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Abstract Background Malaria parasites (Plasmodium sp.), including new species, have recently been discovered as low grade mixed infections in three wild chimpanzees (Pan troglodytes schweinfurthii) sampled randomly in Kibale National Park, Uganda. This suggested a high prevalence of malaria infection in this community. The clinical course of malaria in chimpanzees and the species of the vectors that transmit their parasites are not known. The fact that these apes display a specific behaviour in which they consume plant parts of low nutritional value but that contain compounds with anti-malarial properties suggests that the apes health might be affected by the parasite. The avoidance of the night-biting anopheline mosquitoes is another potential behavioural adaptation that would lead to a decrease in the number of infectious bites and consequently malaria. Methods Mosquitoes were collected over two years using suction-light traps and yeast-generated CO2 traps at the nesting and the feeding sites of two chimpanzee communities in Kibale National Park. The species of the female Anopheles caught were then determined and the presence of Plasmodium was sought in these insects by PCR amplification. Results The mosquito catches yielded a total of 309 female Anopheles specimens, the only known vectors of malaria parasites of mammalians. These specimens belonged to 10 species, of which Anopheles implexus, Anopheles vinckei and Anopheles demeilloni dominated. Sensitive DNA amplification techniques failed to detect any Plasmodium-positive Anopheles specimens. Humidity and trap height influenced the Anopheles capture success, and there was a negative correlation between nest numbers and mosquito abundance. The anopheline mosquitoes were also less diverse and numerous in sites where chimpanzees were nesting as compared to those where they were feeding. Conclusions These observations suggest that the sites where chimpanzees build their nests every night might be selected, at least in part, in order to minimize contact with anopheline mosquitoes, which might lead to a reduced risk in acquiring malaria infections.

Abstract Background Timing the origin of human malarias has been a focus of great interest. Previous studies on the mitochondrial genome concluded that Plasmodium in primates, including those parasitic to humans, radiated relatively recently during a process where host switches were common. Those investigations, however, assumed constant rate of evolution and tightly bound (fixed) calibration points based on host fossils or host distribution. We investigate the effect of such assumptions using different molecular dating methods. We include parasites from Lemuroidea since their distribution provides an external validation to time estimates allowing us to disregard scenarios that cannot explain their introduction in Madagascar. Results We reject the assumption that the Plasmodium mitochondrial genome, as a unit or each gene separately, evolves at a constant rate. Our analyses show that Lemuroidea parasites are a monophyletic group that shares a common ancestor with all Catarrhini malarias except those related to P. falciparum. However, we found no evidence that this group of parasites branched with their hosts early in the evolution of primates. We applied relaxed clock methods and different calibrations points to explore the origin of primate malarias including those found in African apes. We showed that previous studies likely underestimated the origin of malarial parasites in primates. Conclusions The use of fossils from the host as absolute calibration and the assumption of a strict clock likely underestimate time when performing molecular dating analyses on malarial parasites. Indeed, by exploring different calibration points, we found that the time for the radiation of primate parasites may have taken place in the Eocene, a time consistent with the radiation of African anthropoids. The radiation of the four human parasite lineages was part of such events. The time frame estimated in this investigation, together with our phylogenetic analyses, made plausible a scenario where gorillas and humans acquired malaria from a Pan lineage.

