Assessment of therapeutic responses to gametocytocidal drugs in Plasmodium falciparum malaria

Background

Effective mating between laboratory-reared males and wild females is paramount to the success of vector control strategies aiming to decrease disease transmission via the release of sterile or genetically modified male mosquitoes. However mosquito colonization and laboratory maintenance have the potential to negatively affect male genotypic and phenotypic quality through inbreeding and selection, which in turn can decrease male mating competitiveness in the field. To date, very little is known about the impact of those evolutionary forces on the reproductive biology of mosquito colonies and how they ultimately affect male reproductive fitness.

Methods

Here several male reproductive physiological traits likely to be affected by inbreeding and selection following colonization and laboratory rearing were examined. Sperm length, and accessory gland and testes size were compared in male progeny from field-collected females and laboratory strains of Anopheles gambiae sensu stricto colonized from one to over 25 years ago. These traits were also compared in the parental and sequentially derived, genetically modified strains produced using a two-phase genetic transformation system. Finally, genetic crosses were performed between strains in order to distinguish the effects of inbreeding and selection on reproductive traits.

Results

Sperm length was found to steadily decrease with the age of mosquito colonies but was recovered in refreshed strains and crosses between inbred strains therefore incriminating inbreeding costs. In contrast, testes size progressively increased with colony age, whilst accessory gland size quickly decreased in males from colonies of all ages. The lack of heterosis in response to crossing and strain refreshing in the latter two reproductive traits suggests selection for insectary conditions.

Conclusions

These results show that inbreeding and selection differentially affect reproductive traits in laboratory strains overtime and that heterotic ‘supermales’ could be used to rescue some male reproductive characteristics. Further experiments are needed to establish the exact relationship between sperm length, accessory gland and testes size, and male reproductive success in the laboratory and field settings.     

Cloning and characterization of Plasmodium vivax serine hydroxymethyltransferase

Serine hydroxymethyltransferase (SHMT), which catalyzes the reversible reaction of serine and tetrahydrofolate to glycine and methylenetetrahydrofolate, is one of the three enzymes in dTMP synthesis pathway that is highly active during cell division and has been proposed as a potential chemotherapeutic target in infectious diseases and cancer. This is the first study to describe nucleotide and amino acid sequences of SHMT from the malaria parasite Plasmodium vivax. Sequencing of 12 P. vivax isolates revealed limited polymorphisms in 3 noncoding regions. Its biological function is also reported.     

Cloning and heterologous expression of Plasmodium ovale dihydrofolate reductase-thymidylate synthase gene

Plasmodial bifunctional dihydrofolate reductase-thymidylate synthase (DHFR-TS) is a validated antimalarial drug target. In this study, expression of the putative dhfr-ts of Plasmodium ovale rescued the DHFR chemical knockout and a TS null bacterial strain, demonstrating its DHFR and TS catalytic functions. PoDHFR-TS was expressed in Escherichia coli BL21 (DE3) and affinity purified by Methotrexate Sepharose column. Biochemical and enzyme kinetics characterizations indicated that PoDHFR-TS is similar to other plasmodial enzymes, albeit with lower catalytic activity but better tolerance of acidic pH. Importantly, the PoDHFR from Thai isolate EU266602 remains sensitive to the antimalarials pyrimethamine and cycloguanil, in contrast to P. falciparum and P. vivax isolates where resistance to these drugs is widespread.     

Changes in the Treatment Responses to Artesunate-Mefloquine on the Northwestern Border of Thailand during 13 Years of Continuous Deployment

Background

Artemisinin combination treatments (ACT) are recommended as first line treatment for falciparum malaria throughout the malaria affected world. We reviewed the efficacy of a 3-day regimen of mefloquine and artesunate regimen (MAS₃), over a 13 year period of continuous deployment as first-line treatment in camps for displaced persons and in clinics for migrant population along the Thai-Myanmar border.

Methods and Findings

3,264 patients were enrolled in prospective treatment trials between 1995 and 2007 and treated with MAS₃. The proportion of patients with parasitaemia persisting on day-2 increased significantly from 4.5% before 2001 to 21.9% since 2002 (p<0.001). Delayed parasite clearance was associated with increased risk of developing gametocytaemia (AOR = 2.29; 95% CI, 2.00–2.69, p = 0.002). Gametocytaemia on admission and carriage also increased over the years (p = 0.001, test for trend, for both). MAS₃ efficacy has declined slightly but significantly (Hazards ratio 1.13; 95% CI, 1.07–1.19, p<0.001), although efficacy in 2007 remained well within acceptable limits: 96.5% (95% CI, 91.0–98.7). The in vitro susceptibility of P. falciparum to artesunate increased significantly until 2002, but thereafter declined to levels close to those of 13 years ago (geometric mean in 2007: 4.2 nM/l; 95% CI, 3.2–5.5). The proportion of infections caused by parasites with increased pfmdr1 copy number rose from 30% (12/40) in 1996 to 53% (24/45) in 2006 (p = 0.012, test for trend).

