Molecular screening of Plasmodium sp. asymptomatic carriers among transfusion centers from Brazilian Amazon region

Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)

Asymptomatic carriers of Plasmodium spp. as infection source for malaria vector mosquitoes in the Brazilian Amazon

We have described the existence of asymptomatic carriers of Plasmodium vivax and Plasmodium falciparum infections in native Amazon populations. Most of them had low parasitemias, detected only by polymerase chain reaction (PCR). Because they remain symptomless and untreated, we wanted to determine whether they could infect Anopheles darlingi Root, the main Brazilian vector, and act as disease reservoirs. Fifteen adult asymptomatic patients (PCR positive only) were selected, and experimental infections of mosquitoes were performed by direct feeding and by a membrane-feeding system. Seventeen adult symptomatic patients with high parasitemias were used as controls. We found an infection rate in An. darlingi of 1.2% for the asymptomatic carriers and 22% for the symptomatic carriers. Although the asymptomatic group infected mosquitoes at a much lower rate, these patients remain infective longer than treated, symptomatic patients. Also, the prevalence of asymptomatic infections is 4 to 5 times higher than symptomatic infections among natives. These results have implications for the malaria control program in Brazil, which focuses essentially on the treatment of symptomatic patients. © 2005 Entomological Society of America.

Relationship between plasma and red blood cell concentrations of quinine in Brazilian children with uncomplicated Plasmodium falciparummalaria on oral therapy

Neste estudo foi determinada a relação entre as concentrações plasmáticas e eritrocitárias de quinina em crianças com malária falciparum não complicada, oriundas de área endêmica da Região Amazônica. A quinina foi detrminada por cromatografia líquida de alta eficiência. No estado de equilíbrio, a relação foi 1,89 ± 1,25 variando de 1,05 a 2,34. Estes resultados demonstraram que a quinina não se concentra nos eritrócitos das crianças e caracterizaram a ausência de diferença racial nesta relação.

Alterações neuroquímicas no tecido retiniano murino em modelo de malária cerebral induzida pela infecção por Plasmodium berghei (ANKA)

A Malária Cerebral (MC) apresenta-se como uma severa complicação resultante da infecção por Plasmodium falciparum. Esta condição encontra-se comumente associada a disfunções cognitivas, comportamentais e motoras, sendo a retinopatia uma das mais graves conseqüências da doença. Diversos modelos experimentais já foram descritos no intuito de elucidar os mecanismos fisiopatológicos relacionados a esta síndrome, no entanto, estes ainda permanecem pouco compreendidos. Dentro deste contexto, o presente trabalho procurou investigar as alterações neuroquímicas envolvidas na patologia da MC. Os camundongos C57Bl/6 (fêmeas e machos) inoculados com ≈106 eritrócitos parasitados (PbA) apresentaram baixa parasitemia (15-20%) com sinais clínicos evidentes como: deficiência respiratória, ataxia, hemiplegia e coma seguido de morte, condizentes com o quadro de MC. A análise no tecido retiniano demonstrou uma diminuição nos níveis de GSH com 2 dias após a inoculação. Entretanto, essa diminuição não foi tão evidente com o decorrer da infecção (4º e 6º dias após infecção). Concomitante a este aumento durante o processo infeccioso, observamos um progressivo aumento na captação de 3H-glutamato (4º e 6º dia após infecção) por um sistema independente de Na+, sugerindo que o quadro de MC é responsável por um aumento na atividade de uma proteína transportadora. Dados obtidos com a imunofluorescência demonstram que além de aumentar a atividade do sistema de transporte, o quadro de MC também estimula o aumento na expressão do sistema xCG - no tecido retiniano. O presente trabalho demonstra ainda que estes eventos neuroquímicos no tecido retiniano são independentes de ativação inflamatória, visto que os níveis de TNF-α e expressão de NOS-2, apresentam-se alterados somente no tecido retiniano.