Abstract Background Nucleic acid amplification provides the most sensitive and accurate method to detect and identify pathogens. This is primarily useful for epidemiological investigations of malaria because the infections, often with two or more Plasmodium species present simultaneously, are frequently associated with microscopically sub-patent parasite levels and cryptic mixed infections. Numerous distinct equally adequate amplification-based protocols have been described, but it is unclear which to select for epidemiological surveys. Few comparative studies are available, and none that addresses the issue of inter-laboratory variability. Methods Blood samples were collected from patients attending malaria clinics on the Thai-Myanmar border. Frozen aliquots from 413 samples were tested independently in two laboratories by nested PCR assay. Dried blood spots on filter papers from the same patients were also tested by the nested PCR assay in one laboratory and by a multiplex PCR assay in another. The aim was to determine which protocol best detected parasites below the sensitivity level of microscopic examination. Results As expected PCR-based assays detected a substantial number of infected samples, or mixed infections, missed by microscopy (27 and 42 for the most sensitive assay, respectively). The protocol that was most effective at detecting these, in particular mixed infections, was a nested PCR assay with individual secondary reactions for each of the species initiated with a template directly purified from the blood sample. However, a lesser sensitivity in detection was observed when the same protocol was conducted in another laboratory, and this significantly altered the data obtained on the parasite species distribution. Conclusions The sensitivity of a given PCR assay varies between laboratories. Although, the variations are relatively minor, they primarily diminish the ability to detect low-level and mixed infections and are sufficient to obviate the main rationale to use PCR assays rather than microscopy or rapid diagnostic tests. The optimal approach to standardise methodologies is to provide PCR template standards. These will help researchers in different settings to ensure that the nucleic acid amplification protocols they wish to use provide the requisite level of sensitivity, and will permit comparison between sites.

Abstract Chloroquine (CQ) resistant vivax malaria is spreading. In this case, Plasmodium vivax infections during pregnancy and in the postpartum period were not satisfactorily cleared by CQ, despite adequate drug concentrations. A growth restricted infant was delivered. Poor susceptibility to CQ was confirmed in-vitro and molecular genotyping was strongly suggestive of true recrudescence of P. vivax. This is the first clinically and laboratory confirmed case of two high-grade CQ resistant vivax parasite strains from Thailand.

Abstract Background The efficacy of anti-malarial drugs is assessed over a period of 28-63 days (depending on the drugs' residence time) following initiation of treatment in order to capture late failures. However, prolonged follow-up increases the likelihood of new infections depending on transmission intensity. Therefore, molecular genotyping of highly polymorphic regions of Plasmodium falciparum msp1, msp2 and glurp loci is usually carried out to distinguish recrudescence (true failures) from new infections. This tool has now been adopted as an integral part of anti-malarial efficacy studies and clinical trials. However, there are concerns over its utility and reliability because conclusions drawn from molecular typing depend on the genetic profile of the respective parasite populations, but this profile is not systematically documented in most endemic areas. This study presents the genetic diversity of P. falciparum msp1, msp2 and glurp markers in selected sub-Saharan Africa countries with varying levels of endemicity namely Malawi, Tanzania, Uganda, Burkina Faso and São Tomé. Methods A total 780 baseline (Day 0) blood samples from children less than seven years, recruited in a randomized controlled clinical trials done between 1996 and 2000 were genotyped. DNA was extracted; allelic frequency and diversity were investigated by PCR followed by capillary electrophoresis for msp2 and fragment sizing by a digitalized gel imager for msp1 and glurp. Results and Conclusion Plasmodium falciparum msp1, msp2 and glurp markers were highly polymorphic with low allele frequencies. A total of 17 msp1 genotypes [eight MAD20-, one RO33- and eight K1-types]; 116 msp2 genotypes [83 3D7 and 33 FC27- types] and 14 glurp genotypes were recorded. All five sites recorded very high expected heterozygosity (HE) values (0.68 - 0.99). HE was highest in msp2 locus (HE = 0.99), and lowest for msp1 (HE = 0.68) (P < 0.0001). The genetic diversity and allelic frequency recorded were independent of transmission intensity (P = 0.84, P = 0.25 respectively. A few genotypes had particularly high frequencies; however the most abundant showed only a 4% probability that a new infection would share the same genotype as the baseline infection. This is unlikely to confound the distinction of recrudescence from new infection, particularly if more than one marker is used for genotyping. Hence, this study supports the use of msp1, msp2 and glurp in malaria clinical trials in sub-Saharan Africa to discriminate new from recrudescent infections.

