Widjaja H, Basri H, Ohrt C, Taufik T, Tjitra E, Baso S, Fryauff D, Hoffman SL, Richie TL 2008. Changes in the total leukocyte and platelet counts in Papuan and non Papuan adults from northeast Papua infected with acute Plasmodium vivax or uncomplicated Plasmodium falciparum malaria. Malar J 7: 259.

Terrazzano G, Cortese L, Piantedosi D, Zappacosta S, Di Loria A, Santoro D, Ruggiero G, Ciaramella P 2006. Presence of anti-platelet IgM and IgG antibodies in dogs naturally infected by Leishmania infantum. Vet Immunol Immunopathol 110: 331-337.

Thapa R, Biswas B, Mallick D, Sardar S, Modak S 2009. Childhood Plasmodium vivax malaria with severe thrombocytopenia and bleeding manifestations. J Pediatr Hematol Oncol 31: 758-759.

Tjitra E, Anstey NM, Sugiarto P, Warikar N, Kenangalem E, Karyana M, Lampah DA, Price RN 2008. Multidrug-resistant Plasmodi-um vivax associated with severe and fatal malaria: a prospective study in Papua, Indonesia. PLoS Med 5: e128.

Touze JE, Mercier P, Rogier C, Hovette P, Schmoor P, Dabanian C, Campiadgi S, Laroche R 1990. Platelet antibody activity in ma-laria thrombocytopenia. Pathol Biol (Paris) 38: 678-681.

Tribulatti MV, Mucci J, Van Rooijen N, Leguizamon MS, Campetella O 2005. The trans-sialidase from Trypanosoma cruzi induces thrombocytopenia during acute Chagas’ disease by reducing the platelet sialic acid contents. Infect Immun 73: 201-207.

Page 108: UNIVERSIDADE DO ESTADO DO AMAZONAS FUNDAÇÃO DE … · universidade do estado do amazonas . fundaÇÃo de medicina tropical dr. heitor vieira dourado . programa de pÓs-graduaÇÃo

Malaria and thrombocytopenia • Marcus Vinícius Guimarães Lacerda et al. 63

Tyagi P, Biswas S 1999. Naturally occurring plasmodia-specific cir-culating immune complexes in individuals of malaria endemic areas in India. Indian J Malariol 36: 12-18.

Urban BC, Hien TT, Day NP, Phu NH, Roberts R, Pongponratn E, Jones M, Mai NTH, Bethell D, Turner GDH, Ferguson D, White NJ, Roberts DJ 2005. Fatal Plasmodium falciparum malaria causes specific patterns of splenic architectural disorganization. Infect Immun 73: 1986-1994.

van der Heyde HC, Gramaglia I, Sun G, Woods C 2005. Platelet depletion by anti-CD41 (alphaIIb) mAb injection early but not late in the course of disease protects against Plasmodium ber-ghei pathogenesis by altering the levels of pathogenic cytokines. Blood 105: 1956-1963.

Victoria MB, Victoria F, Coelho AHV, Santos LO, Alecrim MGC 1998. Púrpura trombocitopênica em paciente com malária por Plasmodium vivax: relato de caso. Anais do XXXIV Congresso da Sociedade Brasileira de Medicina Tropical, Manaus. Rev Soc Bras Med Trop 31 (Suppl. 1): 55.

Vij AS, Dandona PK, Aggarwal A 2008. Malaria with marked throm-bocytopenia: report of 2 cases. J Indian Med Assoc 106: 123-125.

Warrell DA, Looareesuwan S, Warrell MJ, Kasemsarn P, Intarapra-sert R, Bunnag D, Harinasuta T 1982. Dexamethasone proves deleterious in cerebral malaria. A double-blind trial in 100 coma-tose patients. N Engl J Med 306: 313-319.

Wassmer SC, Combes V, Candal FJ, Juhan-Vague I, Grau GE 2006a. Platelets potentiate brain endothelial alterations induced by Plas-modium falciparum. Infect Immun 74: 645-653.

Wassmer SC, Lepolard C, Traore B, Pouvelle B, Gysin J, Grau GE 2004. Platelets reorient Plasmodium falciparum-infected erythrocyte cy-toadhesion to activated endothelial cells. J Infect Dis 189: 180-189.

