Genetic diversity in the C-terminal 42 kDa region of merozoite surface protein-1 of Plasmodium vivax (PvMSP-1(42)) among Indian isolates

Plasmodium vivax merozoite surface protein 1 (PvMSP-1) is a leading malaria vaccine candidate. This protein is processed to give rise to various sized fragments during merozoite maturation. Here, we describe the analysis of genetic diversity in the 42 kDa C-terminal part of this protein among 33 Indian P. vivax isolates. A total of 27 haplotypes with 72 mutations and 0.0212 ± 0.0005S.D. over all π nucleotide diversity were observed among the isolates. Twenty-six of 27 haplotypes reported here were new as they have not been reported so far from any other country. The difference between non-synonymous (dN) and synonymous (dS) mutations was found to be positive (0.0081 ± 0.0051) for the entire 42 kDa region. Further analysis revealed that 33 kDa (MSP-1₃₃) fragment of the MSP-1₄₂ was highly polymorphic with π nucleotide diversity 0.0290 ± 0.0007S.D. The dN-dS for this region of MSP-1 was also positive (0.0114 ± 0.0071S.E.). On the other hand, there was no non-synonymous mutation in the 19 kDa (MSP-1₁₉) fragment of the MSP-1₄₂ and thus it was highly conserved. In conclusion, MSP-1₃₃ fragment was highly polymorphic and appeared to be under diversifying selection whereas there was no selection at MSP-1₁₉ region among the isolates. Present study will be helpful for the development of PvMSP-1 based vaccine against P. vivax malaria.     

High frequency of genetic diversity of Plasmodium vivax field isolates in Myanmar

Malaria is one of the most serious problems threatening human health in Myanmar. Although the morbidity and mortality rates due to malaria have been gradually declining, Myanmar still contributes to a large proportion of malarial death in the South-East Asia region. However, little is known about the nature and extent of genetic diversity of the malarial parasites circulating in Myanmar. In this study, we investigated the overall infection status of Plasmodium and the population diversity of Plasmodium vivax by analyzing three genetic markers, circumsporozoite protein (CSP), merozoite surface protein-1 (MSP-1), and merozoite surface protein-3 (MSP-3α), of P. vivax field isolates collected from infected individuals. In 349 blood samples collected from the individuals who exhibited clinical symptoms associated with malaria, 63.0% showed a positive result for malaria (220/349). P. vivax was detected in 58.2% (128/220) and Plasmodium falciparum was detected in 29.1% (64/220). Mixed infections with both parasites were detected in 12.7% (28/220). The 116 blood samples in which single infection of P. vivax was confirmed were selected and subjected to further genetic analysis. Genotyping of the CSP gene of P. vivax showed that VK210 type (98.3%, 114/116) is predominant in Myanmar, but a significant level of mixed infections of VK210 and VK247 types (24.1%, 28/116) was also identified. Sequence analyses of MSP-1 and MSP-3α genes revealed a large number of distinguishable alleles: 12 for MSP-1 and 25 for MSP-3α. These results collectively suggest that the P. vivax population in Myanmar is highly diverse and multiple clonal infections are prevalent in the country.     

Genetic structure of Plasmodium vivax isolates from two malaria endemic areas in Afghanistan

In this study, the nature and extent of genetic diversity of Plasmodium vivax populations circulating in Afghanistan have been investigated by analyzing three genetic markers: csp, msp-1, and msp-3α. Blood samples (n = 202) were collected from patients presenting with vivax malaria from south-western (Herat) and south-eastern (Nangarhar) parts of Afghanistan, and analysed using nested-PCR/RFLP and sequencing methods. Genotyping pvmsp-1 revealed type 1, type 2 and recombinant type 3 allelic variants, with type 1 predominant in parasites in both study areas. The sequence analysis of 57 P. vivax isolates identified a total of 26 distinct alleles. Genotyping pvcsp gene showed that VK210 type (86.6%) is predominant in Afghanistan. Moreover, three major types of the pvmsp-3α locus: type A, type B and type C were distinguished among Afghani isolates. The predominant fragments among Nangarhar and Herat parasites were type A (70.8% and 67.9%, respectively). PCR/RFLP products with Hha I and Alu I were detected 52 and 38 distinct variants among Nangarhar and Herat isolates, respectively. These results strongly indicate that the P. vivax populations in Afghanistan are highly diverse.     

