Malaria transmission and naturally acquired immunity to PfEMP-1

Why there are so few gametocytes (the transmission stage of malaria) in the blood of humans infected with Plasmodium spp. is intriguing. This may be due either to reproductive restraint by the parasite or to unidentified gametocyte-specific immune-mediated clearance mechanisms. We propose another mechanism, a cross-stage immunity to Plasmodium falciparum erythrocyte membrane protein 1 (PfEMP-1). This molecule is expressed on the surface of the erythrocyte infected with either trophozoite or early gametocyte parasites. Immunoglobulin G antibodies to PfEMP-1, expressed on both life cycle stages, were measured in residents from an area where malaria is endemic, Papua New Guinea. Anti-PfEMP-1 prevalence increased with age, mirroring the decline in both the prevalence and the density of asexual and transmission stages in erythrocytes. These data led us to propose that immunity to PfEMP-1 may influence malaria transmission by regulation of the production of gametocytes. This regulation may be achieved in two ways: (i) by controlling asexual proliferation and density and (ii) by affecting gametocyte maturation.     

Quantification of Plasmodium falciparum gametocytes in differential stages of development by quantitative nucleic acid sequence-based amplification

Two quantitative nucleic acid sequence-based amplification assays (QT-NASBA) based on Pfs16 and Pfs25, have been developed to quantify sexual stage commitment and mature gametocytes of Plasmodium falciparum. Pfs16 mRNA is expressed in all sexual forms including sexually committed ring stages while expression of Pfs25 mRNA is restricted to late stage gametocytes. Both assays showed a sensitivity of one sexual stage parasite/microl of blood. Blood samples from experimentally infected non-immune human volunteers were tested for Plasmodium falciparum by standard microscopy, a previously developed asexual 18S rRNA QT-NASBA, Pfs16 and Pfs25 mRNA QT-NASBA. Pfs16 QT-NASBA was positive in 9 out of 10 volunteers within 48 h after first detection of 18S rRNA, mostly before or at the day of positive microscopy. In contrast, the Pfs25 mRNA QT-NASBA was negative during the 28 days of follow-up, but consistently positive in gametocyte samples from naturally infected Kenyan patients. These data suggest that sexual stage commitment can occur early in the blood-stage infection without successful maturation into infectious gametocytes. In conclusion, Pfs16 and Pfs25 QT-NASBA assays in combination with a previously developed asexual stage QT-NASBA allow for the separate quantification of all developmental stages present in the circulation. The application of sexual stage QT-NASBA assays may contribute to a better understanding of the biology and epidemiology of malaria transmission.     

Measuring resistant-genotype transmission of malaria parasites: challenges and prospects

Increased gametocytemia in infections with resistant strains of Plasmodium species and their enhanced transmissibility are a matter of concern in planning and evaluating the impact of malaria control strategies. Various studies have determined weekly gametocyte carriage in response to antimalarial drugs in clinical trials. The advent of molecular biology techniques makes it easy to detect and quantify gametocytes, the stages responsible for transmission, and to detect resistant genotypes of the parasite. With the validation of molecular markers of resistance to certain antimalarial drugs, there is a need to devise a simpler formula that could be used with these epidemiological antimalarial resistance tools. Theoretical models for transmission of resistant malaria parasites are difficult to deploy in epidemiological studies. Therefore, devising a simple formula that determines the potential resistant-genotype transmission of malaria parasites should provide further insights into understanding the spread of drug resistance. The present perspective discusses gametocytogenesis in the context of antimalarial treatment and drug resistance. It also highlights the difficulties in applying the available theoretical models of drug resistance transmission and suggests Rashad’s devised formula that could perhaps be used in determining potentially transmissible resistant genotypes as well as in mapping areas with high potential risk for the transmission of drug-resistant malaria. The suggested formula makes use of the data on gametocytes and resistant genotypes of malaria parasites, detected by molecular techniques in a certain geographical area within a particular point in time, to calculate the potential risk of resistant genotype transmission.     

