Interaction of malaria parasites with host late endocytic and autophagic pathways is essential for Plasmodium liver stage development

RESUMO: A Malária é causada por parasitas do género Plasmodium, sendo a doença parasitária mais fatal para o ser humano. Apesar de, durante o século passado, o desenvolvimento económico e a implementação de diversas medidas de controlo, tenham permitido erradicar a doença em muitos países, a Malária continua a ser um problema de saúde grave, em particular nos países em desenvolvimento. A Malária é transmitida através da picada de uma fêmea de mosquito do género Anopheles. Durante a picada, os esporozoítos são injetados na pele do hospedeiro, seguindo-se a fase hepática e obrigatória do ciclo de vida. No fígado, os esporozoítos infetam os hepatócitos onde se replicam, dentro de um vacúolo parasitário (VP) e de uma forma imunitária silenciosa, em centenas de merozoitos. Estas novas formas do parasita são as responsáveis por infetar os eritrócitos, iniciando a fase sanguínea da doença, onde se os primeiros sintomas se manifestam, tais como a característica febre cíclica. A fase hepática da doença é a menos estudada e compreendida. Mais ainda, as interações entre o VP e os organelos da células hospedeira estão ainda pouco caracterizados. Assim, neste estudo, as interações entre os organelos endocíticos e autofágicos da célula hospedeira e o VP foram dissecados, observando-se que os anfisomas, que são organelos resultantes da intersecção do dois processos de tráfego intracelular, interagem com o parasita. Descobrimos que a autofagia tem também uma importante função imunitária durante a fase hepática inicial, ao passo, que durante o desenvolvimento do parasita, já numa fase mais tardia, o parasita depende da interação com os endossomas tardios e anfisomas para crescer. Vesiculas de BSA, EGF e LC3, foram, também, observadas dentro do VP, sugerindo que os parasitas são capazes de internalizar material endocítico e autofágico do hospedeiro. Mais ainda, mostramos que esta interação depende da cinase PIKfyve, responsável pela conversão do fosfoinositidio-3-fosfato no fosfoinositidio-3,5-bifosfato, uma vez que inibindo esta cinase o parasita não é capaz de crescer normalmente. Finalmente, mostramos que a proteína TRPML1, uma proteína efetora do fosfoinositidio-3,5-bifosfato, e envolvida no processo de fusão das membranas dos organelos endocíticos e autofágicos, também é necessária para o crescimento do parasita. Desta forma, o nosso estudo sugere que a membrana do VP funde com vesiculas endocíticas e autofágicas tardias, de uma forma dependente do fositidio-3,5-bifosfato e do seu effetor TRPML1, permitindo a troca de material com a célula hospedeira. Concluindo, os nossos resultados evidenciam que o processo autofágico que ocorre na célula hospedeira tem um papel duplo durante a fase hepática da malaria. Enquanto numa fase inicial os hepatócitos usam o processo autofágico como forma de defesa contra o parasita, já durante a fase de replicação o VP funde com vesiculas autofágicas e endocíticas de forma a obter os nutrientes necessários ao seu desenvolvimento.--------- ABSTRACT: Malaria, which is caused by parasites of the genus Plasmodium, is the most deadly parasitic infection in humans. Although economic development and the implementation of control measures during the last century have erradicated the disease from many areas of the world, it remains a serious human health issue, particularly in developing countries. Malaria is transmitted by female mosquitoes of the genus Anopheles. During the mosquito blood meal, Plasmodium spp. sporozoites are injected into the skin dermis of the vertebrate host, followed by an obligatory liver stage. Upon entering the liver, Plasmodium parasites infect hepatocytes and silently replicate inside a host cell-derived parasitophorous vacuole (PV) into thousands of merozoites. These new parasite forms can infect red blood cells initiating the the blood stage of the disease which shows the characteristic febrile malaria episodes. The liver stage is the least characterized step of the malaria infection. Moreover, the interactions between the Plasmodium spp. PV and the host cell trafficking pathways are poorly understood. We dissected the interaction between Plasmodium parasites and the host cell endocytic and autophagic pathways and we found that both pathways intersect and interconnect in the close vicinity of the parasite PV, where amphisomes are formed and accumulate. Interestingly, we observed a clearance function for autophagy in hepatocytes infected with Plasmodium berghei parasites at early infection times, whereas during late liver stage development late endosomes and amphisomes are required for parasite growth. Moreover, we found the presence of internalized BSA, EGF and LC3 inside parasite vacuoles, suggesting that the parasites uptake endocytic and autophagic cargo. Furthermore, we showed that the interaction between the PV and host traffic pathways is dependent on the kinase PIKfyve, which converts the phosphoinositide PI(3)P into PI(3,5)P2, since PIKfyve inhibition caused a reduction in parasite growth. Finally, we showed that the PI(3,5)P2 effector protein TRPML1, which is involved in late endocytic and autophagic membrane fusion, is also required for parasite development. Thus, our studies suggest that the parasite parasitophorous vacuole membrane (PVM) is able to fuse with late endocytic and autophagic vesicles in a PI(3,5)P2- and TRPML1-dependent manner, allowing the exchange of material between the host cell and the parasites, necessary for the rapid development of the latter that is seen during the liver stage of infection. In conclusion, we present evidence supporting a specific and essential dual role of host autophagy during the course of Plasmodium liver infection. Whereas in the initial hours of infection the host cell uses autophagy as a cell survival mechanism to fight the infection, during the replicative phase the PV fuses with host autophagic and endocytic vesicles to obtain nutrients required for parasite growth.