Estudo da eficácia e tolerância do artesunato oral isolado e em associação com mefloquina, no tratamento da malária falciparum não complicada em área endêmica do Pará, Brasil

Com o objetivo de avaliar a eficácia e tolerância do artesunato no tratamento da malária falciparum não complicada em área endêmica do Estado do Pará, 153 pacientes foram randomizados e estudados em três grupos, distribuídos por esquema terapêutico (I recebeu mefloquina lOOOmg; II utilizou artesunato lOOOmg; III usou a combinação de 600mg de artesunato seguida de 500 de mefloquina). A avaliação constou de exame clínico e parasitológico diariamente nos primeiros 7 dias e semanalmente até o 35o dia do acompanhamento e de análise bioquímica e hematológica realizada antes e no 7o dia, visando o controle de ema e a identificação de possíveis efeitos associados á administração das drogas. Os grupos estudados foram homogêneos quanto ao sexo, parasitemia e presença de febre. O tempo para desaparecimento da parasitemia foi mais curto nos grupos II e III, respectivamente, cujos esquemas terapêuticos empregaram artesunato. O desaparecimento da febre foi mais rápido no grupo tratado com a combinação das drogas. Alterações clínicas e bioquímicas associadas a administração das drogas não mostraram diferenças significativas entre os grupos estudados. O desaparecimento precoce da febre e parasitemia, e a ausência de importantes efeitos indesejáveis, sugerem que artesunato administrado isoladamente ou em combinação com mefloquina constituem medidas terapêuticas capazes de contribuir para o controle da doença na região.

Pilot survey of expressed sequence tags (ESTs) from the asexual blood stages of Plasmodium vivax in human patients

Abstract Background Plasmodium vivax is the most widely distributed human malaria, responsible for 70–80 million clinical cases each year and large socio-economical burdens for countries such as Brazil where it is the most prevalent species. Unfortunately, due to the impossibility of growing this parasite in continuous in vitro culture, research on P. vivax remains largely neglected. Methods A pilot survey of expressed sequence tags (ESTs) from the asexual blood stages of P. vivax was performed. To do so, 1,184 clones from a cDNA library constructed with parasites obtained from 10 different human patients in the Brazilian Amazon were sequenced. Sequences were automatedly processed to remove contaminants and low quality reads. A total of 806 sequences with an average length of 586 bp met such criteria and their clustering revealed 666 distinct events. The consensus sequence of each cluster and the unique sequences of the singlets were used in similarity searches against different databases that included P. vivax, Plasmodium falciparum, Plasmodium yoelii, Plasmodium knowlesi, Apicomplexa and the GenBank non-redundant database. An E-value of <10-30 was used to define a significant database match. ESTs were manually assigned a gene ontology (GO) terminology Results A total of 769 ESTs could be assigned a putative identity based upon sequence similarity to known proteins in GenBank. Moreover, 292 ESTs were annotated and a GO terminology was assigned to 164 of them. Conclusion These are the first ESTs reported for P. vivax and, as such, they represent a valuable resource to assist in the annotation of the P. vivax genome currently being sequenced. Moreover, since the GC-content of the P. vivax genome is strikingly different from that of P. falciparum, these ESTs will help in the validation of gene predictions for P. vivax and to create a gene index of this malaria parasite.

Serological detection of Plasmodium vivax malaria using recombinant proteins corresponding to the 19-kDa C-terminal region of the merozoite surface protein-1

Abstract Background Serological tests to detect antibodies specific to Plasmodium vivax could be a valuable tool for epidemiological studies, for screening blood donors in areas where the malaria is not endemic and for diagnosis of infected individuals. Because P. vivax cannot be easily obtained in vitro, ELISA assays using total or semi-purified antigens are rarely used. Based on this limitation, we tested whether recombinant proteins representing the 19 kDa C-terminal region of the merozoite surface protein-1 of P. vivax (MSP119) could be useful for serological detection of malaria infection. Methods Three purified recombinant proteins produced in Escherichia coli (GST-MSP119, His6-MSP119 and His6-MSP119-PADRE) and one in Pichia pastoris (yMSP119-PADRE) were compared for their ability to bind to IgG antibodies of individuals with patent P. vivax infection. The method was tested with 200 serum samples collected from individuals living in the north of Brazil in areas endemic for malaria, 53 serum samples from individuals exposed to Plasmodium falciparum infection and 177 serum samples from individuals never exposed to malaria. Results Overall, the sensitivity of the ELISA assessed with sera from naturally infected individuals was 95%. The proportion of serum samples that reacted with recombinant proteins GST-MSP119, His6-MSP119, His6-MSP119-PADRE and yMSP119-PADRE was 90%, 93.5%, 93.5% and 93.5%, respectively. The specificity values of the ELISA determined with sera from healthy individuals and from individuals with other infectious diseases were 98.3% (GST-MSP119), 97.7% (His6-MSP119 and His6-MSP119-PADRE) or 100% (yMSP119-PADRE). Conclusions Our study demonstrated that for the Brazilian population, an ELISA using a recombinant protein of the MSP119 can be used as the basis for the development of a valuable serological assay for the detection of P. vivax malaria.

