Vivax Isolates Research Articles

Genetic polymorphism of Duffy binding protein in Pakistan Plasmodium vivax isolates

Plasmodium vivax Duffy binding protein (PvDBP) is crucial for erythrocyte invasion, interacting with the Duffy Antigen Receptor for Chemokines (DARC) on the erythrocyte surface. The amino-terminal cysteine-rich region II of PvDBP (PvDBPII) is a promising blood stage vaccine candidate, yet the genetic polymorphisms of this protein in global P. vivax isolates complicate the design of effective vaccines against vivax malaria. This study analyzed the genetic polymorphism of PvDBPII in Pakistan P. vivax isolates. A total of 29 single nucleotide polymorphisms (SNPs), including 22 nonsynonymous SNPs, were identified in 118 Pakistan PvDBPII. Most amino acid substitutions occurred in subdomains II and III, with six commonly observed in the global PvDBPII population. The amino acid change patterns in Pakistan PvDBPII generally mirrored those in global PvDBPII, although the frequencies of amino acid changes varied by country. Nucleotide diversity in Pakistan PvDBPII was comparable to that found in global PvDBPII. Evidence of natural selection and recombination in Pakistan PvDBPII aligned with observations in global PvDBPII. Analysis of the haplotype network of global PvDBPII revealed a complexed network of 167 haplotypes, but no geographical clustering was observed. The findings are crucial for understanding the genetic characteristics of Pakistan PvDBPII. A comprehensive analysis of nucleotide diversity and evolutionary trends in the global PvDBPII population offers valuable insights for the development of vivax malaria vaccines based on this antigen.

Immunogenicity and transmission-blocking potential of quiescin sulfhydryl oxidase in Plasmodium vivax.

Transmission-blocking vaccines (TBVs) can effectively prevent the community's spread of malaria by targeting the antigens of mosquito sexual stage parasites. At present, only a few candidate antigens have demonstrated transmission-blocking activity (TBA) potential in P. vivax. Quiescin-sulfhydryl oxidase (QSOX) is a sexual stage protein in the rodent malaria parasite Plasmodium berghei and is associated with a critical role in protein folding by introducing disulfides into unfolded reduced proteins. Here, we reported the immunogenicity and transmission-blocking potency of the PvQSOX in P. vivax. The full-length recombinant PvQSOX protein (rPvQSOX) was expressed in the Escherichia coli expression system. The anti-rPvQSOX antibodies were generated following immunization with the rPvQSOX in rabbits. A parasite integration of the pvqsox gene into the P. berghei pbqsox gene knockout genome was developed to express full-length PvQSOX protein in P. berghei (Pv-Tr-PbQSOX). In western blot, the anti-rPvQSOX antibodies recognized the native PvQSOX protein expressed in transgenic P. berghei gametocyte and ookinete. In indirect immunofluorescence assays, the fluorescence signal was detected in the sexual stages, including gametocyte, gamete, zygote, and ookinete. Anti-rPvQSOX IgGs obviously inhibited the ookinetes and oocysts development both in vivo and in vitro using transgenic parasites. Direct membrane feeding assays of anti-rPvQSOX antibodies were conducted using four field P. vivax isolates (named isolates #1-4) in Thailand. Oocyst density in mosquitoes was significantly reduced by 32.00, 85.96, 43.52, and 66.03% with rabbit anti-rPvQSOX antibodies, respectively. The anti-rPvQSOX antibodies also showed a modest reduction of infection prevalence by 15, 15, 20, and 22.22%, respectively, as compared to the control, while the effect was insignificant. The variation in the DMFA results may be unrelated to the genetic polymorphisms. Compared to the P.vivax Salvador (Sal) I strain sequences, the pvqsox in isolate #1 showed no amino acid substitution, whereas isolates #2, #3, and #4 all had the M361I substitution. Our results suggest that PvQSOX could serve as a potential P. vivax TBVs candidate, which warrants further evaluation and optimization.

Population genetic analysis of Plasmodium vivax vir genes in Pakistan.