Alterações clínicolaboratoriais em pacientes com malária por Plasmodium vivax e deficiência de glicose-6-fosfato desidrogenase tratados com 0,50mg/kg/dia de primaquina

O efeito adverso da primaquina na dose de 0,50mg/kg/dia foi investigado em onze pacientes com malária vivax (três com deficiência de glicose-6-fosfato desidrogenase). Alterações clínicas e laboratoriais indicaram hemólise aguda apenas nos enzimopênicos, o que fez com que o tratamento fosse interrompido. Nossos resultados sugerem a necessidade do emprego de um teste de triagem para a deficiência de G6PD em áreas endêmicas de malária vivax a fim de se evitar complicações causadas pelo uso da primaquina.

Efficacy of sulfadoxine-pyrimethamine and mefloquine for the treatment of uncomplicated Plasmodium falciparum malaria in the Amazon basin of Peru

In vivo antimalarial drug efficacy studies of uncomplicated Plasmodium falciparum malaria at an isolated site in the Amazon basin of Peru bordering Brazil and Colombia showed >50% RII/RIII resistance to sulfadoxine-pyrimethamine but no evidence of resistance to mefloquine.

Resistência à sulfadoxina-pirimetamina em Maputo, Moçambique: presença de mutações nos genes dhfr e dhps do Plasmodium falciparum

Foram analisadas a freqüência e distribuição de mutações nos genes dihidrofolato redutase e dihidropteroato sintetase do Plasmodium falciparum, usando a metodologia de reação em cadeia da polimerase e polimorfismos de hidrólise por enzimas de restrição, em amostras de sangue infectado proveniente de crianças moçambicanas, residentes em Maputo. A análise foi feita antes e 7 dias após o tratamento com sulfadoxina-pirimetamina (S/P). Os resultados mostraram a ocorrência de mutações pontuais nos genes estudados e a presença de combinações de três alelos em dhfr (51Ile, 59Arg e 108Asn) e do quintúplo mutante (dhfr 51Ile, 59Arg, 108Asn e dhps 437Gly, 540Glu), ambas situações associadas à falha terapêutica no sétimo dia após tratamento com S/P. Esses achados mostram a importância de se estudar a resistência à S/P em Moçambique, e como os marcadores moleculares de resistência aos antimaláricos podem fornecer dados importantes para a política nacional de controlo da malária.

Stages of in vitro phagocytosis of Plasmodium falciparum-infected erythrocytes by human monocytes

Monocytes/macrophages play a critical role in the defense mechanisms against malaria parasites, and are the main cells responsible for the elimination of malaria parasites from the blood circulation. We carried out a microscope-aided evaluation of the stages of in vitro phagocytosis of Plasmodium falciparum-infected erythrocytes, by human monocytes. These cells were obtained from healthy adult individuals by means of centrifugation through a cushion of Percoll density medium and were incubated with erythrocytes infected with Plasmodium falciparum that had previously been incubated with a pool of anti-plasmodial immune serum. We described the stages of phagocytosis, starting from adherence of infected erythrocytes to the phagocyte membrane and ending with their destruction within the phagolisosomes of the monocytes. We observed that the different erythrocytic forms of the parasite were ingested by monocytes, and that the process of phagocytosis may be completed in around 30 minutes. Furthermore, we showed that phagocytosis may occur continuously, such that different phases of the process were observed in the same phagocyte.