Sex- and stage-specific reporter gene expression in Plasmodium falciparum

For malaria transmission, Plasmodium parasites must successfully complete gametocytogenesis in the vertebrate host. Differentiation into mature male or female Plasmodium falciparum gametocytes takes 9-12 days as the parasites pass through five distinct morphologic stages (I-V). To evaluate the signals controlling the initiation of stage- and/or sex-specific expression, reporter constructs containing the 5'-flanking regions (FR) of seven genes with distinct expression patterns through gametogenesis were developed. The regulatory information present in the 5'-FR of each selected gene was found to be sufficient to drive appropriate sex- and stage-specific reporter gene expression. The transformed parasite lines also provide in vivo markers to identify gametocytes at specific stages, including a subpopulation of schizonts that express early gametocyte markers.     

2-Cys Peroxiredoxin TPx-1 is involved in gametocyte development in Plasmodium berghei

Peroxiredoxins (Prxs) constitute a ubiquitous family of antioxidant enzymes involved in diverse cellular functions including cell proliferation and differentiation. To investigate the physiologic role of typical 2-Cys Prx in malaria parasites (TPx-1), we disrupted this gene in the rodent malaria parasite Plasmodium berghei (pbtpx-1). The gene-disrupted parasite (Prx KO) developed normally in mouse erythrocytes and multiplied at a rate similar to that of the parent strain (WT) during the experimental period. The normal growth rate was not altered after 10 passages, and the level of 8-hydroxy-2'-deoxyguanosine, which accumulates in the parasite genome during the cell cycle, was similar between Prx KO and WT. These results suggest that TPx-1 does not prevent parasite DNA oxidation, in contrast to mammalian Prx, and that it is not essential for asexual parasite growth in mouse erythrocytes. However, Prx KO produced up to 60% fewer gametocytes, sexual-stage parasites involved in the transition between the mammalian host and the mosquito, than WT did. The peak of gametocytemia was also delayed; however, the male/female ratio of gametocytes and the exflagellation activity of male gametocytes were normal. These results suggest that TPx-1 is required for normal gametocyte development but does not affect the male/female gametocyte ratio or male gametogenesis. Although the mechanism by which PbTPx-1 contributes to gametocyte development remains unknown, these findings suggest, for the first time, the involvement of Prx in the sexual development of the malaria parasite.     

Attraction between sexes: male–female gametocyte behaviour within a <Emphasis Type="Italic">Leucocytozoon toddi</Emphasis> (Haemosporida)

Understanding the breeding systems of Plasmodium, and the closely related Haemoproteus and Leucocytozoon (Apicomplexa: Haemosporida), is fundamental to virulence and transmission research. We report an unusual binding behaviour between gametocytes of Leucocytozoon toddi. This aggregative behaviour was notably characterised by a disparity in the likelihood of clustering by female and male gametocytes. Thus, indicating a possible difference in the ‘stickiness’ of gametocytes per sex. Overall, 12% of gametocytes in this high-parasitaemia infection (0.269 gametocytes per 100 red blood cells (RBCs)) were incorporated into aggregations involving substantial contact. The gametocyte sexual combinations within aggregations varied significantly from expected according to the background 0.49 sex ratio within this sample, with female–female contacts occurring more and male–male contacts occurring less frequently than expected. A second L. toddi (identical for 709 bp of the cyt b mitochondrial gene) with lower parasitemia (0.035 gametocytes per 100 RBCs) showed no significant binding. Interestingly, the ratios of male gametocytes in both of these parasites were greater than expected under sex-ratio theory and similar to the 50% observed in species with syzygy breeding strategies. We discuss the ramifications of this observation in terms of sex-ratio theory and breeding strategies and provide speculative explanations for this unusual gametocyte behaviour. Research funding was provided by the Institut de Recherche pour le Développement, l’Institut Pasteur, Grand Programme Horizontal Anophèles de l’Institut Pasteur and World Wide Fund for Nature.     

A model for sequestration of the transmission stages of Plasmodium falciparum: adhesion of gametocyte-infected erythrocytes to human bone marrow cells

With the aim of developing an appropriate in vitro model of the sequestration of developing Plasmodium falciparum sexual-stage parasites, we have investigated the cytoadherence of gametocytes to human bone marrow cells of stromal and endothelial origin. Developing stage III and IV gametocytes, but not mature stage V gametocytes, adhere to bone marrow cells in significantly higher densities than do asexual-stage parasites, although these adhesion densities are severalfold lower than those encountered in classical CD36-dependent assays of P. falciparum cytoadherence. This implies that developing gametocytes undergo a transition from high-avidity, CD36-mediated adhesion during stages I and II to a lower-avidity adhesion during stages III and IV. We show that this adhesion is CD36 independent, fixation sensitive, stimulated by tumor necrosis factor alpha, and dependent on divalent cations and serum components. These data suggest that gametocytes and asexual parasites utilize distinct sets of receptors for adhesion during development in their respective sequestered niches. To identify receptors for gametocyte-specific adhesion of infected erythrocytes to bone marrow cells, we tested a large panel of antibodies for the ability to inhibit cytoadherence. Our results implicate ICAM-1, CD49c, CD166, and CD164 as candidate bone marrow cell receptors for gametocyte adhesion.     