Geographic Structure of Plasmodium vivax: Microsatellite Analysis of Parasite Populations from Sri Lanka, Myanmar, and Ethiopia

Genetic diversity and population structure of Plasmodium vivax parasites can predict the origin and spread of novel variants within a population enabling population specific malaria control measures. We analyzed the genetic diversity and population structure of 425 P. vivax isolates from Sri Lanka, Myanmar, and Ethiopia using 12 trinucleotide and tetranucleotide microsatellite markers. All three parasite populations were highly polymorphic with 3–44 alleles per locus. Approximately 65% were multiple-clone infections. Mean genetic diversity (H_E) was 0.7517 in Ethiopia, 0.8450 in Myanmar, and 0.8610 in Sri Lanka. Significant linkage disequilibrium was maintained. Population structure showed two clusters (Asian and African) according to geography and ancestry. Strong clustering of outbreak isolates from Sri Lanka and Ethiopia was observed. Predictive power of ancestry using two-thirds of the isolates as a model identified 78.2% of isolates accurately as being African or Asian. Microsatellite analysis is a useful tool for mapping short-term outbreaks of malaria and for predicting ancestry.     

Genetic diversity and natural selection of Duffy binding protein of Plasmodium vivax Korean isolates

Plasmodium vivax Duffy binding protein (PvDBP) is a micronemal type I membrane protein that plays an essential role in erythrocyte invasion of merozoites. PvDBP is a prime blood stage vaccine candidate antigen against P. vivax, but its polymorphic nature represents a major obstacle to the successful design of a protective vaccine against vivax malaria. In this study, we analyzed the genetic polymorphism and natural selection at the N-terminal cysteine-rich region of PvDBP (PvDBPII) among 70 P. vivax isolates collected from Korean patients during 2005–2010. Seventeen single nucleotide polymorphisms (SNP), which resulted in 14 non-synonymous and 3 synonymous mutations, were found in PvDBPII among the Korean P. vivax isolates. Sequence analyses revealed that 13 different PvDBPII haplotypes, which were clustered into 3 distinct clades, were identified in Korean P. vivax isolates. The difference between the rates of nonsynomyous and synonymous mutations suggested that the region has evolved under natural selection. High selective pressure preferentially acted on regions identified or predicted to be B- and T-cell epitopes and MHC binding regions of PvDBPII. Recombination may also contribute to genetic diversity of PvDBPII. Our results suggest that PvDBPII of Korean P. vivax isolates display a limited genetic polymorphism and are under selective pressure. These results have significant implications for understanding the nature of the P. vivax population circulating in Korea and provide useful information for development of malaria vaccines based on this antigen.     

IgG subclasses pattern and high-avidity antibody to the C-terminal region of merozoite surface protein 1 of Plasmodium vivax in an unstable hypoendemic region in Iran

The C-terminal region of Plasmodium vivax merozoite surface protein 1 (PvMSP-1₁₉) is a leading vaccine candidate for inclusion in a polyvalent malaria vaccine. In the present study, the IgG subclasses profile and the avidity of IgG to PvMSP-1₁₉ were evaluated in individuals (n = 94) naturally exposed to P. vivax parasite in malaria endemic areas in Chabahar districts, Iran. In individuals with patent P. vivax malaria, 86.1% was sero-positive to PvMSP-1₁₉ and IgG1 (81.9%) was the predominant subclass. In addition, to determine the persistence of specific IgG, IgG1 and IgG3 antibodies to PvMSP-1₁₉, the frequency of antibodies was determined in the infected subjects (n = 74) after treatment with standard chloroquine and it was detected that the frequency of responders was significantly reduced to 51.3%, 51% and 16.2%, respectively. The antigen-binding avidity of IgG antibodies to PvMSP-1₁₉ was measured in sero-positive sera and the high-avidity of IgG, IgG1 and IgG3 was found in 66.6%, 61% and 47% of the infected subjects with P. vivax, respectively. The present result shows that individuals who exposed to vivax malaria in the endemic region in Iran develop antibodies with high-avidity to PvMSP-1₁₉. These results could help to understand the interactions between the host and P. vivax parasite in development of MSP-1₁₉-based vaccine.     

