Multifaceted Role of Heme during Severe Plasmodium falciparum Infections in India

Several immunomodulatory factors are involved in malaria pathogenesis. Among them, heme has been shown to play a role in the pathophysiology of severe malaria in rodents, but its role in human severe malaria remains unclear. Circulating levels of total heme and its main scavenger, hemopexin, along with cytokine/chemokine levels and biological parameters, including hemoglobin and creatinine levels, as well as transaminase activities, were measured in the plasma of 237 Plasmodium falciparum-infected patients living in the state of Odisha, India, where malaria is endemic. All patients were categorized into well-defined groups of mild malaria, cerebral malaria (CM), or severe noncerebral malaria, which included acute renal failure (ARF) and hepatopathy. Our results show a significant increase in total plasma heme levels with malaria severity, especially for CM and malarial ARF. Spearman rank correlation and canonical correlation analyses have shown a correlation between total heme, hemopexin, interleukin-10, tumor necrosis factor alpha, gamma interferon-induced protein 10 (IP-10), and monocyte chemotactic protein 1 (MCP-1) levels. In addition, canonical correlations revealed that heme, along with IP-10, was associated with the CM pathophysiology, whereas both IP-10 and MCP-1 together with heme discriminated ARF. Altogether, our data indicate that heme, in association with cytokines and chemokines, is involved in the pathophysiology of both CM and ARF but through different mechanisms.

A large proportion of asymptomatic Plasmodium infections with low and sub-microscopic parasite densities in the low transmission setting of Temotu Province, Solomon Islands : challenges for malaria diagnostics in an elimination setting

Background Many countries are scaling up malaria interventions towards elimination. This transition changes demands on malaria diagnostics from diagnosing ill patients to detecting parasites in all carriers including asymptomatic infections and infections with low parasite densities. Detection methods suitable to local malaria epidemiology must be selected prior to transitioning a malaria control programme to elimination. A baseline malaria survey conducted in Temotu Province, Solomon Islands in late 2008, as the first step in a provincial malaria elimination programme, provided malaria epidemiology data and an opportunity to assess how well different diagnostic methods performed in this setting. Methods During the survey, 9,491 blood samples were collected and examined by microscopy for Plasmodium species and density, with a subset also examined by polymerase chain reaction (PCR) and rapid diagnostic tests (RDTs). The performances of these diagnostic methods were compared. Results A total of 256 samples were positive by microscopy, giving a point prevalence of 2.7%. The species distribution was 17.5% Plasmodium falciparum and 82.4% Plasmodium vivax. In this low transmission setting, only 17.8% of the P. falciparum and 2.9% of P. vivax infected subjects were febrile (≥38°C) at the time of the survey. A significant proportion of infections detected by microscopy, 40% and 65.6% for P. falciparum and P. vivax respectively, had parasite density below 100/μL. There was an age correlation for the proportion of parasite density below 100/μL for P. vivax infections, but not for P. falciparum infections. PCR detected substantially more infections than microscopy (point prevalence of 8.71%), indicating a large number of subjects had sub-microscopic parasitemia. The concordance between PCR and microscopy in detecting single species was greater for P. vivax (135/162) compared to P. falciparum (36/118). The malaria RDT detected the 12 microscopy and PCR positive P. falciparum, but failed to detect 12/13 microscopy and PCR positive P. vivax infections. Conclusion Asymptomatic malaria infections and infections with low and sub-microscopic parasite densities are highly prevalent in Temotu province where malaria transmission is low. This presents a challenge for elimination since the large proportion of the parasite reservoir will not be detected by standard active and passive case detection. Therefore effective mass screening and treatment campaigns will most likely need more sensitive assays such as a field deployable molecular based assay.