Cellular and humoral immune responses against the Plasmodium vivax MSP-119 malaria vaccine candidate in individuals living in an endemic area in north-eastern Amazon region of Brazil

Abstract Background Plasmodium vivax merozoite surface protein-1 (MSP-1) is an antigen considered to be one of the leading malaria vaccine candidates. PvMSP-1 is highly immunogenic and evidences suggest that it is target for protective immunity against asexual blood stages of malaria parasites. Thus, this study aims to evaluate the acquired cellular and antibody immune responses against PvMSP-1 in individuals naturally exposed to malaria infections in a malaria-endemic area in the north-eastern Amazon region of Brazil. Methods The study was carried out in Paragominas, Pará State, in the Brazilian Amazon. Blood samples were collected from 35 individuals with uncomplicated malaria. Peripheral blood mononuclear cells were isolated and the cellular proliferation and activation was analysed in presence of 19 kDa fragment of MSP-1 (PvMSP-119) and Plasmodium falciparum PSS1 crude antigen. Antibodies IgE, IgM, IgG and IgG subclass and the levels of TNF, IFN-γ and IL-10 were measured by enzyme-linked immunosorbent assay. Results The prevalence of activated CD4+ was greater than CD8+ T cells, in both ex-vivo and in 96 h culture in presence of PvMSP-119 and PSS1 antigen. A low proliferative response against PvMSP-119 and PSS1 crude antigen after 96 h culture was observed. High plasmatic levels of IFN-γ and IL-10 as well as lower TNF levels were also detected in malaria patients. However, in the 96 h supernatant culture, the dynamics of cytokine responses differed from those depicted on plasma assays; in presence of PvMSP-119 stimulus, higher levels of TNF were noted in supernatant 96 h culture of malaria patient’s cells while low levels of IFN-γ and IL-10 were verified. High frequency of malaria patients presenting antibodies against PvMSP-119 was evidenced, regardless class or IgG subclass.PvMSP-119-induced antibodies were predominantly on non-cytophilic subclasses. Conclusions The results presented here shows that PvMSP-119 was able to induce a high cellular activation, leading to production of TNF and emphasizes the high immunogenicity of PvMSP-119 in naturally exposed individuals and, therefore, its potential as a malaria vaccine candidate.

Argemone mexicana decoction for the treatment of uncomplicated falciparum malaria

A prospective, dose-escalating, quasi-experimental clinical trial was conducted with a traditional healer using a decoction of Argemone mexicana for the treatment of malaria in Mali. The remedy was prescribed in three regimens: once daily for 3 days (Group A; n=23); twice daily for 7 days (Group B; n=40); and four times daily for the first 4 days followed by twice daily for 3 days (Group C; n=17). Thus, 80 patients were included, of whom 80% were aged<5 years and 25% were aged<1 year. All presented to the traditional healer with symptoms of malaria and had a Plasmodium falciparum parasitaemia>2000/microl but no signs of severe malaria. The proportions of adequate clinical response (ACR) at Day 14 were 35%, 73% and 65% in Groups A, B and C, respectively (P=0.011). At Day 14, overall proportions of ACR were lower in children aged<1 year (45%) and higher in patients aged>5 years (81%) (P=0.027). Very few patients had complete parasite clearance, but at Day 14, 67% of patients with ACR had a parasitaemia<2000/microl. No patient needed referral for severe disease. Only minor side effects were observed. Further research should determine whether this local resource could represent a first-aid home treatment in remote areas.