Genetic diversity of transmission blocking vaccine candidate (Pvs25 and Pvs28) antigen in Plasmodium vivax clinical isolates from Iran

The leading candidates for a Transmission Blocking Vaccine (TBV) in Plasmodium vivax parasite are the ookinete surface protein 25 (Pvs25) and Pvs28, which their phase I clinical trial is ongoing. Therefore, we carried out survey of polymorphisms of the pvs25 and pvs28 genes in P. vivax populations that are circulating in the two malaria areas of contrasting endemicity in Iran, before field application of the TBV. To characterize the polymorphisms of pvs25 and pvs28 genes, 50 isolates were analyzed by sequencing method and their gene structure was compared with parasite populations from India, Bangladesh, Indonesia, Thailand, Mexico and Brazil. Three mutations were detected in pvs25 and pvs28 including Q87K, E97Q, I130T and M52L, T65K, T140S with two and four distinct haplotypes, in comparison with the Sal I sequence type, respectively. Both haplotypes of Pvs25 were found among northern and southern P. vivax isolates; however, only two and three of the Pvs28 variants were observed among the northern and southern isolates, respectively. In conclusion, the present results show the limited sequence polymorphism of the pvs25 and pvs28 genes among field P. vivax population in Iran. These results highly encourage with respect to applicability of Pvs25 and Pvs28-based vaccine against P. vivax infection in the region, where these parasites are prevalent, whether these occur in the temperate or tropical zones.     

Non-allele specific antibody responses to genetically distinct variant forms of Plasmodium vivax Duffy binding protein (PvDBP-II) in Iranians exposed to seasonal malaria transmission

Duffy binding protein (DBP) is a leading vaccine candidate of Plasmodium vivax. The binding domain of DBP (DBP-II) is polymorphic, that may be a major challenge for development of a broadly effective vaccine against vivax malaria. The present investigation was undertaken to explore whether the sequence diversity of DBP-II causes variation in naturally acquired anti-DBP-II antibodies. In this study, the five genetically distinct variants were expressed, and anti-DBP-II responses were measured in P. vivax-infected individuals (n = 202). Finally, by performing immune-depletion ELISA experiments, antibody responses to the conserved sites of all allelic forms were evaluated using the corresponding and non-corresponding patients’ sera (n = 20). In this study, natural P. vivax infection produces IgG against all five examined variant forms of PvDBP-II with no statistically difference. Sequence analysis in the 20 selected samples (for antibody depletion experiment) showed eight distinct haplotypes, DBPI (n = 1), DBPIII (n = 3), DBPIV (n = 1), DBPV (n = 1), DBPVI (n = 5), DBPIX (n = 6), DBPX (n = 1), and DBP XI (n = 2). The results showed the presence of the cross-reactive antibody responses to heterologous variants of PvDBP-II in Iranian individuals who were infected with distinct allelic forms of the PvDBP-II. Therefore, it is proposed that the majority of antibodies recognized sharing B-cell epitopes and this could overcome the PvDBP-II variation as a one of the biggest challenges of PvDBP-II-based vaccine development.     

Distribution of dihydrofolate reductase (dhfr) and dihydropteroate synthase (dhps) mutant alleles in Plasmodium vivax isolates from Thailand

The analysis of prevalence and distribution of pvdhfr and pvdhps mutations were performed in 169 samples collected from patients with Plasmodium vivax infection who attended the malaria clinics in the provinces along the three international borders of Thailand (Thai-Myanmar, Thai-Cambodian, and Thai-Malaysian borders). SNP-haplotypes of the pvdhfr at amino acid positions 13, 33, 57, 58, 61, 117, and 173 and of the pvdhps at positions 383 and 553 were examined by nested PCR-RFLP. Significant differences in the prevalence and distribution of pvdhfr and pvdhps combination alleles were observed in P. vivax isolates collected from all the three border areas. The most prevalent combination alleles were triple mutant pvdhfr 57L/58R/117T alleles/double wild-type pvdhps alleles (n = 18), double mutant pvdhfr 58R/117N alleles/double wild-type pvdhps alleles (n = 10), and triple mutant pvdhfr 58R/61M/117N alleles/double wild-type pvdhps alleles (n = 52) or with single mutant pvdhps 383G allele (n = 28), respectively. These information on prevalence and patterns of pvdhfr and pvdhps polymorphisms obtained from the present study suggest the presence of SP pressure on P. vivax isolates in Thailand which could be linked to the introduction of malaria from neighboring countries. Results did not support the application of SP for P. vivax control program in Thailand as well as the neighboring countries.     