Abstract Background Investigations of Plasmodium vivax are restricted to samples collected from infected persons or primates, because this parasite cannot be maintained in in vitro cultures. Contamination of P. vivax isolates with host leukocytes and platelets is detrimental to a range of ex vivo and molecular investigations. Easy-to-produce CF11 cellulose filters have recently provided us with an inexpensive method for the removal of leukocytes and platelets. This contrasted with previous reports of unacceptably high levels of infected red blood cell (IRBC) retention by CF11. The aims of this study were to compare the ability of CF11 cellulose filters and the commercial filter Plasmodipur at removing leukocyte and platelet, and to investigate the retention of P. vivax IRBCs by CF11 cellulose filtration. Methods and Results Side-by-side comparison of six leukocyte removal methods using blood samples from five healthy donor showed that CF11 filtration reduced the mean initial leukocyte counts from 9.4 × 103 per μl [95%CI 5.2–13.5] to 0.01 × 103 [95%CI 0.01–0.03]. The CF11 was particularly effective at removing neutrophils. CF11 treatment also reduced initial platelet counts from 211.6 × 103 per μl [95%CI 107.5–315.7] to 0.8 × 103 per μl [95%CI -0.7–2.2]. Analysis of 30 P. vivax blood samples before and after CF11 filtration showed only a minor loss in parasitaemia (≤ 7.1% of initial counts). Stage specific retention of P. vivax IRBCs was not observed. Conclusion CF11 filtration is the most cost and time efficient method for the production of leukocyte- and platelet-free P. vivax-infected erythrocytes from field isolates.

Abstract Background Plasmodium vivax is the second most prevalent malaria parasite affecting more than 75 million people each year, mostly in South America and Asia. In addition to major morbidity this parasite is associated with relapses and a reduction in birthweight. The emergence and spread of drug resistance in Plasmodium falciparum is a major factor in the resurgence of this parasite. P. vivax resistance to drugs has more recently emerged and monitoring the situation would be helped, as for P. falciparum, by molecular methods that can be used to characterize parasites in field studies and drug efficacy trials. Methods Practical PCR genotyping protocols based on polymorphic loci present in two P. vivax genetic markers, Pvcs and Pvmsp1, were developed. The methodology was evaluated using 100 P. vivax isolates collected in Thailand. Results and Discussion Analysis revealed that P. vivax populations in Thailand are highly diverse genetically, with mixed genotype infections found in 26 % of the samples (average multiplicity of infection = 1.29). A large number of distinguishable alleles were found for the two markers, 23 for Pvcs and 36 for Pvmsp1. These were generally randomly distributed amongst the isolates. A total of 68 distinct genotypes could be enumerated in the 74 isolates with a multiplicity of infection of 1. Conclusion These results indicate that the genotyping protocols presented can be useful in the assessment of in vivo drug efficacy clinical trials conducted in endemic areas and for epidemiological studies of P. vivax infections.