Wassmer SC, Souza JB, Frere C, Candal FJ, Juhan-Vague I, Grau GE 2006b. TGF-beta1 released from activated platelets can in-duce TNF-stimulated human brain endothelium apoptosis: a new mechanism for microvascular lesion during cerebral malaria. J Immunol 176: 1180-1184.

Watier H, Verwaerde C, Landau I, Werner E, Fontaine J, Capron A, Auriault C 1992. T-cell-dependent immunity and thrombocy-topenia in rats infected with Plasmodium chabaudi. Infect Im-mun 60: 136-142.

WHO - World Health Organization 2010. Guidelines for the treatment of malaria [homepage on the Internet]: 2010. Available from: whqlibdoc.who.int/publications/2010/9789241547925_eng.pdf.

Wickramasinghe SN, Abdalla SH 2000. Blood and bone marrow chang-es in malaria. Baillieres Best Pract Res Clin Haematol 13: 277-299.

Yamaguchi S, Kubota T, Yamagishi T, Okamoto K, Izumi T, Takada M, Kanou S, Suzuki M, Tsuchiya J, Naruse T 1997. Severe throm-bocytopenia suggesting immunological mechanisms in two cases of vivax malaria. Am J Hematol 56: 183-186.

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113

ANEXO F: Normas da revista PLOS ONE

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PLOS ONE Manuscript Guidelines

1. Format requirements

PLOS ONE does not consider presubmission inquiries. All submissions should be

prepared as follows:

Cover letter

You should supply an approximately one page cover letter that:

Concisely summarizes why your paper is a valuable addition to the scientific

literature

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Lists any recommended or opposed reviewers

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process. See the PLOS ONE Editorial Policy regarding publication fees for more

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Manuscript Organization

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encourage a concise and accessible writing style.

Most articles published in PLOS ONE begin with the ordered sections:

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Authors

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Abstract

Introduction

and end with the sections of:

Acknowledgments

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Figure Legends

Tables

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of the manuscript file.

Between these beginning and ending sections, articles may be organized in different

ways according to what authors think is the best way to present and discuss their

science. In most cases, internal sections include:

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Materials and Methods

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Discussion

Conclusions (optional)

PLOS ONE has no specific requirements for the order of these sections, and in some

cases it may be appropriate to combine sections. Guidelines for individual sections can

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Abbreviations should be kept a minimum and defined upon first use in the text. Non-

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Appropriate standardized nomenclature should be used as appropriate, including

appropriate usage of species names and SI units.

File type requirements

Authors may submit their manuscript files in Word (as .doc or .docx), LaTeX (as .pdf), or

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If you would like to submit your manuscript using LaTeX, you must author your article

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file. Please consult our LaTeX guidelines for a list of what will be required.

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If your manuscript is or will be in .docx format and contains equations, you must follow

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production.

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.docx file remain editable in .doc by enabling "Compatibility Mode" before you begin. To

do this, open a new document and save as Word 97-2003 (*.doc). Several features of

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If you have already composed your article as .docx and used its built-in equation editing

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this problem, re-key your equations in one of the two following ways.

1. Use MathType to create the equation. MathType is the recommended method for

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If, when saving your final document, you see a message saying "Equations will be

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2. Guidelines for Standard Sections

Title

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Manuscripts must be submitted with both a full title and a short title, which will appear at

the top of the PDF upon publication if accepted. Only the full title should be included in

the manuscript file; the short title will be entered during the online submission process.

The full title must be 150 characters or fewer. It should be specific, descriptive, concise,

and comprehensible to readers outside the subject field. Avoid specialist abbreviations if

possible. Where appropriate, authors should include the species or model system used

(for biological papers) or type of study design (for clinical papers).

Examples:

Impact of Cigarette Smoke Exposure on Innate Immunity: A Caenorhabditis

elegans Model

Solar Drinking Water Disinfection (SODIS) to Reduce Childhood Diarrhoea in

Rural Bolivia: A Cluster-Randomized, Controlled Trial

The short title must be 50 characters or fewer and should state the topic of the paper.