DNA barcoding of Schistosoma haematobium on Zanzibar reveals substantial genetic diversity and two major phylogenetic groups

To shed light on the genetic diversity of Schistosoma haematobium on Zanzibar a DNA barcoding study was performed on parasite material isolated from different time-points 4 years apart. Substantive sequence variation was found within the mitochondrial cytochrome oxidase subunit I (cox1) and the NADH-dehydrogenase subunit 1 (nad1) with 27 and 22 unique haplotypes identified respectively and 38 when both gene regions were considered. Upon phylogenetic analysis and comparison with other S. haematobium isolates, haplotypes or barcode types partitioned into two discrete major groups, designated Group 1 and Group 2. Whilst Group 1 isolates were recovered from both Zanzibar and the African mainland, Group 2 isolates were exclusive to Zanzibar. A mixture of Group 1 and 2 parasites were recovered from individual children with no child shedding parasites of a single group haplotype alone. Whilst changes in general levels of genetic diversity between the two parasite isolation time-points were observed, no obvious change in genetic diversity was detected, despite large-scale drug distribution of praziquantel during the intervening period and there was no biased of Group 1 or 2 parasites persisting at the different time-points. To assist in future genetic screening of schistosome larval stages e.g. eggs, miracidia or cercariae, two new DNA-typing assays based on group-specific PCR primers and SNaPshot™ probes have been developed to distinguish Group 1 and 2 haplotypes.     

Preservation of Wild Isolates of Human Malaria Parasites in Wet Ice and Adaptation Efficacy to In Vitro Culture

Wild isolates of malaria parasites were preserved in wet ice for 2–12 days and cultivated by a candle jar method. In four isolates of Plasmodium falciparum collected from Myanmar and preserved for 12 days, all failed to grow. In 31 isolates preserved for 5–10 days, nine were transformed to young gametocytes, but 22 isolates grew well. From Ranong, Thailand, nine isolates preserved for 7 days were examined, and six grew well. On the other hand, all of the 59 isolates collected from eastern Indonesian islands failed to establish as culture-adapted isolates, even most of them were preserved only for 2–3 days: 10 isolates stopped to grow, and 49 isolates were transformed to sexual stages by Day 10. These results indicated that a great difference in adaptation to in vitro culture may exist between wild isolates distributed in continental Southeast Asia and in eastern Indonesia and that gametocytogenesis might be easily switched on in Indonesian isolates. In wild isolates of P. vivax, P. malariae and P. ovale preserved for 2–9 days, ring forms or young trophozoites survived, but adaptation to in vitro culture failed. These results indicate that wild isolates can be preserved in wet ice for 9–10 days.     

Evidence for genetic linkage between a polymorphism in the GNAS gene and malaria in South Indian population

The complex imprinted GNAS locus which encodes G-alpha subunit (Gαs) is involved in a number of G-protein coupled signaling pathways in eukaryotic cells. Erythrocyte invasion by Plasmodium falciparum parasites is significantly regulated by protein of GNAS gene. This study was designed to evaluate the association between single nucleotide polymorphisms (SNPs) present in GNAS locus and susceptibility to malaria. In this case control study, individuals affected by P. falciparum malaria (n = 230), Plasmodium vivax malaria (n = 230) and normal controls (n = 230) were tested for the association of eighteen (18) known SNPs to evaluate their role in the onset of the disease. There was no significant difference in genotype frequencies of all the SNPs tested between P. falciparum and P. vivax affected individuals. However, when Bonferroni correction for multiple comparisons were performed as a control, our results demonstrated alleles and genotypes of rs7121: C > T (NC_000020.10:g.57478807C > T), a silent polymorphism situated in the exon 5, were significantly (p < 0.05) associated with susceptibility to malaria in the South Indians participants. Our results demonstrate that population specific polymorphisms that exist in GNAS gene may alter the risk of occurrence of malaria.     

