Impact of Plasmodium falciparum gene deletions on malaria rapid diagnostic test performance.

BACKGROUND: Malaria rapid diagnostic tests (RDTs) have greatly improved access to diagnosis in endemic countries. Most RDTs detect Plasmodium falciparum histidine-rich protein 2 (HRP2), but their sensitivity is seriously threatened by the emergence of pfhrp2-deleted parasites. RDTs detecting P. falciparum or pan-lactate dehydrogenase (Pf- or pan-LDH) provide alternatives. The objective of this study was to systematically assess the performance of malaria RDTs against well-characterized pfhrp2-deleted P. falciparum parasites. METHODS: Thirty-two RDTs were tested against 100 wild-type clinical isolates (200 parasites/µL), and 40 samples from 10 culture-adapted and clinical isolates of pfhrp2-deleted parasites. Wild-type and pfhrp2-deleted parasites had comparable Pf-LDH concentrations. Pf-LDH-detecting RDTs were also tested against 18 clinical isolates at higher density (2,000 parasites/µL) lacking both pfhrp2 and pfhrp3. RESULTS: RDT positivity against pfhrp2-deleted parasites was highest (> 94%) for the two pan-LDH-only RDTs. The positivity rate for the nine Pf-LDH-detecting RDTs varied widely, with similar median positivity between double-deleted (pfhrp2/3 negative; 63.9%) and single-deleted (pfhrp2-negative/pfhrp3-positive; 59.1%) parasites, both lower than against wild-type P. falciparum (93.8%). Median positivity for HRP2-detecting RDTs against 22 single-deleted parasites was 69.9 and 35.2% for HRP2-only and HRP2-combination RDTs, respectively, compared to 96.0 and 92.5% for wild-type parasites. Eight of nine Pf-LDH RDTs detected all clinical, double-deleted samples at 2,000 parasites/µL. CONCLUSIONS: The pan-LDH-only RDTs evaluated performed well. Performance of Pf-LDH-detecting RDTs against wild-type P. falciparum does not necessarily predict performance against pfhrp2-deleted parasites. Furthermore, many, but not all HRP2-based RDTs, detect pfhrp2-negative/pfhrp3-positive samples, with implications for the HRP2-based RDT screening approach for detection and surveillance of HRP2-negative parasites.

Identification and Characterization of the Rhoptry Neck Protein 2 in Babesia divergens and B. microti.

Apicomplexan parasites include those of the genera Plasmodium, Cryptosporidium, and Toxoplasma and those of the relatively understudied zoonotic genus Babesia In humans, babesiosis, particularly transfusion-transmitted babesiosis, has been emerging as a major threat to public health. Like malaria, the disease pathology is a consequence of the parasitemia which develops through cyclical replication of Babesia parasites in host erythrocytes. However, there are no exoerythrocytic stages in Babesia, so targeting of the blood stage and associated proteins to directly prevent parasite invasion is the most desirable option for effective disease control. Especially promising among such molecules are the rhoptry neck proteins (RONs), whose homologs have been identified in many apicomplexan parasites. RONs are involved in the formation of the moving junction, along with AMA1, but no RON has been identified and characterized in any Babesia spp. Here we identify the RON2 proteins of Babesia divergens (BdRON2) and B. microti (BmRON2) and show that they are localized apically and that anti-BdRON2 antibodies are significant inhibitors of parasite invasion in vitro Neither protein is immunodominant, as both proteins react only marginally with sera from infected animals. Further characterization of the direct role of both BdRON2 and BmRON2 in parasite invasion is required, but knowledge of the level of conformity of RON2 proteins within the apicomplexan phylum, particularly that of the AMA1-RON2 complex at the moving junction, along with the availability of an animal model for B. microti studies, provides a key to target this complex with a goal of preventing the erythrocytic invasion of these parasites and to further our understanding of the role of these conserved ligands in invasion.

Independent origin of plasmodium falciparum antifolate super-resistance, Uganda, Tanzania, and Ethiopia.

