Colin J. Sutherland

Plasmodium falciparum Associated with Severe Childhood Malaria Preferentially Expresses PfEMP1 Encoded by Group A var Genes

Parasite-encoded variant surface antigens (VSAs) like the var gene–encoded Plasmodium falciparum erythrocyte membrane protein 1 (PfEMP1) family are responsible for antigenic variation and infected red blood cell (RBC) cytoadhesion in P. falciparum malaria. Parasites causing severe malaria in nonimmune patients tend to express a restricted subset of VSA (VSASM) that differs from VSA associated with uncomplicated malaria and asymptomatic infection (VSAUM). We compared var gene transcription in unselected P. falciparum clone 3D7 expressing VSAUM to in vitro–selected sublines expressing VSASM to identify PfEMP1 responsible for the VSASM phenotype. Expression of VSASM was accompanied by up-regulation of Group A var genes. The most prominently up-regulated Group A gene (PFD1235w/MAL7P1.1) was translated into a protein expressed on the infected RBC surface. The proteins encoded by Group A var genes, such as PFD1235w/MAL7P1.1, appear to be involved in the pathogenesis of severe disease and are thus attractive candidates for a vaccine against life-threatening P. falciparum malaria.

Analysis of Plasmodium falciparum diversity in natural infections by deep sequencing

Malaria elimination strategies require surveillance of the parasite population for genetic changes that demand a public health response, such as new forms of drug resistance. Here we describe methods for the large-scale analysis of genetic variation in Plasmodium falciparum by deep sequencing of parasite DNA obtained from the blood of patients with malaria, either directly or after short-term culture. Analysis of 86,158 exonic single nucleotide polymorphisms that passed genotyping quality control in 227 samples from Africa, Asia and Oceania provides genome-wide estimates of allele frequency distribution, population structure and linkage disequilibrium. By comparing the genetic diversity of individual infections with that of the local parasite population, we derive a metric of within-host diversity that is related to the level of inbreeding in the population. An open-access web application has been established for the exploration of regional differences in allele frequency and of highly differentiated loci in the P. falciparum genome.

Artesunate Reduces but Does Not Prevent Posttreatment Transmission of Plasmodium falciparum to Anopheles gambiae

Combination therapy that includes artemisinin derivatives cures most falciparum malaria infections. Lowering transmission by reducing gametocyte infectivity would be an additional benefit. To examine the effect of such therapy on transmission, Gambian children with Plasmodium falciparum malaria were treated with standard regimens of chloroquine or pyrimethamine-sulfadoxine alone or in combination with 1 or 3 doses of artesunate. The infectivity to mosquitoes of gametocytes in peripheral blood was determined 4 or 7 days after treatment. Infection of mosquitoes was observed in all treatment groups and was positively associated with gametocyte density. The probability of transmission was lowest in those who received pyrimethamine-sulfadoxine and 3 doses of artesunate, and it was 8-fold higher in the group that received pyrimethamine-sulfadoxine alone. Artesunate reduced posttreatment infectivity dramatically but did not abolish it completely. The study raises questions about any policy to use pyrimethamine-sulfadoxine alone as the first-line treatment for malaria.

Two Nonrecombining Sympatric Forms of the Human Malaria Parasite Plasmodium ovale Occur Globally

BACKGROUND Malaria in humans is caused by apicomplexan parasites belonging to 5 species of the genus Plasmodium. Infections with Plasmodium ovale are widely distributed but rarely investigated, and the resulting burden of disease is not known. Dimorphism in defined genes has led to P. ovale parasites being divided into classic and variant types. We hypothesized that these dimorphs represent distinct parasite species. METHODS Multilocus sequence analysis of 6 genetic characters was carried out among 55 isolates from 12 African and 3 Asia-Pacific countries. RESULTS Each genetic character displayed complete dimorphism and segregated perfectly between the 2 types. Both types were identified in samples from Ghana, Nigeria, São Tomé, Sierra Leone, and Uganda and have been described previously in Myanmar. Splitting of the 2 lineages is estimated to have occurred between 1.0 and 3.5 million years ago in hominid hosts. CONCLUSIONS We propose that P. ovale comprises 2 nonrecombining species that are sympatric in Africa and Asia. We speculate on possible scenarios that could have led to this speciation. Furthermore, the relatively high frequency of imported cases of symptomatic P. ovale infection in the United Kingdom suggests that the morbidity caused by ovale malaria has been underestimated.