Molecular epidemiology of genital Chlamydia trachomatis infection in high-risk women in Senegal, West Africa

The prevalence and heterogeneity of Chlamydia trachomatis infections in a cohort of female sex workers in Dakar (Senegal) were determined by using endocervical-swab-based PCR DNA amplification assays. The overall prevalence of cervical chlamydial infection was 28.5% (206 of 722), and most of these infections were asymptomatic. An increased number of sexual partners was significantly associated with infection (adjusted odds ratio [AOR] = 1.37; 95% confidence interval [CI] = 1.06 to 1.77), while the presence of a yeast infection was negatively associated with chlamydial infection (AOR = 0.28; 95% CI = 0.10 to 0.83). Six different C. trachomatis genotypes were identified based on phylogenetic analysis of the omp1 gene sequences. Interestingly, genotype E predominated (47.6%) and was not associated with visible signs of cervical inflammation compared to non-E genotypes (P < 0.05). Overall, the high rate of asymptomatic C. trachomatis infection by genotype E may suggest genotype-specific properties that confer a transmission advantage in this high risk population.     

Impact of mosquito bites on asexual parasite density and gametocyte prevalence in asymptomatic chronic Plasmodium falciparum infections and correlation with IgE and IgG titers

An immunomodulatory role of arthropod saliva has been well documented, but evidence for an effect on Plasmodium sp. infectiousness remains controversial. Mosquito saliva may orient the immune response toward a Th2 profile, thereby priming a Th2 response against subsequent antigens, including Plasmodium. Orientation toward a Th1 versus a Th2 profile promotes IgG and IgE proliferation, respectively, where the former is crucial for the development of an efficient antiparasite immune response. Here we assessed the direct effect of mosquito bites on the density of Plasmodium falciparum asexual parasites and the prevalence of gametocytes in chronic, asymptomatic infections in a longitudinal cohort study of seasonal transmission. We additionally correlated these parasitological measures with IgE and IgG antiparasite and anti-salivary gland extract titers. The mosquito biting density was positively correlated with the asexual parasite density but not asexual parasite prevalence and was negatively correlated with gametocyte prevalence. Individual anti-salivary gland IgE titers were also negatively correlated with gametocyte carriage and were strongly positively correlated with antiparasite IgE titers, consistent with the hypothesis that mosquito bites predispose individuals to develop an IgE antiparasite response. We provide evidence that mosquito bites have an impact on asymptomatic infections and differentially so for the production of asexual and sexual parasites. An increased research focus on the immunological impact of mosquito bites during asymptomatic infections is warranted, to establish whether strategies targeting the immune response to saliva can reduce the duration of infection and the onward transmission of the parasite.     

Development and Validation of a Quantitative PCR Assay Using Multiplexed Hydrolysis Probes for Detection and Quantification of Theileria orientalis Isolates and Differentiation of Clinically Relevant Subtypes

Theileria orientalis is an emerging pathogen of cattle in Asia, Australia, and New Zealand. This organism is a vector-borne hemoprotozoan that causes clinical disease characterized by anemia, abortion, and death, as well as persistent subclinical infections. Molecular methods of diagnosis are preferred due to their sensitivity and utility in differentiating between pathogenic and apathogenic genotypes. Conventional PCR (cPCR) assays for T. orientalis detection and typing are laborious and do not provide an estimate of parasite load. Current real-time PCR assays cannot differentiate between clinically relevant and benign genotypes or are only semiquantitative without a defined clinical threshold. Here, we developed and validated a hydrolysis probe quantitative PCR (qPCR) assay which universally detects and quantifies T. orientalis and identifies the clinically associated Ikeda and Chitose genotypes (UIC assay). Comparison of the UIC assay results with previously validated universal and genotype-specific cPCR results demonstrated that qPCR detects and differentiates T. orientalis with high sensitivity and specificiy. Comparison of quantitative results based on percent parasitemia, determined via blood film analysis and packed cell volume (PCV) revealed significant positive and negative correlations, respectively. One-way analysis of variance (ANOVA) indicated that blood samples from animals with clinical signs of disease contained statistically higher concentrations of T. orientalis DNA than animals with subclinical infections. We propose clinical thresholds to assist in classifying high-, moderate-, and low-level infections and describe how parasite load and the presence of the Ikeda and Chitose genotypes relate to disease.     