Conclusion

Artesunate-mefloquine remains a highly efficacious antimalarial treatment in this area despite 13 years of widespread intense deployment, but there is evidence of a modest increase in resistance. Of particular concern is the slowing of parasitological response to artesunate and the associated increase in gametocyte carriage.     

Population genetic analysis of Plasmodium falciparum parasites using a customized Illumina GoldenGate genotyping assay

The diversity in the Plasmodium falciparum genome can be used to explore parasite population dynamics, with practical applications to malaria control. The ability to identify the geographic origin and trace the migratory patterns of parasites with clinically important phenotypes such as drug resistance is particularly relevant. With increasing single-nucleotide polymorphism (SNP) discovery from ongoing Plasmodium genome sequencing projects, a demand for high SNP and sample throughput genotyping platforms for large-scale population genetic studies is required. Low parasitaemias and multiple clone infections present a number of challenges to genotyping P. falciparum. We addressed some of these issues using a custom 384-SNP Illumina GoldenGate assay on P. falciparum DNA from laboratory clones (long-term cultured adapted parasite clones), short-term cultured parasite isolates and clinical (non-cultured isolates) samples from East and West Africa, Southeast Asia and Oceania. Eighty percent of the SNPs (n = 306) produced reliable genotype calls on samples containing as little as 2 ng of total genomic DNA and on whole genome amplified DNA. Analysis of artificial mixtures of laboratory clones demonstrated high genotype calling specificity and moderate sensitivity to call minor frequency alleles. Clear resolution of geographically distinct populations was demonstrated using Principal Components Analysis (PCA), and global patterns of population genetic diversity were consistent with previous reports. These results validate the utility of the platform in performing population genetic studies of P. falciparum.     

Malaria in the Post-Partum Period; a Prospective Cohort Study

Background

Several studies have shown a prolonged or increased susceptibility to malaria in the post-partum period. A matched cohort study was conducted to evaluate prospectively the susceptibility to malaria of post-partum women in an area where P.falciparum and P.vivax are prevalent.

Methods

In an area of low seasonal malaria transmission on the Thai-Myanmar border pregnant women attending antenatal clinics were matched to a non-pregnant, non-post-partum control and followed up prospectively until 12 weeks after delivery.

Results

Post-partum women (n = 744) experienced significantly less P.falciparum episodes than controls (hazard ratio (HR) 0.39 (95%CI 0.21–0.72) p = 0.003) but significantly more P.vivax (HR 1.34 (1.05–1.72) p = 0.018). The reduced risk of falciparum malaria was accounted for by reduced exposure, whereas a history of P.vivax infection during pregnancy was a strong risk factor for P.vivax in post-partum women (HR 13.98 (9.13–21.41), p<0.001). After controlling for effect modification by history of P.vivax, post-partum women were not more susceptible to P.vivax than controls (HR: 0.33 (0.21–0.51), p<0.001). Genotyping of pre-and post-partum infections (n⊕ = ⊕10) showed that each post-partum P.falciparum was a newly acquired infection.

Conclusions

In this area of low seasonal malaria transmission post-partum women were less likely to develop falciparum malaria but more likely to develop vivax malaria than controls. This was explained by reduced risk of exposure and increased risk of relapse, respectively. There was no evidence for altered susceptibility to malaria in the post-partum period. The treatment of vivax malaria during and immediately after pregnancy needs to be improved.     

Dihydrofolate-Reductase Mutations in Plasmodium knowlesi Appear Unrelated to Selective Drug Pressure from Putative Human-To-Human Transmission in Sabah,Malaysia

Background

Malaria caused by zoonotic Plasmodium knowlesi is an emerging threat in Eastern Malaysia. Despite demonstrated vector competency, it is unknown whether human-to-human (H-H) transmission is occurring naturally. We sought evidence of drug selection pressure from the antimalarial sulfadoxine-pyrimethamine (SP) as a potential marker of H-H transmission.

Methods

The P. knowlesi dihdyrofolate-reductase (pkdhfr) gene was sequenced from 449 P. knowlesi malaria cases from Sabah (Malaysian Borneo) and genotypes evaluated for association with clinical and epidemiological factors. Homology modelling using the pvdhfr template was used to assess the effect of pkdhfr mutations on the pyrimethamine binding pocket.