Systematic review of sub-microscopic P. vivax infections : prevalence and determining factors

Background Sub-microscopic (SM) Plasmodium infections represent transmission reservoirs that could jeopardise malaria elimination goals. A better understanding of the epidemiology of these infections and factors contributing to their occurrence will inform effective elimination strategies. While the epidemiology of SM P. falciparum infections has been documented, that of SM P. vivax infections has not been summarised. The objective of this study is to address this deficiency. Methodology/Principal Findings A systematic search of PubMed was conducted, and results of both light microscopy (LM) and polymerase chain reaction (PCR)-based diagnostic tests for P. vivax from 44 cross-sectional surveys or screening studies of clinical malaria suspects were analysed. Analysis revealed that SM P. vivax is prevalent across different geographic areas with varying transmission intensities. On average, the prevalence of SM P. vivax in cross-sectional surveys was 10.9%, constituting 67.0% of all P. vivax infections detected by PCR. The relative proportion of SM P. vivax is significantly higher than that of the sympatric P. falciparum in these settings. A positive relationship exists between PCR and LM P. vivax prevalence, while there is a negative relationship between the proportion of SM P. vivax and the LM prevalence for P. vivax. Amongst clinical malaria suspects, however, SM P. vivax was not identified. Conclusions/Significance SM P. vivax is prevalent across different geographic areas, particularly areas with relatively low transmission intensity. Diagnostic tools with sensitivity greater than that of LM are required for detecting these infection reservoirs. In contrast, SM P. vivax is not prevalent in clinical malaria suspects, supporting the recommended use of quality LM and rapid diagnostic tests in clinical case management. These findings enable malaria control and elimination programs to estimate the prevalence and proportion of SM P. vivax infections in their settings, and develop appropriate elimination strategies to tackle SM P. vivax to interrupt transmission.

Pan-Plasmodium band sensitivity for Plasmodium falciparum detection in combination malaria rapid diagnostic tests and implications for clinical management

Background: Malaria rapid diagnostic tests (RDTs) are appropriate for case management, but persistent antigenaemia is a concern for HRP2-detecting RDTs in endemic areas. It has been suggested that pan-pLDH test bands on combination RDTs could be used to distinguish persistent antigenaemia from active Plasmodium falciparum infection, however this assumes all active infections produce positive results on both bands of RDTs, an assertion that has not been demonstrated. Methods: In this study, data generated during the WHO-FIND product testing programme for malaria RDTs was reviewed to investigate the reactivity of individual test bands against P. falciparum in 18 combination RDTs. Each product was tested against multiple wild-type P. falciparum only samples. Antigen levels were measured by quantitative ELISA for HRP2, pLDH and aldolase. Results: When tested against P. falciparum samples at 200 parasites/μL, 92% of RDTs were positive; 57% of these on both the P. falciparum and pan bands, while 43% were positive on the P. falciparum band only. There was a relationship between antigen concentration and band positivity; ≥4 ng/mL of HRP2 produced positive results in more than 95% of P. falciparum bands, while ≥45 ng/mL of pLDH was required for at least 90% of pan bands to be positive. Conclusions: In active P. falciparum infections it is common for combination RDTs to return a positive HRP2 band combined with a negative pan-pLDH band, and when both bands are positive, often the pan band is faint. Thus active infections could be missed if the presence of a HRP2 band in the absence of a pan band is interpreted as being caused solely by persistent antigenaemia.

Plasmodium vivax: Microsatellite analysis of multiple-clone infections

We used mixtures of genomic DNA from two genetically distinct isolates from Brazil, 42M and 312M, to investigate how accurately 12-locus microsatellite typing describes the overall genetic diversity and characterizes multilocus haplotypes in multiple-clone Plasmodium vivax infections. We found varying PCR amplification efficiencies of microsatellite alleles; for example, from the same 1:1 mixture of 42M and 312M DNA we amplified predominantly 312M-type alleles at 10 loci and 42M-type alleles at 2 loci. All microsatellite alleles were accurately scored in 1:0.5 and 1:0.25 312M:42M DNA mixtures, even when minor peak heights did not meet previously suggested criteria for minor allele detection in multiple-clone infections. Relative proportions of major and minor alleles were unaffected by multiple displacement amplification of template DNA prior to PCR-based microsatellite typing. Although microsatellite typing may detect minor alleles in clone mixtures, amplification biases may lead to inaccurate assignment of predominant haplotypes in multiple-clone P. vivax infections. (C) 2008 Elsevier Inc. All rights reserved.