Highly Sensitive Detection of Malaria Parasitemia in a Malaria-Endemic Setting: Performance of a New Loop-Mediated Isothermal Amplification Kit in a Remote Clinic in Uganda

Background. Current malaria diagnostic tests, including microscopy and antigen-detecting rapid tests, cannot reliably detect low-density infections. Molecular methods such as polymerase chain reaction (PCR) are highly sensitive but remain too complex for field deployment. A new commercial molecular assay based on loop-mediated isothermal amplification (LAMP) was assessed for field use. Methods. Malaria LAMP (Eiken Chemical, Japan) was evaluated for samples from 272 outpatients at a rural Ugandan clinic and compared with expert microscopy, nested PCR, and quantitative PCR (qPCR). Two technicians performed the assay after 3 days of training, using 2 alternative blood sample–preparation methods and visual interpretation of results by fluorescence assay. Results. Compared with 3-well nested PCR, the sensitivity of both LAMP and single-well nested PCR was 90%; the microscopy sensitivity was 51%. For samples with a Plasmodium falciparum qPCR titer of ≥2 parasites/µL, LAMP sensitivity was 97.8% (95% confidence interval, 93.7%–99.5%). Most false-negative LAMP results involved samples with parasitemia levels detectable by 3-well nested PCR but very low or undetectable by qPCR. Conclusions. Malaria LAMP in a remote Ugandan clinic achieved sensitivity similar to that of single-well nested PCR in a United Kingdom reference laboratory. LAMP dramatically lowers the detection threshold achievable in malaria-endemic settings, providing a new tool for diagnosis, surveillance, and screening in elimination strategies.

Reduction of Malaria Transmission to Anopheles Mosquitoes with a Six-Dose Regimen of Co-Artemether

Background Resistance of malaria parasites to chloroquine (CQ) and sulphadoxine-pyrimethamine (SP) is increasing in prevalence in Africa. Combination therapy can both improve treatment and provide important public health benefits if it curbs the spread of parasites harbouring resistance genes. Thus, drug combinations must be identified which minimise gametocyte emergence in treated cases, and so prevent selective transmission of parasites resistant to any of the partner drugs. Methods and Findings In a randomised controlled trial, 497 children with uncomplicated falciparum malaria were treated with CQ and SP (three doses and one dose respectively; n = 91), or six doses of artemether in fixed combination with lumefantrine (co-artemether [Coartem, Riamet]) (n = 406). Carriage rates of Plasmodium falciparum gametocytes and trophozoites were measured 7, 14, and 28 d after treatment. The infectiousness of venous blood from 29 children carrying P. falciparum gametocytes 7 d after treatment was tested by membrane-feeding of Anopheles mosquitoes. Children treated with co-artemether were significantly less likely to carry gametocytes within the 4 weeks following treatment than those receiving CQ/SP (30 of 378 [7.94%] versus 42 of 86 [48.8%]; p < 0.0001). Carriers in the co-artemether group harboured gametocytes at significantly lower densities, for shorter periods (0.3 d versus 4.2 d; p < 0.0001) and were less infectious to mosquitoes at day 7 (p < 0.001) than carriers who had received CQ/SP. Conclusions Co-artemether is highly effective at preventing post-treatment transmission of P. falciparum. Our results suggest that co-artemether has specific activity against immature sequestered gametocytes, and has the capacity to minimise transmission of drug-resistant parasites.

Seasonal intermittent preventive treatment with artesunate and sulfadoxine-pyrimethamine for prevention of malaria in Senegalese children: a randomised placebo-controlled double-blind trial.

BACKGROUND In the Sahel and sub-Sahelian regions of Africa, malaria transmission is highly seasonal. During a short period of high malaria transmission, mortality and morbidity are high in children under age 5 years. We assessed the efficacy of seasonal intermittent preventive treatment-a full dose of antimalarial treatment given at defined times without previous testing for malaria infection. METHODS We did a randomised, placebo-controlled, double-blind trial of the effect of intermittent preventive treatment on morbidity from malaria in three health-care centres in Niakhar, a rural area of Senegal. 1136 children aged 2-59 months received either one dose of artesunate plus one dose of sulfadoxine-pyrimethamine or two placebos on three occasions during the malaria transmission season. The primary outcome was a first or single episode of clinical malaria detected through active or passive case detection. Primary analysis was by intention-to-treat. This study is registered with , number NCT00132561. FINDINGS During 13 weeks of follow-up, the intervention led to an 86% (95% CI 80-90) reduction in the occurrence of clinical episodes of malaria. With passive case detection, protective efficacy against malaria was 86% (77-92), and when detected actively was 86% (78-91). The incidence of malaria in children on active drugs was 308 episodes per 1000 person-years at risk, whereas in those on placebo it was 2250 episodes per 1000 person-years at risk. 13 children were not included in the intention-to-treat analysis, which was restricted to children who received a first dose of antimalarial or placebo. There was an increase in vomiting in children who received the active drugs, but generally the intervention was well tolerated. INTERPRETATION Intermittent preventive treatment could be highly effective for prevention of malaria in children under 5 years of age living in areas of seasonal malaria infection.