Super-resistant Plasmodium falciparum threatens the effectiveness of sulfadoxine-pyrimethamine in intermittent preventive treatment for malaria during pregnancy. It is characterized by the A581G Pfdhps mutation on a background of the double-mutant Pfdhps and the triple-mutant Pfdhfr. Using samples collected during 2004-2008, we investigated the evolutionary origin of the A581G mutation by characterizing microsatellite diversity flanking Pfdhps triple-mutant (437G+540E+581G) alleles from 3 locations in eastern Africa and comparing it with double-mutant (437G+540E) alleles from the same area. In Ethiopia, both alleles derived from 1 lineage that was distinct from those in Uganda and Tanzania. Uganda and Tanzania triple mutants derived from the previously characterized southeastern Africa double-mutant lineage. The A581G mutation has occurred multiple times on local Pfdhps double-mutant backgrounds; however, a novel microsatellite allele incorporated into the Tanzania lineage since 2004 illustrates the local expansion of emergent triple-mutant lineages.

A malaria vaccine candidate based on an epitope of the Plasmodium falciparum RH5 protein.

BACKGROUND: The Plasmodium falciparum protein RH5 is an adhesin molecule essential for parasite invasion of erythrocytes. Recent studies show that anti-PfRH5 sera have potent invasion-inhibiting activities, supporting the idea that the PfRH5 antigen could form the basis of a vaccine. Therefore, epitopes recognized by neutralizing anti-PfRH5 antibodies could themselves be effective vaccine immunogens if presented in a sufficiently immunogenic fashion. However, the exact regions within PfRH5 that are targets of this invasion-inhibitory activity have yet to be identified. METHODS: A battery of anti-RH5 monoclonal antibodies (mAbs) were produced and screened for their potency by inhibition of invasion assays in vitro. Using an anti-RH5 mAb that completely inhibited invasion as the selecting mAb, affinity-selection using random sequence peptide libraries displayed on virus-like particles of bacteriophage MS2 (MS2 VLPs) was performed. VLPs were sequenced to identify the specific peptide epitopes they encoded and used to raise specific antisera that was in turn tested for inhibition of invasion. RESULTS: Three anti-RH5 monoclonals (0.1 mg/mL) were able to inhibit invasion in vitro by >95%. Affinity-selection with one of these mAbs yielded a VLP which yielded a peptide whose sequence is identical to a portion of PfRH5 itself. The VLP displaying the peptide binds strongly to the antibody, and in immunized animals elicits an anti-PfRH5 antibody response. The resulting antisera against the specific VLP inhibit parasite invasion of erythrocytes more than 90% in vitro. CONCLUSIONS: Here, data is presented from an anti-PfRH5 mAb that completely inhibits erythrocyte invasion by parasites in vitro, one of the few anti-malarial monoclonal antibodies reported to date that completely inhibits invasion with such potency, adding to other studies that highlight the potential of PfRH5 as a vaccine antigen. The specific neutralization sensitive epitope within RH5 has been identified, and antibodies against this epitope also elicit high anti-invasion activity, suggesting this epitope could form the basis of an effective vaccine against malaria.

Global and local genetic diversity at two microsatellite loci in Plasmodium vivax parasites from Asia, Africa and South America.

BACKGROUND: Even though Plasmodium vivax has the widest worldwide distribution of the human malaria species and imposes a serious impact on global public health, the investigation of genetic diversity in this species has been limited in comparison to Plasmodium falciparum. Markers of genetic diversity are vital to the evaluation of drug and vaccine efficacy, tracking of P. vivax outbreaks, and assessing geographical differentiation between parasite populations. METHODS: The genetic diversity of eight P. vivax populations (n=543) was investigated by using two microsatellites (MS), m1501 and m3502, chosen because of their seven and eight base-pair (bp) repeat lengths, respectively. These were compared with published data of the same loci from six other P. vivax populations. RESULTS: In total, 1,440 P. vivax samples from 14 countries on three continents were compared. There was highest heterozygosity within Asian populations, where expected heterozygosity (He) was 0.92-0.98, and alleles with a high repeat number were more common. Pairwise FST revealed significant differentiation between most P. vivax populations, with the highest divergence found between Asian and South American populations, yet the majority of the diversity (~89%) was found to exist within rather than between populations. CONCLUSIONS: The MS markers used were informative in both global and local P. vivax population comparisons and their seven and eight bp repeat length facilitated population comparison using data from independent studies. A complex spatial pattern of MS polymorphisms among global P. vivax populations was observed which has potential utility in future epidemiological studies of the P. vivax parasite.

A community-randomized evaluation of the effect of intermittent preventive treatment in infants on antimalarial drug resistance in southern Tanzania.