Modeling the development of acquired clinical immunity to Plasmodium falciparum malaria

Individuals living in regions where malaria is endemic develop an acquired immunity to malaria which enables them to remain asymptomatic while still carrying parasites. Field studies indicate that cumulative exposure to a variety of diverse Plasmodium parasites is required for the transition from symptomatic to asymptomatic malaria. This study used a simulation model of the within-host dynamics of P. falciparum to investigate the development of acquired clinical immunity under different transmission conditions and levels of parasite diversity. Antibodies developed to P. falciparum erythrocyte membrane protein 1 (PfEMP1), a clonally variant molecule, were assumed to be a key human immunological response to P. falciparum infection, along with responses to clonally conserved but polymorphic antigens. The time to the development of clinical immunity was found to be proportional to parasite diversity and inversely proportional to transmission intensity. The effect of early termination of symptomatic infections by chemotherapy was investigated and found not to inhibit the host's ability to develop acquired immunity. However, the time required to achieve this state was approximately double that compared to when no treatment was administered. This study demonstrates that an immune response primarily targeted against PfEMP1 has the ability to reduce clinical symptoms of infections irrespective of whether treatment is administered, supporting its role in the development of acquired clinical immunity. The results also illustrate a novel use for simulation models of P. falciparum infections, investigation of the influence of intervention strategies on the development of naturally acquired clinical immunity.

Potencies of human immunodeficiency virus protease inhibitors in vitro against Plasmodium falciparum and in vivo against murine malaria

Parasite resistance to antimalarial drugs is a serious threat to human health, and novel agents that act on enzymes essential for parasite metabolism, such as proteases, are attractive targets for drug development. Recent studies have shown that clinically utilized human immunodeficiency virus (HIV) protease inhibitors can inhibit the in vitro growth of Plasmodium falciparum at or below concentrations found in human plasma after oral drug administration. The most potent in vitro antimalarial effects have been obtained for parasites treated with saquinavir, ritonavir, or lopinavir, findings confirmed in this study for a genetically distinct P. falciparum line (3D7). To investigate the potential in vivo activity of antiretroviral protease inhibitors (ARPIs) against malaria, we examined the effect of ARPI combinations in a murine model of malaria. In mice infected with Plasmodium chabaudi AS and treated orally with ritonavir-saquinavir or ritonavir-lopinavir, a delay in patency and a significant attenuation of parasitemia were observed. Using modeling and ligand docking studies we examined putative ligand binding sites of ARPIs in aspartyl proteases of P. falciparum (plasmepsins II and IV) and P. chabaudi (plasmepsin) and found that these in silico analyses support the antimalarial activity hypothesized to be mediated through inhibition of these enzymes. In addition, in vitro enzyme assays demonstrated that P. falciparum plasmepsins II and IV are both inhibited by the ARPIs saquinavir, ritonavir, and lopinavir. The combined results suggest that ARPIs have useful antimalarial activity that may be especially relevant in geographical regions where HIV and P. falciparum infections are both endemic.

Peroxide-based hybrid and prodrug antimalarials to deliver cysteine protease inhibitors into the parasitic food vacuole

Tese de doutoramento, Farmácia (Química Farmacêutica e Terapêutica), Universidade de Lisboa, Faculdade de Farmácia, 2014

Avaliação da incidência da deficiência de Glicose-6-Fosfato Desidrogenase (G6PD) e perfil hematológico em indivíduos de uma região de Rondônia