Authors and Affiliations

All author names should be listed in the following order:

First names (or initials, if used),

Middle names (or initials, if used), and

Last names (surname, family name)

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Each author should list an associated department, university, or organizational affiliation

and its location, including city, state/province (if applicable), and country. If the article

has been submitted on behalf of a consortium, all author names and affiliations should

be listed at the end of the article.

This information cannot be changed after initial submission, so please ensure

that it is correct.

PLOS ONE bases its criteria for authorship on those outlined by the International

Committee of Medical Journal Editors (ICMJE), summarized below:

Authors should meet conditions 1, 2, and 3 below to be assigned credit for

authorship:

1. substantial contributions to conception and design of the work, acquisition

of data, or analysis and interpretation of data

2. drafting the article or revising it critically for important intellectual content;

and

3. final approval of the version to be published.

All persons designated as authors should qualify for authorship, and all those

who qualify should be listed.

When a large, multicenter group has conducted the work, the group should

identify the individuals who accept direct responsibility for the manuscript. These

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individuals should fully meet the criteria for authorship/contributorship defined

above. Each author should have participated sufficiently in the work to take

public responsibility for appropriate portions of the content.

When submitting a manuscript authored by a group, the corresponding author

should clearly indicate the preferred citation and identify all individual authors as

well as the group name. The contributions of all authors must be described. Note

that acquisition of funding, collection of data, or general supervision of the

research group alone does not constitute authorship. Contributions that fall short

of authorship should be mentioned in the Acknowledgments section of the paper.

The National Library of Medicine (NLM) indexes the group name and the names of

individuals the group has identified as being directly responsible for the

manuscript. The NLM also lists the names of collaborators if they are listed in

Acknowledgments.

One author should be designated as the corresponding author, and his or her email

address or other contact information should be included on the manuscript cover page.

This information will be published with the article if accepted.

See the PLOS ONE Editorial Policy regarding authorship criteria for more information.

Abstract

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The abstract should not exceed 300 words. It should:

Describe the main objective(s) of the study

Explain how the study was done, including any model organisms used, without

methodological detail

Summarize the most important results and their significance

Abstracts should not include:

Citations

Specialist abbreviations, if possible

Introduction

The introduction should:

Provide some background to put the manuscript into context and allow readers

outside the field to understand the purpose and significance of the study

Define the problem addressed and why it is important

Include a brief review of the key literature

Note any relevant controversies or disagreements in the field

Conclude with a brief statement of the overall aim of the work and a comment

about whether that aim was achieved

Materials and Methods

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This section should provide enough detail to allow suitably skilled investigators to fully

replicate your study. Specific information and/or protocols for new methods should be

included in detail. If materials, methods, and protocols are well established, authors may

refer to other papers where those protocols are described in detail, but the submission

should include sufficient information to be understood independent of these references.

We encourage authors to submit detailed protocols for newer or less well-established

methods as Supporting Information. These are published online only, but are linked to

the article and are fully searchable. For more information about formatting Supporting

Information files, click here.

Methods sections of papers on research using human or animal subjects and/or

tissue or field sampling must include required ethics statements. See the Reporting

Guidelines for human research, clinical trials, animal research, and observational and

field studies for more information.

Methods sections of papers with data that should be deposited in a publicly

available database should specify where the data have been deposited and provide

the relevant accession numbers and version numbers, if appropriate. Accession

numbers should be provided in parentheses after the entity on first use. If the accession

numbers have not yet been obtained at the time of submission, please state that they

will be provided during review. They must be provided prior to publication.

Methods sections of papers using cell lines must state the origin of the cell lines used.

See the Reporting Guidelines for cell line research for more information.

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Methods sections of papers adding new taxon names to the literature must follow the

Reporting Guidelines below for a new zoological taxon, botanical taxon, or fungal taxon.

Results, Discussion, and Conclusions

These sections may all be separate, or may be combined to create a mixed

Results/Discussion section (commonly labeled "Results and Discussion") or a mixed

Discussion/Conclusions section (commonly labeled "Discussion"). These sections may

be further divided into subsections, each with a concise subheading, as appropriate.

These sections have no word limit, but the language should be clear and concise.