The Plasmodium Class XIV Myosin, MyoB, Has a Distinct Subcellular Location in Invasive and Motile Stages of the Malaria Parasite and an Unusual Light Chain

Myosin B (MyoB) is one of the two short class XIV myosins encoded in the Plasmodium genome. Class XIV myosins are characterized by a catalytic "head," a modified "neck," and the absence of a "tail" region. Myosin A (MyoA), the other class XIV myosin in Plasmodium, has been established as a component of the glideosome complex important in motility and cell invasion, but MyoB is not well characterized. We analyzed the properties of MyoB using three parasite species as follows: Plasmodium falciparum, Plasmodium berghei, and Plasmodium knowlesi. MyoB is expressed in all invasive stages (merozoites, ookinetes, and sporozoites) of the life cycle, and the protein is found in a discrete apical location in these polarized cells. In P. falciparum, MyoB is synthesized very late in schizogony/merogony, and its location in merozoites is distinct from, and anterior to, that of a range of known proteins present in the rhoptries, rhoptry neck or micronemes. Unlike MyoA, MyoB is not associated with glideosome complex proteins, including the MyoA light chain, myosin A tail domain-interacting protein (MTIP). A unique MyoB light chain (MLC-B) was identified that contains a calmodulin-like domain at the C terminus and an extended N-terminal region. MLC-B localizes to the same extreme apical pole in the cell as MyoB, and the two proteins form a complex. We propose that MLC-B is a MyoB-specific light chain, and for the short class XIV myosins that lack a tail region, the atypical myosin light chains may fulfill that role.

Emergence of FY*Anull in a Plasmodium vivax-endemic region of Papua New Guinea

In Papua New Guinea (PNG), numerous blood group polymorphisms and hemoglobinopathies characterize the human population. Human genetic polymorphisms of this nature are common in malarious regions, and all four human malaria parasites are holoendemic below 1500 meters in PNG. At this elevation, a prominent condition characterizing Melanesians is α+-thalassemia. Interestingly, recent epidemiological surveys have demonstrated that α+-thalassemia is associated with increased susceptibility to uncomplicated malaria among young children. It is further proposed that α+-thalassemia may facilitate so-called “benign” Plasmodium vivax infection to act later in life as a “natural vaccine” against severe Plasmodium falciparum malaria. Here, in a P. vivax-endemic region of PNG where the resident Abelam-speaking population is characterized by a frequency of α+-thalassemia ≥0.98, we have discovered the mutation responsible for erythrocyte Duffy antigen-negativity (Fy[a−b−]) on the FY*A allele. In this study population there were 23 heterozygous and no homozygous individuals bearing this new allele (allele frequency, 23/1062 = 0.022). Flow cytometric analysis illustrated a 2-fold difference in erythroid-specific Fy-antigen expression between heterozygous (FY*A/FY*Anull) and homozygous (FY*A/FY*A) individuals, suggesting a gene-dosage effect. In further comparisons, we observed a higher prevalence of P. vivax infection in FY*A/FY*A (83/508 = 0.163) compared with FY*A/FY*Anull (2/23 = 0.087) individuals (odds ratio = 2.05, 95% confidence interval = 0.47–8.91). Emergence of FY*Anull in this population suggests that P. vivax is involved in selection of this erythroid polymorphism. This mutation would ultimately compromise α+-thalassemia/P. vivax-mediated protection against severe P. falciparum malaria.

Mapping regions containing binding residues within functional domains of Plasmodium vivax and Plasmodium knowlesi erythrocyte-binding proteins

Invasion of erythrocytes by malaria parasites is mediated by specific molecular interactions. Whereas Plasmodium vivax and Plasmodium knowlesi use the Duffy blood group antigen, Plasmodium falciparum uses sialic acid residues of glycophorin A as receptors to invade human erythrocytes. P. knowlesi uses the Duffy antigen as well as other receptors to invade rhesus erythrocytes by multiple pathways. Parasite ligands that bind these receptors belong to a family of erythrocyte-binding proteins (EBP). The EBP family includes the P. vivax and P. knowlesi Duffy-binding proteins, P. knowlesi β and γ proteins, which bind alternate receptors on rhesus erythrocytes, and P. falciparum erythrocyte-binding antigen (EBA-175), which binds sialic acid residues of human glycophorin A. Binding domains of each EBP lie in a conserved N-terminal cysteine-rich region, region II, which contains around 330 amino acids with 12 to 14 conserved cysteines. Regions containing binding residues have now been mapped within P. vivax and P. knowlesi β region II. Chimeric domains containing P. vivax region II sequences fused to P. knowlesi β region II sequences were expressed on the surface of COS cells and tested for binding to erythrocytes. Binding residues of P. vivax region II lie in a 170-aa stretch between cysteines 4 and 7, and binding residues of P. knowlesi β region II lie in a 53-aa stretch between cysteines 4 and 5. Mapping regions responsible for receptor recognition is an important step toward understanding the structural basis for the interaction of these parasite ligands with host receptors.