O estudo compreendeu a avaliação da deficiência de Glicose-6-Fosfato Desidrogenase (G6PD) e perfil hematológico em 122 indivíduos (69 homens e 53 mulheres), com idade variando entre 3 a 84 anos, selecionados conforme a aceitação em participação no estudo, residentes na área urbana e rural do município de Porto Velho, Rondônia, Brasil, no período de julho de 2003 a agosto de 2004. A análise foi realizada utilizando-se o método da glicose NaNO2, e hemograma completo. Foram detectados quatro indivíduos do sexo masculino com deficiência da G6PD, sendo 5,8% entre os homens e 3,3% do total analisado. Dos indivíduos com deficiência da G6PD nenhum apresentava malária, através de diagnóstico realizado pela gota espessa corado pelo Giemsa. Entre os homens, 19 (27,5%) apresentaram malária, sendo 15 por Plasmodium vivax e quatro por Plasmodium falciparum; 48 (69,5%) apresentaram valores de hemoglobina abaixo de 14,0 g/dl, e 26 (37,6%) apresentaram valores eritrocitários abaixo do 4,5 milhões/mm³. Entre as mulheres apenas duas (3,7%) apresentaram malária por Plasmodium vivax; 24 (45,2%) apresentaram valores de hemoglobina abaixo de 12,0 g/dl, e 12 (22,6%) apresentaram massa eritrocitária abaixo de 4,0 milhões/mm³. A eosinofilia esteve presente em 47 (68,1%) dos homens e em 34 (64,1%) das mulheres. A incidência de deficiência da G6PD foi significativa na população masculina que procurou assistência médica devido a sintomas febris. Considerando que a primaquina é utilizada para o tratamento da malária vivax e falciparum, o risco de ocorrência de hemólise intravascular grave entre os indivíduos é significante. O teste utilizado é muito simples e de baixo custo e sugerimos a adoção desta metodologia na rotina dos laboratórios de atendimento público em áreas endêmicas de malária.

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.

On the pathogenesis of Plasmodium vivax malaria: Perspectives from the Brazilian field

Life-threatening Plasmodium vivax malaria cases, while uncommon, have been reported since the early 20th century. Unfortunately, the pathogenesis of these severe vivax malaria cases is still poorly understood. In Brazil, the proportion of vivax malaria cases has been steadily increasing, as have the number of cases presenting serious clinical complications. The most frequent syndromes associated with severe vivax malaria in Brazil are severe anaemia and acute respiratory distress. Additionally, P. vivax infection may also result in complications associated with pregnancy. Here, we review the latest findings on severe vivax malaria in Brazil. We also discuss how the development of targeted field research infrastructure in Brazil is providing clinical and ex vivo experimental data that benefits local and international efforts to understand the pathogenesis of P. vivax. (C) 2012 Australian Society for Parasitology Inc. Published by Elsevier Ltd. All rights reserved.

Amazonian malaria: Asymptomatic human reservoirs, diagnostic challenges, environmentally driven changes in mosquito vector populations, and the mandate for sustainable control strategies

Across the Americas and the Caribbean, nearly 561,000 slide-confirmed malaria infections were reported officially in 2008. The nine Amazonian countries accounted for 89% of these infections; Brazil and Peru alone contributed 56% and 7% of them, respectively. Local populations of the relatively neglected parasite Plasmodium vivax, which currently accounts for 77% of the regional malaria burden, are extremely diverse genetically and geographically structured. At a time when malaria elimination is placed on the public health agenda of several endemic countries, it remains unclear why malaria proved so difficult to control in areas of relatively low levels of transmission such as the Amazon Basin. We hypothesize that asymptomatic parasite carriage and massive environmental changes that affect vector abundance and behavior are major contributors to malaria transmission in epidemiologically diverse areas across the Amazon Basin. Here we review available data supporting this hypothesis and discuss their implications for current and future malaria intervention policies in the region. Given that locally generated scientific evidence is urgently required to support malaria control interventions in Amazonia, we briefly describe the aims of our current field-oriented malaria research in rural villages and gold-mining enclaves in Peru and a recently opened agricultural settlement in Brazil. (C) 2011 Elsevier B.V. All rights reserved.