Strategic use of antimalarial drugs that block falciparum malaria parasite transmission to mosquitoes to achieve local malaria elimination

The ultimate aim of malaria chemotherapy is not only to treat symptomatic infection but also to reduce transmission potential. With the absence of clinically proven vaccines, drug-mediated blocking of malaria transmission gains growing interest in the research agenda for malaria control and elimination. In addition to the limited arsenal of antimalarials available, the situation is further complicated by the fact that most commonly used antimalarials are being extensively resisted by the parasite and do not assist in blocking its transmission to vectors. Most antimalarials do not exhibit gametocytocidal and/ or sporontocidal activity against the sexual stages of Plasmodium falciparum but may even enhance gametocytogenesis and gametocyte transmissibility. Artemisinin derivatives and 8-aminoquinolines are useful transmission-blocking antimalarials whose optimal actions are on different stages of gametocytes. Transmission control interventions that include gametocytocides covering the spectrum of gametocyte development should be used to reduce and, if possible, stop transmission and infectivity of gametocytes to mosquitoes. Potent gametocytocidal drugs could also help deter the spread of antimalarial drug resistance. Novel proof-of-concept compounds with gametocytocidal activity, such as trioxaquines, synthetic endoperoxides, and spiroindolone, should be further tested for possible clinical utility before investigating the possibility of integrating them in transmission-reducing interventions. Strategic use of potent gametocytocides at appropriate timing with artemisinin-based combination therapies should be given attention, at least, in the short run. This review highlights the role that antimalarials could play in blocking gametocyte transmission and infectivity to mosquitoes and, hence, in reducing the potential of falciparum malaria transmissibility and drug resistance spread.     

Improved synchronous production of Plasmodium falciparum gametocytes in vitro

The sexual stages of the Plasmodium falciparum life cycle are attractive targets for vaccines and transmission blocking drugs. Difficulties in culturing and obtaining large amounts of sexual stage P. falciparum parasites, particularly early stages, have often limited research progress in this area. We present a new protocol which simplifies the process of stimulating gametocytogenesis leading to improved synchronous gametocyte production. This new method can be adapted to enrich for early stage gametocytes (I and II) with a higher degree of purity than has previously been achieved, using MACS magnetic affinity columns. The protocol described lends itself to large scale culturing and harvesting of synchronous parasites suitable for biochemical assays, northern blots, flow cytometry, microarrays and proteomic analysis.     

Cytokine-mediated inactivation of malarial gametocytes is dependent on the presence of white blood cells and involves reactive nitrogen intermediates.

Supernatants of human peripheral blood mononuclear cells (PBMC) incubated for 24 hr in the presence of extracts of freeze-thawed blood stage parasites of Plasmodium vivax or P. falciparum mediate inactivation of gametocytes of either species when incubated in vitro with whole human blood cells. Cultured P. falciparum gametocytes incubated with these malaria extract-stimulated PBMC supernatants in the presence of human blood from which white blood cells (WBC) had been removed were not inactivated. Thus the effects of the PBMC supernatants on gametocyte infectivity were dependent upon the presence of WBC. The suppressive effects mediated in the presence of WBC could be partially reversed in the presence of concentrations of 1 mM or higher of the L-arginine analogue NGL-monomethyl arginine acetate (L-NMMA). Our results indicate that the effects of WBC in inactivating gametocytes are due, at least in part, to a mechanism involving an L-arginine-dependent pathway. Previous studies have shown that the mediators of gametocyte inactivation in the stimulated PBMC supernatants comprised tumour necrosis factor (TNF) acting in conjunction with unidentified, but essential, 'complementary factors'. In the present study we show that these mediators, TNF and complementary factors, affect gametocytes indirectly through their interaction with WBC.     