Results

Fourteen non-synonymous mutations were detected, with the most common being at codon T91P (10.2%) and R34L (10.0%), resulting in 21 different genotypes, including the wild-type, 14 single mutants, and six double mutants. One third of the P. knowlesi infections were with pkdhfr mutants; 145 (32%) patients had single mutants and 14 (3%) had double-mutants. In contrast, among the 47 P. falciparum isolates sequenced, three pfdhfr genotypes were found, with the double mutant 108N+59R being fixed and the triple mutants 108N+59R+51I and 108N+59R+164L occurring with frequencies of 4% and 8%, respectively. Two non-random spatio-temporal clusters were identified with pkdhfr genotypes. There was no association between pkdhfr mutations and hyperparasitaemia or malaria severity, both hypothesized to be indicators of H-H transmission. The orthologous loci associated with resistance in P. falciparum were not mutated in pkdhfr. Subsequent homology modelling of pkdhfr revealed gene loci 13, 53, 120, and 173 as being critical for pyrimethamine binding, however, there were no mutations at these sites among the 449 P. knowlesi isolates.

Conclusion

Although moderate diversity was observed in pkdhfr in Sabah, there was no evidence this reflected selective antifolate drug pressure in humans.     

Genotyping of Plasmodium vivax reveals both short and long latency relapse patterns in Kolkata

Background

The Plasmodium vivax that was once prevalent in temperate climatic zones typically had an interval between primary infection and first relapse of 7–10 months, whereas in tropical areas P.vivax infections relapse frequently at intervals of 3–6 weeks. Defining the epidemiology of these two phenotypes from temporal patterns of illness in endemic areas is difficult or impossible, particularly if they overlap.

Methods

A prospective open label comparison of chloroquine (CQ) alone versus CQ plus unobserved primaquine for either 5 days or 14 days was conducted in patients presenting with acute vivax malaria in Kolkata. Patients were followed for 15 months and primary and recurrent infections were genotyped using three polymorphic antigen and up to 8 microsatellite markers.

Results

151 patients were enrolled of whom 47 (31%) had subsequent recurrent infections. Recurrence proportions were similar in the three treatment groups. Parasite genotyping revealed discrete temporal patterns of recurrence allowing differentiation of probable relapse from newly acquired infections. This suggested that 32 of the 47 recurrences were probable relapses of which 22 (69%) were genetically homologous. The majority (81%) of probable relapses occurred within three months (16 homologous, 10 heterologous) and six genetically homologous relapses (19%) were of the long latency (8–10 month interval) phenotype.

Conclusions

With long follow-up to assess temporal patterns of vivax malaria recurrence, genotyping of P.vivax can be used to assess relapse rates. A 14 day unobserved course of primaquine did not prevent relapse. Genotyping indicates that long latency P.vivax is prevalent in West Bengal, and that the first relapses after long latent periods are genetically homologous.

Trial Registration

Controlled-Trials.com ISRCTN14027467     

The genetic diversity of Plasmodium vivax populations

Little is known of the genetic diversity and population structure of Plasmodium vivax, a debilitating and highly prevalent malaria parasite of humans. This article reviews the known polymorphic genetic markers, summarizes current data on the population structure of this parasite and discusses future prospects for using knowledge of the genetic diversity to improve control measures.     

Sequence variation does not confound the measurement of plasma PfHRP2 concentration in African children presenting with severe malaria

ABSTRACT: BACKGROUND: Plasmodium falciparum histidine-rich protein PFHRP2 measurement is used widely for diagnosis, and more recently for severity assessment in falciparum malaria. The Pfhrp2 gene is highly polymorphic, with deletion of the entire gene reported in both laboratory and field isolates. These issues potentially confound the interpretation of PFHRP2 measurements. METHODS: Studies designed to detect deletion of Pfhrp2 and its paralog Pfhrp3 were undertaken with samples from patients in seven countries contributing to the largest hospital-based severe malaria trial (AQUAMAT). The quantitative relationship between sequence polymorphism and PFHRP2 plasma concentration was examined in samples from selected sites in Mozambique and Tanzania. RESULTS: There was no evidence for deletion of either Pfhrp2 or Pfhrp3 in the 77 samples with lowest PFHRP2 plasma concentrations across the seven countries. Pfhrp2 sequence diversity was very high with no haplotypes shared among 66 samples sequenced. There was no correlation between Pfhrp2 sequence length or repeat type and PFHRP2 plasma concentration. CONCLUSIONS: These findings indicate that sequence polymorphism is not a significant cause of variation in PFHRP2 concentration in plasma samples from African children. This justifies the further development of plasma PFHRP2 concentration as a method for assessing African children who may have severe falciparum malaria. The data also add to the existing evidence base supporting the use of rapid diagnostic tests based on PFHRP2 detection.