BackgroundThe emergence and spread of multidrug-resistant Plasmodium falciparum in the Greater Mekong Subregion (GMS) threatens global malaria elimination efforts. Mass drug administration (MDA), the presumptive antimalarial treatment of an entire population to clear the subclinical parasite reservoir, is a strategy to accelerate malaria elimination. We report a cluster randomised trial to assess the effectiveness of dihydroartemisinin-piperaquine (DP) MDA in reducing falciparum malaria incidence and prevalence in 16 remote village populations in Myanmar, Vietnam, Cambodia, and the Lao People's Democratic Republic, where artemisinin resistance is prevalent.Methods and findingsAfter establishing vector control and community-based case management and following intensive community engagement, we used restricted randomisation within village pairs to select 8 villages to receive early DP MDA and 8 villages as controls for 12 months, after which the control villages received deferred DP MDA. The MDA comprised 3 monthly rounds of 3 daily doses of DP and, except in Cambodia, a single low dose of primaquine. We conducted exhaustive cross-sectional surveys of the entire population of each village at quarterly intervals using ultrasensitive quantitative PCR to detect Plasmodium infections. The study was conducted between May 2013 and July 2017. The investigators randomised 16 villages that had a total of 8,445 residents at the start of the study. Of these 8,445 residents, 4,135 (49%) residents living in 8 villages, plus an additional 288 newcomers to the villages, were randomised to receive early MDA; 3,790 out of the 4,423 (86%) participated in at least 1 MDA round, and 2,520 out of the 4,423 (57%) participated in all 3 rounds. The primary outcome, P. falciparum prevalence by month 3 (M3), fell by 92% (from 5.1% [171/3,340] to 0.4% [12/2,828]) in early MDA villages and by 29% (from 7.2% [246/3,405] to 5.1% [155/3,057]) in control villages. Over the following 9 months, the P. falciparum prevalence increased to 3.3% (96/2,881) in early MDA villages and to 6.1% (128/2,101) in control villages (adjusted incidence rate ratio 0.41 [95% CI 0.20 to 0.84]; p = 0.015). Individual protection was proportional to the number of completed MDA rounds. Of 221 participants with subclinical P. falciparum infections who participated in MDA and could be followed up, 207 (94%) cleared their infections, including 9 of 10 with artemisinin- and piperaquine-resistant infections. The DP MDAs were well tolerated; 6 severe adverse events were detected during the follow-up period, but none was attributable to the intervention.ConclusionsAdded to community-based basic malaria control measures, 3 monthly rounds of DP MDA reduced the incidence and prevalence of falciparum malaria over a 1-year period in areas affected by artemisinin resistance. P. falciparum infections returned during the follow-up period as the remaining infections spread and malaria was reintroduced from surrounding areas. Limitations of this study include a relatively small sample of villages, heterogeneity between villages, and mobility of villagers that may have limited the impact of the intervention. These results suggest that, if used as part of a comprehensive, well-organised, and well-resourced elimination programme, DP MDA can be a useful additional tool to accelerate malaria elimination.Trial registrationClinicalTrials.gov NCT01872702.

The global strategies for malaria control and elimination have led to substantial decreases in malaria incidence worldwide (1). In countries outside of Africa, Plasmodium vivax often predominates, making this species [...]

Author(s): Jean-Marc Chavatte 1,*, Georges Snounou 2,* Experimental human infections have contributed significantly to knowledge about infectious diseases [1]. In a recent report in PLOS Medicine , Rebecca Watts and [...]

The lack of a long-term in vitro culture method has severely restricted the study of Plasmodium vivax , in part because it limits genetic manipulation and reverse genetics. We used the recently optimized Plasmodium cynomolgi Berok in vitro culture model to investigate the putative P. vivax drug resistance marker MDR1 Y976F. Introduction of this mutation using clustered regularly interspaced short palindromic repeats–CRISPR-associated protein 9 (CRISPR-Cas9) increased sensitivity to mefloquine, but had no significant effect on sensitivity to chloroquine, amodiaquine, piperaquine, and artesunate. To our knowledge, this is the first reported use of CRISPR-Cas9 in P. cynomolgi , and the first reported integrative genetic manipulation of this species. [ABSTRACT FROM AUTHOR]

Malaria relapses, resulting from the activation of quiescent hepatic hypnozoites of Plasmodium vivax and Plasmodium ovale, hinder global efforts to control and eliminate malaria. As primaquine, the only drug capable of eliminating hypnozoites, is unsuitable for mass administration, an alternative drug is needed urgently. Currently, analyses of hypnozoites, including screening of compounds that would eliminate them, can only be made using common macaque models, principally Macaca rhesus and Macaca fascicularis, experimentally infected with the relapsing Plasmodium cynomolgi. Here, we present a protocol for long-term in vitro cultivation of P. cynomolgi--infected M. fascicularis primary hepatocytes during which hypnozoites persist and activate to resume normal development. In a proof-of-concept experiment, we obtained evidence that exposure to an inhibitor of histone modification enzymes implicated in epigenetic control of gene expression induces an accelerated rate of hypnozoite activation. The protocol presented may further enable investigations of hypnozoite biology and the search for compounds that kill hypnozoites or disrupt their quiescence., True malaria relapses are the reappearance of parasites in the blood weeks to years after complete clearance of blood-stage parasites in individuals who had no further infectious mosquito bites. Relapses [...]