A preliminary study on pro- and anti-inflammatory cytokine profiles in Plasmodium vivax malaria patients from central zone of India

The aim of this preliminary study was to investigate the plasma cytokine profiles in a group of patients suffering from Plasmodium vivax malaria during the peak of its transmission season. Plasma samples of 173 P. vivax patients and 34 healthy individuals were analyzed for IFN-γ, TNF-α, IL-10 and IP-10 levels by ELISA. Levels of both pro- and anti-inflammatory cytokines were significantly higher in P. vivax patients compared to controls. Children with P. vivax infection had significantly higher levels of IFN-γ than adults (P = 0.017). Asexual parasitaemia versus TNF-α (r = −0.31, P = 0.01), IL-10 (r = −0.30, P = 0.015) and gametocytaemia versus IFN-γ (r = −0.26; P = 0.034) levels showed significant negative correlation in children compared to adults. The median concentrations of IFN-γ (P = 0.001), IL-10 (P = 0.032) and IP-10 (P ≤ 0.05) were higher in children reported with chills and rigors, whereas in adults only IFN-γ levels was higher (P < 0.0001). The median plasma concentrations of IFN- γ (P = 0.02), IL-10 (P < 0.0001) and IP-10 (P = 0.068) were higher in patients with mild anaemia compared to non-anaemic patients. The results indicated that both pro- and anti-inflammatory cytokine responses are associated with clinical signs of mild anaemia and paroxysm during symptomatic P. vivax malaria in Central India.     

Plasmodium vivax Isolates from Cambodia and Thailand Show High Genetic Complexity and Distinct Patterns of P. vivax Multidrug Resistance Gene 1 (pvmdr1) Polymorphisms

Plasmodium vivax accounts for an increasing fraction of malaria infections in Thailand and Cambodia. We compared P. vivax genetic complexity and antimalarial resistance patterns in the two countries. Use of a heteroduplex tracking assay targeting the merozoite surface protein 1 gene revealed that vivax infections in both countries are frequently polyclonal (84%), with parasites that are highly diverse (H_E = 0.86) but closely related (G_ST = 0.18). Following a history of different drug policies in Thailand and Cambodia, distinct patterns of antimalarial resistance have emerged: most Cambodian isolates harbor the P. vivax multidrug resistance gene 1 (pvmdr1) 976F mutation associated with chloroquine resistance (89% versus 8%, P < 0.001), whereas Thai isolates more often display increased pvmdr1 copy number (39% versus 4%, P < 0.001). Finally, genotyping of paired isolates from individuals suspected of suffering relapse supports a complex scheme of relapse whereby recurrence of multiple identical variants is sometimes accompanied by the appearance of novel variants.     

Therapeutic efficacy of chloroquine and chloroquine plus primaquine for the treatment of Plasmodium vivax in Ethiopia

Plasmodium vivax is the second most important cause of morbidity in Ethiopia. There is, however, little information on P. vivax resistance to chloroquine and chloroquine plus primaquine treatment although these drugs have been used as the first line treatment for over 50 years. We assessed the efficacy of standard chloroquine and chloroquine plus primaquine treatment for P. vivax infections in a randomized open-label comparative study in Debre Zeit and Nazareth in East Shoa, Ethiopia.A total of 290 patients with microscopically confirmed P. vivax malaria who presented to the outpatient settings of the two laboratory centers were enrolled: 145 patients were randomized to receive CQ and 145 to receive CQ + PQ treatment. Participants were followed-up for 28-157 days according to the WHO procedures. There were 12 (6.5%) lost to follow-up patients and 9 (3.1%) withdrawals. In all, 96% (277/290) of patients were analysed at day 28. Baseline characteristics were similar in all treatment groups. In all, 98.6% (275/277) of patients had cleared their parasitemia on day 3 with no difference in mean parasite clearance time between regimens (48.34 ± 17.68, 50.67 ± 15.70 h for the CQ and CQ + PQ group, respectively, P = 0.25). The cumulative incidence of therapeutic failure at day 28 by a life-table analysis method was 5.76% (95% CI: 2.2-14.61) and 0.75% (95% CI: 0.11-5.2%) in the CQ and CQ + PQ group, respectively (P = 0.19). The relapse rate was 8% (9/108) for the CQ group and 3% (4/132) for the comparison group (P = 0.07). The cumulative risk of relapse at day 157 by a life-table method was 61.8% (95% CI: 20.1-98.4%) in the CQ group, compared with 26.3% (95% CI: 7.5-29.4%) in the CQ + PQ group (P = 0.0038).The study confirms the emergence of CQ and PQ resistance/treatment failure in P. vivax malaria in Ethiopia. Although treatment failures were detected, they were similar between the treatment groups. We recommend regular monitoring and periodic evaluation of the efficacy of these antimalarial drugs in systematically selected sentinel sites to detect further development of resistance and to make timely national antimalarial drug policy changes.     