Developmental gene expression of a 230-kilodalton macrogamete-specific protein of the avian coccidial parasite, Eimeria maxima.

We prepared a cDNA library from gametocytes of Eimeria maxima and screened it using antibodies raised against an 82-kDa gametocyte antigen. One cDNA clone designated pEM230 was isolated and characterized. It encodes a portion of a 230-kDa gametocyte protein and its DNA sequence shows the presence of several tandem repeats of 42 bp. In order to determine the stage and sex specificity of the mRNA for the 230-kDa protein, Northern blotting and in situ hybridization studies were performed. The 230-kDa protein is encoded for by a 7 kb mRNA, which is expressed exclusively during the macrogamete stage with no detectable expression seen in any other stage of parasite development.     

Parasitemia characteristics of Plasmodium vivax malaria patients in the Republic of Korea

Parasitemia characteristics of Plasmodium vivax malaria in temperate regions may differ from those in tropical zones. However, most parasitological and clinical features of P. vivax malaria have been investigated in the latter. In this study, we investigated 383 malaria patients to clarify the parasitemia characteristics of a P. vivax strain in the Republic of Korea (ROK). The mean parasitemia (8,396/μL) was less than half of tropical P. vivax malaria, and multiple invasions of erythrocytes were not rare (53.5% of the patients, 2.4% of the total investigated RBCs), but less than the observations in tropical zones. The intervals between the first symptom onset and diagnosis were significantly longer in gametocyte (+) patients than in gametocyte (-) patients. Only half of the total patients had both genders of gametocytes (191 of 353), and the male gametocyte density (169/μL) was lower than that of P. vivax strains of a previous study. Multiple invasions of erythrocytes and gametocytemia were coincident factors of the degree of anemia in P. vivax malaria. The present findings demonstrate the P. vivax strain in ROK reveals relatively low parasitemia and low male to female gametocyte ratio. The low ratio may be related with low transmission efficacy.     

Epidemiology of malaria in a village of Sudanese savannah area in Mali (Bancoumana). 2. Entomo-parasitological and clinical study

We carried out five cross sectional surveys between 1993 and 1994 to assess the epidemiology of malaria in the village of Bancoumana, located in the Sudanese savannah areas of Mali. Each survey included a collection of entomological, clinical, parasitological and immunological data. The study population involved 1600 children from six months to 9 years of age. The main vector was Anopheles gambiae s.l., man bite rate and entomological inoculation rate were maximum respectively in August (peak of the transmission season) and October (end of transmission season). Plasmodium. falciparum was the main parasite species observed. Spleen enlargement rate, parasite rate, gametocyte rate and parasite density varied significantly with age and season. The parasite rate, gametocyte rate and parasite density were significantly low in October 1994 compared with October 1993 while the entomologic parameter did not show any variation over the two years. This reduction of parasitologic index between 1993 and 1994 may be related to an increase of anti-malarial drug use in the population. Our results show that malaria is hyperendemic in the village of Bancoumana.     

Molecular analysis of Plasmodium falciparum infections in man]

Plasmodium falciparum diversity has been analysed in two Senegalese villages with different transmission conditions and distinct kinetics of immunity acquisition. A very large allelic polymorphism was observed in both villages, with a similar number of alleles but quite distinct allelic frequencies, indicating a substantial micro-geographical heterogeneity of malaria parasite populations. In addition, the molecular characteristics of the infections differed in both villages. As in most endemic areas, many infected subjects carry multiple parasite clones. In Dielmo, the number of distinct clones hosted decreases at the age of acquisition of an efficient immunity. There was no influence of age on the number of clones hosted in Ndiop where adults experience clinical attacks. This indicates that complexity reflects acquired immunity. The precise longitudinal follow-up of parasitaemia, clinical signs and parasite genetic characteristics showed a rapid turn over of parasite populations in the peripheral blood during the transmission season, suggesting that immunity does not prevent infection but restricts multiplication of numerous genotypes at the erythrocytic stage. Clinical malaria occurs after a rapid, apparently unrestricted growth of recently inoculated parasites. The successive clinical attacks experienced by children are associated with genotypes different for each attack and different from those that the child carried during preceding asymptomatic phases. These data indicate that parasite diversity contributes to the pathology of infection and that control of parasite density, which is at least in part strain-specific, is an essential element of protection against malaria clinical attacks.