BACKGROUND: Intermittent preventive treatment in infants (IPTi) is the administration of sulfadoxine-pyrimethamine (SP) at 2, 3, and 9 months of age to prevent malaria. We investigated the influence of IPTi on drug resistance. METHODS: Twenty-four areas were randomly assigned to receive or not receive IPTi. Blood collected during representative household surveys at baseline and 15 and 27 months after implementation was tested for SP and resistance markers. RESULTS: The frequency of SP in blood was similar in the IPTi and comparison areas at baseline and at 15 months. dhfr and dhps mutations were also similar at baseline and then increased similarly in both arms after 15 months of SP-IPTi. First-line treatment was switched from SP to artemether-lumefantrine before the final survey, when SP positivity fell among infants in comparison areas but increased in IPTi areas. This was accompanied by an increase in dhfr but not dhps mutations in IPTi areas (P = .004 and P = .18, respectively). CONCLUSIONS: IPTi did not increase drug pressure or the selection on dhfr and dhps mutants, when SP was the first-line malaria treatment. Introduction of artemether-lumefantrine was followed by an increase in dhfr mutations, consistent with weak selection attributable to SP-IPTi, but not by an increase in dhps mutations, suggesting a fitness cost of this mutation.

High prevalence of dhfr triple mutant and correlation with high rates of sulphadoxine-pyrimethamine treatment failures in vivo in Gabonese children.

BACKGROUND: Drug resistance contributes to the global malaria burden. Plasmodium falciparum dihydrofolate reductase (dhfr) and dihydropteroate synthase (dhps) polymorphisms confer resistance to sulphadoxine-pyrimethamine (SP). METHODS: The study assessed the frequency of SP resistance-conferring polymorphisms in Plasmodium falciparum-positive samples from two clinical studies in Lambaréné. Their role on treatment responses and transmission potential was studied in an efficacy open-label clinical trial with a 28-day follow-up in 29 children under five with uncomplicated malaria. RESULTS: SP was well tolerated by all subjects in vivo. Three subjects were excluded from per-protocol analysis. PCR-corrected, 12/26 (46%) achieved an adequate clinical and parasitological response, 13/26 (50%) were late parasitological failures, while 1/26 (4%) had an early treatment failure, resulting in early trial discontinuation. Of 106 isolates, 98 (92%) carried the triple mutant dhfr haplotype. Three point mutations were found in dhps in a variety of haplotypic configurations. The 437G + 540E double mutant allele was found for the first time in Gabon. CONCLUSIONS: There is a high prevalence of dhfr triple mutant with some dhps point mutations in Gabon, in line with treatment failures observed, and molecular markers of SP resistance should be closely monitored.

Epidemic of Plasmodium falciparum malaria involving substandard antimalarial drugs, Pakistan, 2003.

Because of instability in eastern Afghanistan, new refugees crossed into the federally administered tribal areas of northwestern Pakistan in 2002. In 2003, we investigated an epidemic of Plasmodium falciparum malaria in 1 of the camps. Incidence was 100.4 cases/1,000 person-years; in other nearby camps it was only 2.1/1,000 person-years. Anopheline mosquitoes were found despite an earlier spray campaign. Documented clinical failures at the basic health unit prompted a drug resistance survey of locally manufactured sulfadoxine-pyrimethamine used for routine treatment. The in vivo failure rate was 28.5%. PCR analysis of the P. falciparum dihydrofolate reductase and dihyropteroate synthase genes showed no mutations associated with clinical failure. However, chemical analysis of the drug showed that it was substandard. As global incidence decreases and epidemics become more of a threat, enhanced quality assurance of control interventions is essential.

Multiple origins and regional dispersal of resistant dhps in African Plasmodium falciparum malaria.

BACKGROUND: Although the molecular basis of resistance to a number of common antimalarial drugs is well known, a geographic description of the emergence and dispersal of resistance mutations across Africa has not been attempted. To that end we have characterised the evolutionary origins of antifolate resistance mutations in the dihydropteroate synthase (dhps) gene and mapped their contemporary distribution. METHODS AND FINDINGS: We used microsatellite polymorphism flanking the dhps gene to determine which resistance alleles shared common ancestry and found five major lineages each of which had a unique geographical distribution. The extent to which allelic lineages were shared among 20 African Plasmodium falciparum populations revealed five major geographical groupings. Resistance lineages were common to all sites within these regions. The most marked differentiation was between east and west African P. falciparum, in which resistance alleles were not only of different ancestry but also carried different resistance mutations. CONCLUSIONS: Resistant dhps has emerged independently in multiple sites in Africa during the past 10-20 years. Our data show the molecular basis of resistance differs between east and west Africa, which is likely to translate into differing antifolate sensitivity. We have also demonstrated that the dispersal patterns of resistance lineages give unique insights into recent parasite migration patterns.