Plasmodium induces swelling-activated ClC-2 anion channels in the host erythrocyte

Intraerythrocytic growth of the human malaria parasite Plasmodium falciparum depends on delivery of nutrients. Moreover, infection challenges cell volume constancy of the host erythrocyte requiring enhanced activity of cell volume regulatory mechanisms. Patch clamp recording demonstrated inwardly and outwardly rectifying anion channels in infected but not in control erythrocytes. The molecular identity of those channels remained elusive. We show here for one channel type that voltage dependence, cell volume sensitivity, and activation by oxidation are identical to ClC-2. Moreover, Western blots and FACS analysis showed protein and functional ClC-2 expression in human erythrocytes and erythrocytes from wild type (Clcn2(+/+)) but not from Clcn2(-/-) mice. Finally, patch clamp recording revealed activation of volume-sensitive inwardly rectifying channels in Plasmodium berghei-infected Clcn2(+/+) but not Clcn2(-/-) erythrocytes. Erythrocytes from infected mice of both genotypes differed in cell volume and inhibition of ClC-2 by ZnCl(2) (1 mm) induced an increase of cell volume only in parasitized Clcn2(+/+) erythrocytes. Lack of ClC-2 did not inhibit P. berghei development in vivo nor substantially affect the mortality of infected mice. In conclusion, activation of host ClC-2 channels participates in the altered permeability of Plasmodium-infected erythrocytes but is not required for intraerythrocytic parasite survival.

The proteasome inhibitor MLN-273 blocks exoerythrocytic and erythrocytic development of Plasmodium parasites.

Protein degradation is regulated during the cell cycle of all eukaryotic cells and is mediated by the ubiquitin-proteasome pathway. Potent and specific peptide-derived inhibitors of the 20S proteasome have been developed recently as anti-cancer agents, based on their ability to induce apoptosis in rapidly dividing cells. Here, we tested a novel small molecule dipeptidyl boronic acid proteasome inhibitor, named MLN-273 on blood and liver stages of Plasmodium species, both of which undergo active replication, probably requiring extensive proteasome activity. The inhibitor blocked Plasmodium falciparum erythrocytic development at an early ring stage as well as P. berghei exoerythrocytic progression to schizonts. Importantly, neither uninfected erythrocytes nor hepatocytes were affected by the drug. MLN-273 caused an overall reduction in protein degradation in P. falciparum, as demonstrated by immunoblots using anti-ubiquitin antibodies to label ubiquitin-tagged protein conjugates. This led us to conclude that the target of the drug was the parasite proteasome. The fact that proteasome inhibitors are presently used as anti-cancer drugs in humans forms a solid basis for further development and makes them potentially attractive drugs also for malaria chemotherapy.

The Plasmodium serine-type SERA proteases display distinct expression patterns and non-essential in vivo roles during life cycle progression of the malaria parasite

Parasite proteases play key roles in several fundamental steps of the Plasmodium life cycle, including haemoglobin degradation, host cell invasion and parasite egress. Plasmodium exit from infected host cells appears to be mediated by a class of papain-like cysteine proteases called 'serine repeat antigens' (SERAs). A SERA subfamily, represented by Plasmodium falciparum SERA5, contains an atypical active site serine residue instead of a catalytic cysteine. Members of this SERAser subfamily are abundantly expressed in asexual blood stages, rendering them attractive drug and vaccine targets. In this study, we show by antibody localization and in vivo fluorescent tagging with the red fluorescent protein mCherry that the two P. berghei serine-type family members, PbSERA1 and PbSERA2, display differential expression towards the final stages of merozoite formation. Via targeted gene replacement, we generated single and double gene knockouts of the P. berghei SERAser genes. These loss-of-function lines progressed normally through the parasite life cycle, suggesting a specialized, non-vital role for serine-type SERAs in vivo. Parasites lacking PbSERAser showed increased expression of the cysteine-type PbSERA3. Compensatory mechanisms between distinct SERA subfamilies may thus explain the absence of phenotypical defect in SERAser disruptants, and challenge the suitability to develop potent antimalarial drugs based on specific inhibitors of Plasmodium serine-type SERAs.