Amodiaquine and Artemether-Lumefantrine Select Distinct Alleles of the Plasmodium falciparum mdr1 Gene in Tanzanian Children Treated for Uncomplicated Malaria

ABSTRACT The artemisinin-based combination therapies artemether-lumefantrine (AL) and amodiaquine (AQ) plus artesunate have been adopted for treatment of Plasmodium falciparum malaria in many African countries. Molecular markers of parasite resistance suitable for surveillance have not been established for any of the component drugs in either of these combinations. We assessed P. falciparum mdr1 (Pfmdr1) alleles present in 300 Tanzanian children presenting with uncomplicated falciparum malaria, who were enrolled in a clinical trial of antimalarial therapy. Pfmdr1 genotype analysis was also performed with isolates from 182 children who failed AQ monotherapy and 54 children who failed AL treatment. Pfmdr1 alleles 86Y, 184Y, and 1246Y were more common among treatment failures in the AQ group than among pretreatment infections. The converse was found in the AL-treated group. Children presenting with the 86Y/184Y/1246Y Pfmdr1 haplotype and treated with AQ were significantly more likely to retain this haplotype if they were parasite positive during posttreatment follow-up than were children treated with AL (odds ratio, 33.25; 95% confidence interval, 4.17 to 1441; P, <0.001). We conclude that AL and AQ exert opposite within-host selective effects on the Pfmdr1 gene of P. falciparum.

Revisiting the circulation time of Plasmodium falciparum gametocytes: molecular detection methods to estimate the duration of gametocyte carriage and the effect of gametocytocidal drugs.

BackgroundThere is renewed acknowledgement that targeting gametocytes is essential for malaria control and elimination efforts. Simple mathematical models were fitted to data from clinical trials in order to determine the mean gametocyte circulation time and duration of gametocyte carriage in treated malaria patients.MethodsData were used from clinical trials from East Africa. The first trial compared non-artemisinin combination therapy (non-ACT: sulphadoxine-pyrimethamine (SP) plus amodiaquine) and artemisinin-based combination therapy (ACT: SP plus artesunate (AS) or artemether-lumefantrine). The second trial compared ACT (SP+AS) with ACT in combination with a single dose of primaquine (ACT-PQ: SP+AS+PQ). Mature gametocytes were quantified in peripheral blood samples by nucleic acid sequence based amplification. A simple deterministic compartmental model was fitted to gametocyte densities to estimate the circulation time per gametocyte; a similar model was fitted to gametocyte prevalences to estimate the duration of gametocyte carriage after efficacious treatment.ResultsThe mean circulation time of gametocytes was 4.6-6.5 days. After non-ACT treatment, patients were estimated to carry gametocytes for an average of 55 days (95% CI 28.7 - 107.7). ACT reduced the duration of gametocyte carriage fourfold to 13.4 days (95% CI 10.2-17.5). Addition of PQ to ACT resulted in a further fourfold reduction of the duration of gametocyte carriage.ConclusionsThese findings confirm previous estimates of the circulation time of gametocytes, but indicate a much longer duration of (low density) gametocyte carriage after apparently successful clearance of asexual parasites. ACT shortened the period of gametocyte carriage considerably, and had the most pronounced effect on mature gametocytes when combined with PQ.

Moderate Effect of Artemisinin-Based Combination Therapy on Transmission of Plasmodium falciparum

Background. Artemisinin-based combination therapy (ACT) reduces microscopically confirmed gametocytemia and mosquito infection. However, molecular techniques have recently revealed high prevalences of submicroscopic gametocytemia. Our objective here was to determine the effect of sulfadoxine-pyrimethamine (SP) monotherapy and treatment with SP plus amodiaquine (AQ), SP plus artesunate (AS), and artemether-lumefantrine (AL; Coartem) on submicroscopic gametocytemia and infectiousness.Methods. Kenyan children (n=528) 6 months-10 years of age were randomized to 4 treatment arms. Gametocytemia was determined by both microscopy and Pfs25 RNA-based quantitative nucleic acid sequence-based amplification (Pfs25 QT-NASBA). Transmission was determined by membrane-feeding assays.Results. Gametocyte prevalence, as determined by Pfs25 QT-NASBA, was 89.4% (219/245) at enrollment and decreased after treatment with SP plus AS, SP plus AQ, and AL. Membrane-feeding assays for a group of randomly selected children revealed that the proportion of infectious children was as much as 4-fold higher than expected when based on microscopy. ACT did not significantly reduce the proportion of infectious children but did reduce the proportion of infected mosquitoes.Conclusions. Submicroscopic gametocytemia is common after treatment and contributes considerably to mosquito infection. Our findings should be interpreted in the context of transmission intensity, but the effect of ACT on malaria transmission appears to be moderate and restricted to the duration of gametocyte carriage and the proportion of mosquitoes that are infected by carriers.