Monitoring for multidrug-resistant Plasmodium falciparum isolates and analysis of pyrimethamine resistance evolution in Uige province, Angola.

OBJECTIVES: To assess the extent of drug resistance in Uige through molecular genetic analysis and to test whether the dhfr triple mutant alleles present in Angola are of southeast Asian origin. METHODS: Seventy-one samples of blood from children admitted to the Pediatric Emergency Unit of Uige Provincial Hospital in 2004 were screened for resistance mutations at pfcrt, pfmdr1, pfdhfr, pfdhps and pfATPase6. RESULTS: Mutations in pfcrt (codon76), pfmdr1 (codon86), pfdhfr (codons 51, 59, 108) and pfdhps (codons 436, 437) were common. Among the 66 isolates for which we were able to determine complete genetic information 13.7% carried all seven of these mutations. Flanking microsatellite analysis revealed the triple mutant pfdhfr was derived from the southeast Asian lineage, while the N51I+S108N double mutant pfdhfr alleles are a local origin. pfATPase6 mutations were rare and S769N was not found. CONCLUSION: The parasite population of Uige Angola has high frequency mutations in pfcrt, dhfr and dhps associated with resistance to chloroquine and sulphadoxine pyrimethamine, reflecting past reliance on these two drugs which were the mainstay of treatment until recently. Our findings show that drug resistance in Uige has occurred through a combination of local drug pressure and the regional and international dispersal of resistance mutant alleles.

Cluster of falciparum malaria cases in UK airport.

A cluster of 6 cases of Plasmodium falciparum malaria occurred in a UK airport among 30 travelers returning to the United States from East Africa. Molecular genotyping analysis indicated that all had been exposed to different parasites. The travelers' use of chemoprophylaxis was poor; their perception of risk was limited.

Sustained use of insecticide-treated curtains is not associated with greater circulation of drug-resistant malaria parasites, or with higher risk of treatment failure among children with uncomplicated malaria in Burkina Faso.

The impact of vector control measures on the evolution of antimalarial drug resistance is an important issue for malaria control programs. We investigated whether the in vivo efficacy of chloroquine (CQ) in children aged 6-59 months with uncomplicated malaria differed in 9 villages that had benefited from long-term use of insecticide-treated curtains (ITCs) and in 9 nearby non-ITC villages. We also compared the prevalence of genetic markers of resistance to CQ and sulfadoxine-pyrimethamine (SP) between the two groups of villages. The study enrolled 1,035 children with uncomplicated malaria and 231 infected but asymptomatic children. After taking account of re-infections, the proportions of children who experienced clinical failure after treatment with CQ were 14% and 19% in ITC and non-ITC villages, respectively (OR = 0.68; 95% CI: 0.39, 1.18). Parasitologic failure was observed in 49% of children in ITC villages and 58% of children in non-ITC villages (OR = 0.71 95%CI: 0.44, 1.13). The proportion of symptomatic children who harbored parasites carrying the pfcrt-76T allele was 43% in ITC villages and 40% in non-ITC villages (OR = 1.09; 95%CI: 0.80, 1.50). The pfmdr1-86Y allele was detected in 31% and 29% of children in the two groups of villages (OR = 1.14; 95%CI: 0.75, 1.72). Triple mutations in the dhfr gene were observed in 12% of children in both groups. No double mutations in the dhps gene were observed. Similar results were observed in asymptomatic children. In this setting, ITC use was not associated with increased circulation of parasites resistant to standard antimalarial drugs, or with a greater risk of treatment failure among children less than 5 years of age.

Comparison of the therapeutic efficacy of chloroquine and sulphadoxine-pyremethamine in children and pregnant women.