Prevalence and risk factors associated to pruritus in Plasmodium vivax patients using chloroquine in the Brazilian Amazon

Chloroquine-induced pruritus has been described as a common adverse event in African patients being treated for Plasmodium falciparum malaria, and has been associated with treatment discontinuation in this setting. In Latin America, where Plasmodium vivax is the most common species causing malaria and chloroquine is still used as the first-line schizonticidal for treating this parasite infection, there are no reports on chloroquine-induced pruritus. This study aimed to estimate the frequency of pruritus and associated risk factors in P. vivax-infected patients treated with chloroquine in a reference centre in the Brazilian Amazon. In this cross-sectional study, patients who were prescribed with chloroquine for the treatment of microscopy-confirmed P. vivax infection in the past five days were actively asked about the occurrence of any level of pruritus and potential risk factors were investigated. Univariable and multivariable logistic regression was performed for the analysis of possible risk factors in two sets of patients: (1) all the patients interviewed and (2) restricted to patients with previous use of chloroquine. Among the 510 patients interviewed, 20.4% (95%CI: 16.9–23.9%) developed any level of pruritus during treatment with chloroquine. Most episodes of pruritus occurred during the first two days of treatment and the most common location was hands and feet. In multivariate analysis performed in the entire population, the only risk factors independently associated to pruritus were allergy history (adjusted odds ratio [AOR]: 1.83; 95%CI 1.02–3.31; p = 0.044) and high parasitaemia (AOR: 1.96: 95%CI 1.22–3.13; p = 0.005). In the analysis restricted to the 215 patients with previous use of chloroquine, previous chloroquine-induced pruritus was a strong predictor of pruritus occurrence (AOR: 11.84: 95%CI 3.15–44.47; p < 0.001). Two patients (0.4%) interrupted treatment due to the severity of pruritus. Pruritus is a common adverse event in patients being treated with chloroquine for P. vivax malaria in the Brazilian Amazon. Host–parasite interaction may play a relevant role in the development of pruritus and concurs with the finding of strong association of pruritus with high parasitaemia and allergy history. Patients with previous chloroquine-induced pruritus had a high risk for developing pruritus. Due to its high frequency, this side effect cannot be neglected as it can have major implications on patients’ compliance to treatment hampering elimination efforts in the region.     

Mutations in the Antifolate-Resistance-Associated Genes Dihydrofolate Reductase and Dihydropteroate Synthase in Plasmodium vivax Isolates from Malaria-Endemic Countries

Parasite dihydrofolate reductase (DHFR) and dihydropteroate synthase (DHPS) are known target enzymes of antifolate drugs used for the treatment and prophylaxis of persons with malaria. We sequenced the Plasmodium vivax dihydrofolate reductase (pvdhfr) and dihydropteroate synthase (pvdhps) genes to examine the prevalence and extent of point mutations in isolates from malaria-endemic countries. Double mutations (S58R and S117N) or quadruple mutations (F57L/I, S58R, T61M, and S117T) in the pvdhfr gene were found in isolates from Thailand (96.4%) and Myanmar (71.4%), but in only one isolate (1.0%) from Korea, where sulfadoxine-pyrimethamine has never been used. The pvdhfr point mutations correlated strongly with the pvdhps point mutations and ranged from single to triple mutations (S382A, A383G, and A553G), among isolates from Thailand, Myanmar, and Korea. These findings suggests that the prevalence of mutations in pvdhfr and pvdhps in P. vivax isolates from different malaria-endemic countries is associated with selection pressure imposed by sulfadoxine-pyrimethamine.     