Heat Shock Protein 90 as a Drug Target against Protozoan Infections Biochemical characterization of HSP90 From plasmodium falciparum and trypanosoma evansi and evaluation of its inhibitor as a candidate drug

Using a pharmacological inhibitor of Hsp90 in cultured malarial parasite, we have previously implicated Plasmodium falciparum Hsp90 (PfHsp90) as a drug target against malaria. In this study, we have biochemically characterized PfHsp90 in terms of its ATPase activity and interaction with its inhibitor geldanamycin (GA) and evaluated its potential as a drug target in a preclinical mouse model of malaria. In addition, we have explored the potential of Hsp90 inhibitors as drugs for the treatment of Trypanosoma infection in animals. Our studies with full-length PfHsp90 showed it to have the highest ATPase activity of all known Hsp90s; its ATPase activity was 6 times higher than that of human Hsp90. Also, GA brought about more robust inhibition of PfHsp90 ATPase activity as compared with human Hsp90. Mass spectrometric analysis of PfHsp90 expressed in P. falciparum identified a site of acetylation that overlapped with Aha1 and p23 binding domain, suggesting its role in modulating Hsp90 multichaperone complex assembly. Indeed, treatment of P. falciparum cultures with a histone deacetylase inhibitor resulted in a partial dissociation of PfHsp90 complex. Furthermore, we found a well known, semisynthetic Hsp90 inhibitor, namely 17-(allylamino)-17-demethoxygeldanamycin, to be effective in attenuating parasite growth and prolonging survival in a mouse model of malaria. We also characterized GA binding to Hsp90 from another protozoan parasite, namely Trypanosoma evansi. We found 17-(allylamino)-17-demethoxygeldanamycin to potently inhibit T. evansi growth in a mouse model of trypanosomiasis. In all, our biochemical characterization, drug interaction, and animal studies supported Hsp90 as a drug target and its inhibitor as a potential drug against protozoan diseases.

Expression of Plasmodium falciparum 3D7 STEVOR proteins for evaluation of antibody responses following malaria infections in naïve infants

Clinical immunity to Plasmodium falciparum malaria develops after repeated exposure to the parasite. At least 2 P. falciparum variant antigens encoded by multicopy gene families (var and rif) are targets of this adaptive antibody-mediated immunity. A third multigene family of variant antigens comprises the stevor genes. Here, 4 different stevor sequences were selected for cloning and expression in Escherichia coli and His6-tagged fusion proteins were used for assessing the development of immunity. In a cross-sectional analysis of clinically immune adults living in a malaria endemic area in Ghana, high levels of anti-STEVOR IgG antibody titres were determined in ELISA. A cross-sectional study of 90 nine-month-old Ghanaian infants using 1 recombinant STEVOR showed that the antibody responses correlated positively with the number of parasitaemia episodes. In a longitudinal investigation of 17 immunologically naïve 9-month-old infants, 3 different patterns of anti-STEVOR antibody responses could be distinguished (high, transient and low). Children with high anti-STEVOR-antibody levels exhibited an elevated risk for developing parasitaemia episodes. Overall, a protective effect could not be attributed to antibodies against the STEVOR proteins chosen for the study presented here.

THE CLINICAL AND PARASITOLOGIC COURSES OF PLASMODIUM FALCIPARUM AND PLASMODIUM VIVAX INFECTIONS IN HUMANS: AN ANALYSIS OF 432 EPISODES OF INDUCED MALARIA IN THE JESSUP VOLUNTEER STUDIES, 1966-1975

The clinical records of 432 P. falciparum and P. vivax infected volunteer male inmates of the Maryland House of Corrections in Jessup, Maryland, were studied to determine (1) the clinical and parasitologic courses of infections in both parasite species, and (2) the influence of previous homologous and/or heterologous strain exposures on subsequent infections. The clinical and parasitologic courses of infection with both P. falciparum and P. vivax species indicated that: (a) there were characteristic strain related differences between P. falciparum and P. vivax. P. falciparum strains were more apt to cause severe infections than P. vivax strains. (b) Blood-induced infections produced significantly shorter prepatent and incubation periods than mosquito-induced. (c) Blacks tolerated the infections better than whites and, (d) homologous and heterologous strain immunities persisted with previous malaria history. In previously exposed cases, clinical manifestations were moderate, peak fever lowered, and peak parasitemias limited. (e) Anti-malarial drugs were effective in reducing sexual and asexual forms of the malaria parasite, and limiting peak fevers, irrespective of method of induction, race, parasite strain and species, and drug type used.^ Given these findings, and the current worldwide resurgence of malaria, this study has major implications in terms of setting malaria control and public health policies in both developed and developing countries.^