Caracterização de genótipos de Plasmodium vivax na Ilha de São Luís, Estado do Maranhão

O estudo foi desenvolvido com o objetivo de caracterizar os genótipos da proteína circunsporozoíta de Plasmodium vivax, circulantes em área periférica da Ilha de São Luís, Maranhão. Foram obtidas amostras de sangue para exame parasitológico direto (gota espessa) de 126 indivíduos, dentre os quais, foram coletadas também 109 amostras para diagnóstico molecular, por reação em cadeia da polimerase. O exame parasitológico demonstrou a presença de Plasmodium vivax em 2 indivíduos, sintomáticos, enquanto o estudo molecular foi positivo para o Plasmodium vivax em 7 indivíduos (2 sintomáticos e positivos na gota espessa e 5 assintomáticos e negativos na gota espessa). Em dois havia associação com Plasmodium falciparum. A genotipagem das amostras de Plasmodium vivax revelou a variante VK 210, havendo associação com a variante VK 247 em duas delas.

Metemoglobinemia em pacientes com malária por Plasmodium vivax em uso oral de primaquina

INTRODUÇÃO: A primaquina pode acarretar sérios eventos adversos, com destaque para a toxicidade ao sangue. O objetivo deste trabalho é determinar a metemoglobinemia de 20 pacientes com malária por Plasmodium vivax tratados com primaquina, comparando-os segundo o sexo e a expressão da glicose-6-fosfato desidrogenase. MÉTODOS: Quantificação da metemoglobina por espectrofotometria visível e avaliação qualitativa da glicose-6-fosfato desidrogenase. RESULTADOS: A metemoglobinemia variou de 2,85 a 5,45% nos pacientes do sexo masculino e de 3,77 a 7,34% no feminino. CONCLUSÕES: A instituição da terapia aumentou de maneira significativa os teores de metemoglobina, sem manifestação clínica evidente e independente do sexo e da atividade enzimática.

Plasmodium falciparum infection in pregnant women attending antenatal care in Luanda, Angola

INTRODUCTION: Malaria during pregnancy remains a serious public health problem. The aim of this study was to establish the prevalence and possible risk factors for malaria in pregnant women attending antenatal care at Augusto Ngangula Specialized General Hospital in Luanda, Angola. METHODS: Pregnant women (679 total) who attended antenatal care from April to September 2008 were included in the study after signing informed consent. For each participant, the social-demographic profile and malaria and obstetric histories were investigated via a questionnaire. Diagnosis was made by optic microscopy, and hemoglobin concentration measured. The associations between age, parity, gestational age, residence, schooling, malaria during gravity, anemia and treatment with incidence of Plasmodium falciparum infection were analyzed through logistic regression. RESULTS: During the period of study, 74 (10.9%) out of 679 women were infected by P. falciparum. The average concentration of hemoglobin was 11.1 ± 0.07g/dL, and there were significant associations between the history of malaria during pregnancy, P. falciparum infection (p<0.01) and anemia at the time of observation (p<0.001). CONCLUSIONS: Previous history of malaria during pregnancy represents a risk factor for current infection and anemia was an important complication associated with malaria, even in women who were treated with sulfadoxine-pyrimethamine during pregnancy.