Subtle changes in Plasmodium falciparum infection complexity following enhanced intervention in Malawi

With support from the Global Fund, the United States President's Malaria Initiative (PMI) and other cooperating partners, Malawi is implementing a comprehensive malaria control programme involving indoor residual spraying in targeted districts, universal coverage with insecticide-treated bed nets, use of rapid diagnostic tests to confirm the clinical diagnosis of malaria and use of the highly effective artemisinin-based combination therapy, artemether-lumefantrine (AL), as the first-line treatment for malaria. We genotyped 24 genome-wide single nucleotide polymorphisms (SNPs) in Plasmodium falciparum infections (n = 316) sampled from a single location in Malawi before (2006 and 2007) and after enhanced intervention (2008 and 2012). The SNP data generated were used to examine temporal changes in the proportion of multiple-genotype infections (MIs), mean number of heterozygous SNPs within MIs, parasite genetic diversity (expected heterozygosity and genotypic richness), multilocus linkage disequilibrium and effective population size (N_e). While the proportion of MIs, expected heterozygosity, genotypic richness, multilocus linkage disequilibrium and N_e were unchanged over time, the mean number (±standard deviation) of heterozygous SNPs within MIs decreased significantly (p = 0.01) from 9(±1) in 2006 to 7(±1) in 2012. These findings indicate that the genetic diversity of P. falciparum malaria parasites in this area remains high, suggesting that only subtle gains, if any, have been made in reducing malaria transmission. Continued surveillance is required to evaluate the impact of malaria control interventions in this area and the rest of Malawi, and to better target control interventions.     

Plasmodium falciparum populations from northeastern Myanmar display high levels of genetic diversity at multiple antigenic loci

Levels of genetic diversity of the malaria parasites and multiclonal infections are correlated with transmission intensity. In order to monitor the effect of strengthened malaria control efforts in recent years at the China–Myanmar border area, we followed the temporal dynamics of genetic diversity of three polymorphic antigenic markers msp1, msp2, and glurp in the Plasmodium falciparum populations. Despite reduced malaria prevalence in the region, parasite populations exhibited high levels of genetic diversity. Genotyping 258 clinical samples collected in four years detected a total of 22 PCR size alleles. Multiclonal infections were detected in 45.7% of the patient samples, giving a minimum multiplicity of infection of 1.41. The majority of alleles experienced significant temporal fluctuations through the years. Haplotype diversity based on the three-locus genotypes ranged from the lowest in 2009 at 0.33 to the highest in 2010 at 0.80. Sequencing of msp1 fragments from 36 random samples of five allele size groups detected 13 different sequences, revealing an additional layer of genetic complexity. This study suggests that despite reduced prevalence of malaria infections in this region, the parasite population size and transmission intensity remained high enough to allow effective genetic recombination of the parasites and continued maintenance of genetic diversity.     

Extensive heterozygosity in flanking microsatellites of Plasmodium falciparum Na/H exchanger (pfnhe-1) gene among Indian isolates

Plasmodium falciparum Na⁺/H⁺ exchanger-1 (pfnhe-1) gene has been proposed to be a possible marker for quinine resistance. Here, we describe the sequence analysis of the flanking microsatellites of the pfnhe-1 gene among 108 Indian P.falciparum isolates. Among the parasite population, a high degree of polymorphism was observed at all the 10 microsatellite loci within ±40 kb region of the pfnhe-1 gene where the number of alleles varied from 2 to 16 with a high expected heterozygosity ranging from 0.43 to 0.91 at these loci. Also, higher levels of heterozygosity have been observed in P.falciparum isolates collected from both low and high transmission and drug resistant areas. Furthermore, there was no association between QN resistance associated DNNND repeats in PFNHE-1 and the flanking microsatellite haplotypes. In conclusion, the observed high level of microsatellite polymorphism and absence of selective sweep in the flanking ±40 kb region of the pfnhe-1 gene could be an indication that there is no strong selection pressure on this target gene.