Together, these sections should describe the results of the experiments, the

interpretation of these results, and the conclusions that can be drawn. Authors should

explain how the results relate to the hypothesis presented as the basis of the study and

provide a succinct explanation of the implications of the findings, particularly in relation

to previous related studies and potential future directions for research.

PLOS ONE editorial decisions do not rely on the novelty or perceived impact, so

authors should avoid overstating their conclusions. See the PLOS ONEPublication

Criteria for more information.

Acknowledgements

People who contributed to the work but do not fit the PLOS ONE authorship

criteria should be listed in the acknowledgments, along with their contributions. You

must ensure that anyone named in the acknowledgments agrees to being so named.

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Funding sources should not be included in the acknowledgments, or anywhere in the

manuscript file. You will provide this information during the manuscript submission

process.

References

Only published or accepted manuscripts should be included in the reference list. Papers that have been submitted but not yet accepted should not be cited. Limited citation of unpublished work should be included in the body of the text only as “unpublished data.” All “personal communications” citations should be supported by a letter from the relevant authors.

Style information:

PLOS uses the numbered citation (citation-sequence) method and first five

authors, et al.

References are listed and numbered in the order that they appear in the text.

In the text, citations should be indicated by the reference number in brackets.

The parts of the manuscript should be in the correct order before ordering the

citations: body, boxes, figure captions, tables, and supporting information

captions.

Abstracts and author summaries may not contain citations.

Journal name abbreviations should be those found in the NCBI

databases: http://www.ncbi.nlm.nih.gov/nlmcatalog/journals.

Because all references will be linked electronically as much as possible to the papers

they cite, proper formatting of the references is crucial. For convenience, a number of

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reference software companies supply PLOS style files (e.g., Reference

Manager, EndNote).

Published Papers

1. Hou WR, Hou YL, Wu GF, Song Y, Su XL, et al. (2011) cDNA, genomic sequence

cloning and overexpression of ribosomal protein gene L9 (rpL9) of the giant panda

(Ailuropoda melanoleuca). Genet Mol Res 10: 1576-1588.

Note: Use of a DOI number for the full-text article is acceptable as an alternative to or in

addition to traditional volume and page numbers.

Accepted, unpublished papers

Same as above, but “In press” appears instead of the page numbers.

Electronic Journal Articles

1. Huynen MMTE, Martens P, Hilderlink HBM (2005) The health impacts of

globalisation: a conceptual framework. Global Health 1: 14. Available:

http://www.globalizationandhealth.com/content/1/1/14. Accessed 25 January 2012.

Books

1. Bates B (1992) Bargaining for life: A social history of tuberculosis. Philadelphia:

University of Pennsylvania Press. 435 p.

Book Chapters

1. Hansen B (1991) New York City epidemics and history for the public. In: Harden VA,

Risse GB, editors. AIDS and the historian. Bethesda: National Institutes of Health. pp.

21-28.

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Figure legends

Figures should not be included in the manuscript file, but figure legends should be.

Figure legends should describe the key messages of a figure. Legends should have a

short title of 15 words or less. The full legend should have a description of the figure and

allow readers to understand the figure without referring to the text. The legend itself

should be succinct, avoid lengthy descriptions of methods, and define all non-standard

symbols and abbreviations.

Further information can be found in the Figure Guidelines.

Tables

Tables should be included at the end of the manuscript. All tables should have a

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.

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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

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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:

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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

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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

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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

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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

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New Zoological Taxon

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Until acceptance of this amendment, the ICZN has proposed an interim solution for

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obtainable. Therefore, for all papers that include the naming of a new zoological taxon,

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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

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In the Results section, the globally unique identifier (GUID), currently in the form of a

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Anochetus boltoni Fisher sp. nov. urn:lsid:zoobank.org:act:B6C072CF-1CA6-40C7-

8396-534E91EF7FBB

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

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responsibility to provide us with this information so we can include it in the final

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Please also insert the following text into the Methods section, in a sub-section to be

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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

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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

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online version of this work is archived and available from the following digital

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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,

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that any scientific botanical name published by us is considered effectively published

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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

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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.

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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.

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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.

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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):

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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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professional editing are encouraged to use language-editing and copyediting services.

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Obtaining this service is the responsibility of the author, and should be done before

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