Artesunate + amodiaquine versus artemether-lumefantrine for the treatment of uncomplicated Plasmodium falciparum malaria in the Colombian Pacific region: a noninferiority trial

INTRODUCTION: In Colombia, there are no published studies for the treatment of uncomplicated Plasmodium falciparum malaria comparing artemisinin combination therapies. Hence, it is intended to demonstrate the non-inferior efficacy/safety profiles of artesunate + amodiaquine versus artemether-lumefantrine treatments. METHODS: A randomized, controlled, open-label, noninferiority (Δ≤5%) clinical trial was performed in adults with uncomplicated P. falciparum malaria using the 28‑day World Health Organization validated design/definitions. Patients were randomized 1:1 to either oral artesunate + amodiaquine or artemether-lumefantrine. The primary efficacy endpoint: adequate clinical and parasitological response; secondary endpoints: - treatment failures defined per the World Health Organization. Safety: assessed through adverse events. RESULTS: A total of 105 patients was included in each group: zero censored observations. Mean (95%CI - Confidence interval) adequate clinical and parasitological response rates: 100% for artesunate + amodiaquine and 99% for artemether-lumefantrine; the noninferiority criteria was met (Δ=1.7%). There was one late parasitological therapeutic failure (1%; artemether-lumefantrine group), typified by polymerase chain reaction as the MAD20 MSP1 allele. The fever clearance time (artesunate + amodiaquine group) was significantly shorter (p=0.002). Respectively, abdominal pain for artesunate + amodiaquine and artemether-lumefantrine was 1.9% and 3.8% at baseline (p=0.68) and 1% and 13.3% after treatment (p<0.001). CONCLUSIONS: Uncomplicated P. falciparum malaria treatment with artesunate + amodiaquine is noninferior to the artemether-lumefantrine standard treatment. The efficacy/safety profiles grant further studies in this and similar populations.

Plasmodium vivax malaria: related factors to severity in the State of Maranhão, Brazil

INTRODUCTION: Malaria caused by Plasmodium vivax species has shown signs of severity, recorded with increasing frequency in the medical literature. This study aimed to characterize the signs of severe malaria by Plasmodium vivax in the State of Maranhão, Brazil. METHODS: A descriptive cohort study of patients assisted in the field and a historical and concurrent study of a series of cases among hospitalized patients were undertaken to identify the clinical and laboratory signs of severity. RESULTS: A total of 153 patients were included in the study, 13 of whom were hospitalized. Males made up the majority, numbering 103 (67.3%). The age of the patients ranged from 10 to 70 years, 92.2% were natives of the State of Maranhão, and 65% of the patients had had malaria before. The average time elapsed between symptom onset and diagnosis among outpatients was three days, while among hospitalized patients this average reached 15.5 days, a statistically significant difference (p=0.001). The parasitemia ranged from 500 to 10,000 parasites/µl in 92.8% of cases. The clinical and laboratory manifestations of severity were vomiting and diarrhea, jaundice, drowsiness, mental confusion, seizures, loss of consciousness, agitation, bleeding, pale skin, coughing and dyspnea, thrombocytopenia, anemia, elevation of nitrogenous compounds, and elevated transaminases and bilirubin. CONCLUSIONS: The monitoring of malaria patients with Plasmodium vivax showed the possibility of aggravation, the intensity of which varied in different circumstances, especially the interval time between falling ill and diagnostic confirmation.

In vitro antimalarial activity of tigecycline against Plasmodium falciparum culture-adapted reference strains and clinical isolates from the Brazilian Amazon

Introduction: We evaluated the in vitro antimalarial activity of tigecycline as an alternative drug for the treatment of severe malaria. Methods: A chloroquine-sensitive Plasmodium falciparum reference strain, a chloroquine-resistant reference strain, and three clinical isolates were tested for in vitro susceptibility to tigecycline. A histidine-rich protein in vitro assay was used to evaluate antimalarial activity. Results: The geometric-mean 50% effective concentration (EC50%) of tigecycline was 535.5 nM (confidence interval (CI): 344.3-726.8). No significant correlation was found between the EC50% of tigecycline and that of any other tested antimalarial drug. Conclusions: Tigecycline may represent an alternative drug for the treatment of patients with severe malaria.

An unusual case of heart failure due to Plasmodium vivax infection with a favorable outcome

Although malaria is one of the oldest types of parasitic infection, we have recently witnessed substantial changes in the outcome of malarial infections. Severe Plasmodium vivax infections have recently become more frequent, and are occasionally associated with fatal outcomes. Cardiac arrhythmia and myocardial failure have also been reported, typically in association with Plasmodium falciparum infections. We report a case of myocarditis and heart failure, due to Plasmodium vivaxinfection, along with the favorable outcome.