OBJECTIVE: To compare the parasitological failure rates of under-fives and pregnant women with parasitaemia treated with chloroquine (CQ) or sulphadoxine-pyrimethamine (SP). METHODS: During a clinical trial of CQ, SP, amodiaquine (AQ) and SP plus AQ combination for malaria treatment in pregnant women in Ghana, a parallel study of treatment of children below 5 years of age with symptomatic malaria with CQ and SP was undertaken. Four hundred and fifty pregnant women with malaria parasitaemia and 203 children with malaria parasitaemia were randomized to receive CQ or SP. They were followed up and parasitological failure by days 14 and 28 after the start of treatment was assessed. RESULTS: Polymerase chain reaction (PCR)-uncorrected parasitological failure rates by day 28 after the start of treatment with CQ were 58.5% (55/94), 38.5% (45/117), 31% (13/42) and 8.2% (4/49) in children, primigravidae, secundigravidae and multigravidae, respectively. For those treated with SP the rates by day 28 were 36.4% (32/88), 27.1% (29/107), 6.1% (3/49) and 3.8% (2/52) in children, primigravidae, secundigravidae and multigravidae, respectively. In both CQ and SP treatment arms, children were twice as likely to experience recrudescence as pregnant women (RR 2.1 [95% CI 1.6-2.6] P < 0.0001) by day 28 after the start of treatment. CONCLUSIONS: Parasitological failure rates were significantly lower in asymptomatic pregnant women, particularly in multigravidae, compared with symptomatic children. Reliance on drug sensitivity results observed in children only to decide on antimalarial regimes for pregnant women may not be appropriate.

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.

High sequence diversity and evidence of balancing selection in the Pvmsp3alpha gene of Plasmodium vivax in the Venezuelan Amazon.

The genetic diversity of a defined Plasmodium vivax population from the Venezuelan Amazon was evaluated by direct sequencing of the gene encoding the P. vivax merozoite surface protein-3alpha, Pvmsp3alpha. Three allele sizes (1.9, 1.4 and 1.1kb) were amplified from 58 isolates with frequencies of 59.3%, 21.9% and 18.8%, respectively. 27 Pvmsp3alpha nucleotide sequences were determined, with nine distinct haplotypes observed. The genetic diversity (h) at this single locus was 0.774. The P. vivax population in this region exhibits significant diversity in contrast to the genetically restricted diversity of the sympatric P. falciparum population. Despite sharing vector and human hosts, different control strategies may be required for these two species in this region. Substitution patterns in the conserved C-terminus of Pvmsp3alpha showed a significant departure from neutrality, suggesting these polymorphisms are being maintained by frequency-dependent selection as the result of an effective immune response from the host. Our findings support the use of Pvmsp3alpha genotyping as a tool for monitoring interventions aimed at control of P. vivax.

Human Babesiosis: Pathogens, Prevalence, Diagnosis and Treatment.

Human babesiosis is a zoonotic disease caused by protozoan parasites of the Babesia genus, primarily in the Northeastern and Midwest United States due to B. microti, and Western Europe due to B. divergens. Parasites are transmitted by the bite of the ixodid tick when the vector takes a blood meal from the vertebrate host, and the economic importance of bovine babesiosis is well understood. The pathology of human disease is a direct result of the parasite's ability to invade host's red blood cells. The current understanding of human babesiosis epidemiology is that many infections remain asymptomatic, especially in younger or immune competent individuals, and the burden of severe pathology resides within older or immunocompromised individuals. However, transfusion-transmitted babesiosis is an emerging threat to public health as asymptomatic carriers donate blood and there are as yet no licensed or regulated tests to screen blood products for this pathogen. Reports of tick-borne cases within new geographical regions such as the Pacific Northwest of the US, through Eastern Europe, and into China are also on the rise. Further, new Babesia spp. have been identified globally as agents of severe human babesiosis, suggesting that the epidemiology of this disease is rapidly changing, and it is clear that human babesiosis is a serious public health concern that requires close monitoring and effective intervention measure.

Malaria invasion ligand RH5 and its prime candidacy in blood-stage malaria vaccine design.

With drug resistance to available therapeutics continuing to develop against Plasmodium falciparum malaria, the development of an effective vaccine candidate remains a major research goal. Successful interruption of invasion of parasites into erythrocytes during the blood stage of infection will prevent the severe clinical symptoms and complications associated with malaria. Previously studied blood stage antigens have highlighted the hurdles that are inherent to this life-cycle stage, namely that highly immunogenic antigens are also globally diverse, resulting in protection only against the vaccine strain, or that naturally acquired immunity to blood stage antigens do not always correlate with actual protection. The blood stage antigen reticulocyte binding homolog RH5 is essential for parasite viability, has globally limited diversity, and is associated with protection from disease. Here we summarize available information on this invasion ligand and recent findings that highlight its candidacy for inclusion in a blood-stage malaria vaccine.

Gambian children successfully treated with chloroquine can harbor and transmit Plasmodium falciparum gametocytes carrying resistance genes.

Polymorphisms in two genes of Plasmodium falciparum (P. falciparum multidrug resistance 1 [pfmdr1] and P. falciparum chloroquine [CQ] resistance transporter [pfcrt]) are associated with CQ treatment failure. We found significant linkage disequilibrium between these loci among isolates from symptomatic Gambian children (P = 0.026) and strong selection for the resistance-associated alleles pfmdr1-86Tyr and pfcrt-76Thr in children with persistent or re-emerging P. falciparum trophozoites during post-treatment follow-up (P = 1.9 x 10(-7)). Therefore, this genotype is characteristic of resistant infections among our study population. Since the long-term public health impact of parasites carrying such resistant genotypes depends upon their transmissibility, we examined the prevalence of pfmdr1-86Tyr and pfcrt-76Thr among Gambian children harboring sexual stage parasites during post-treatment follow-up. Gametocytes that emerged after successful treatment with CQ were significantly more likely to be of this genotype than were those emerging after other treatments (P = 4.83 x 10(-4)), and were infective to Anopheles mosquitoes. Therapeutic success may thus be accompanied by public health failure as cured children pass resistance genes on to mosquitoes at an enhanced rate.

Genetic complexity of Plasmodium falciparum gametocytes isolated from the peripheral blood of treated Gambian children.

The genetic complexity of Plasmodium falciparum gametocytes isolated from Gambian children participating in a controlled trial of anti-malarial therapy was investigated. RNA and DNA were prepared from gametocyte-positive blood, which was also used in transmission experiments with Anopheles gambiae mosquitoes. Amplification by a reverse transcriptase-polymerase chain reaction (RT-PCR) of transcripts from the genes for the ring-infected erythrocyte surface antigen and the 16-kD antigen, which exhibit asexual and sexual stage-specific expression, was used to identify 30 post-treatment gametocyte isolates in which trophozoites persisted below the threshold of detection by microscopy. These included isolates from children who received sulfadoxine/pyrimethamine plus artesunate. Twenty-nine gametocyte-positive isolates that were free of subpatent trophozoites were examined further by PCR amplification of polymorphic genomic loci. We estimate that an average minimum of 2.3 genotypes occurred in these gametocyte-only isolates, and many of these were shown to be infective to mosquitoes. Thus, meiotic recombination between different genotypes is predicted to be a common event in this study area.

Sympatric Plasmodium falciparum isolates from Venezuela have structured var gene repertoires.

BACKGROUND: The human malaria parasite Plasmodium falciparum expresses adhesins belonging to the erythrocyte membrane protein 1 (PfEMP1) family on the surface of the infected host erythrocyte. These antigens elicit a strain-specific antibody response that is associated with protection from disease. During clonal expansion of blood-stage parasites, the surface phenotype of the infected erythrocyte changes because of transcriptional switching among the 40 to 50 members of the highly polymorphic var multi-gene family which encode PfEMP1 variants. Studies to date have compared var repertoires of natural isolates from various geographical locations but have not addressed any within-population structure that may exist among repertoires. METHODS: Distinct parasite genotypes from a single population co-circulating among a defined group of hosts were selected. PCR products encoding the DBL-alpha domain of PfEMP-1 were cloned and sequenced from each of three isolates. Repertoire similarity was statistically evaluated using combinatorial analysis. The chromosomal location of shared sequences was inferred from similarity to dbl-alpha of known location in the 3D7 genome. RESULTS: Sympatric parasites were found to share few var gene sequences, even when alleles at other polymorphic loci were shared. A number of the sequences shared by at least two of the isolates studied were found to be related to 3D7 genomic sequences with non-telomeric chromosomal locations, or atypical domain structures, which may represent globally conserved loci. CONCLUSION: The parasite population studied is structured, with minimal overlap in PfEMP1 repertoires. The var gene family accumulates diversity more rapidly than other antigen genes examined. This may be facilitated by ectopic recombination among the sub-telomeric regions of P. falciparum chromosomes.