Plasmodium vivax variant interspersed repeats (vir) refer to the key protein used for escaping the host immune system. Knowledge in the genetic variation of vir genes can be used for the development of vaccines or diagnostic methods. Therefore, we evaluated the genetic diversity of the vir genes of P. vivax populations of several Asian countries, including Pakistan, which is a malaria-endemic country experiencing a significant rise in malaria cases in recent years. We analyzed the genetic diversity and population structure of 4 vir genes (vir 4, vir 12, vir 21, and vir 27) in the Pakistan P. vivax population and compared these features to those of the corresponding vir genes in other Asian countries. In Pakistan, vir 4 (S=198, H=9, Hd=0.889, Tajima's D value=1.12321) was the most genetically heterogenous, while the features of vir 21 (S=8, H=7, Hd=0.664, Tajima's D value =-0.63763) and vir 27 (S =25, H =11, Hd =0.682, Tajima's D value=-2.10836) were relatively conserved. Additionally, vir 4 was the most genetically diverse among Asian P. vivax populations, although within population diversity was low. Meanwhile, vir 21 and vir 27 among all Asian populations were closely related genetically. Our findings on the genetic diversity of vir genes and its relationships between populations in diverse geographical locations contribute toward a better understanding of the genetic characteristics of vir. The high level of genetic diversity of vir 4 suggests that this gene can be a useful genetic marker for understanding the P. vivax population structure. Longitudinal genetic diversity studies of vir genes in P. vivax isolates obtained from more diverse geographical areas are needed to better understand the function of vir genes and their use for the development of malaria control measures, such as vaccines.

Extensive genetic diversity in Plasmodium vivax from Sudan and its genetic relationships with other geographical isolates

Plasmodium vivax, traditionally overlooked has experienced a notable increase in cases in East Africa. This study investigated the geographical origin and genetic diversity of P. vivax in Sudan using 14 microsatellite markers. A total of 113 clinical P. vivax samples were collected from two different ecogeographical zones, New Halfa and Khartoum, in Sudan. Additionally, 841 geographical samples from the database were incorporated for a global genetic analysis to discern genetic relationships among P. vivax isolates on regional and worldwide scales. On the regional scale, our findings revealed 91 unique and 8 shared haplotypes among the Sudan samples, showcasing a remarkable genetic diversity compared to other geographical isolates and supporting the hypothesis that P. vivax originated from Africa. On a global scale, distinct genetic clustering of P. vivax isolates from Africa, South America, and Asia (including Papua New Guinea and Solomon Island) was observed, with limited admixture among the three clusters. Principal component analysis emphasized the substantial contribution of African isolates to the observed global genetic variation. The Sudanese populations displayed extensive genetic diversity, marked by significant multi-locus linkage disequilibrium, suggesting an ancestral source of P. vivax variation globally and frequent recombination among the isolates. Notably, the East African P. vivax exhibited similarity with some Asian isolates, indicating potential recent introductions. Overall, our results underscore the effectiveness of utilizing microsatellite markers for implementing robust control measures, given their ability to capture extensive genetic diversity and linkage disequilibrium patterns.

Spatio-temporal analysis of genetic diversity of merozoite surface protein-3 alpha in Myanmar Plasmodium vivax isolates

Myanmar aims to eliminate malaria by 2030. However, recent increase of malaria incidence is a great challenge to archive that goal. Increasing prevalence of Plasmodium vivax also hinders this endeavor. Monitoring genetic structure of the parasite is necessary to understand genetic nature and evolutionary aspect of P. vivax population in Myanmar. Partial fragment flanking blocks I and II of merozoite surface protein-3 alpha of P. vivax (pvmsp-3α) was amplified from P. vivax isolates collected in Pyin Oo Lwin, Mandalay Region, Myanmar in 2013–2015. Sequence analysis of pvmsp-3α was performed to determine genetic diversity and natural selection of this gene. Spatio-temporal genetic changes of pvmsp-3α in Myanmar P. vivax population were also investigated via comparative analysis of gene sequences obtained in this study and previously reported Myanmar pvmsp-3α sequences. Genetic diversity of Myanmar pvmsp-3α was detected in P. vivax isolates analyzed. Size polymorphisms in block I and amino acid changes and recombination events in block II were main factors contributing to the genetic diversity of pvmsp-3α. Comparative spatio-temporal analysis with previously reported Myanmar pvmsp-3α populations revealed the presence of genetic differences by population with moderate genetic differentiation between populations. Similar pattern of natural selection was also detected in Myanmar pvmsp-3α populations. These suggested that enough size of the P. vivax population sufficient to generate or maintain the genetic diversity remains in the population. Thus, continuous molecular surveillance of genetic structure of Myanmar P. vivax is necessary.

Natural genetic diversity of the DBL domain of a novel member of the Plasmodium vivax erythrocyte binding-like proteins (EBP2) in the Amazon rainforest

In malaria parasites, the erythrocyte binding-like proteins (EBL) are a family of invasion proteins that are attractive vaccine targets. In the case of Plasmodium vivax, the widespread malaria parasite, blood-stage vaccines have been largely focused on a single EBL candidate, the Duffy binding-like domain (DBL) of the Duffy binding protein (DBPII), due to its well-characterized role in the reticulocyte invasion. A novel P. vivax EBL family member, the Erythrocyte binding protein (EBP2, also named EBP or DBP2), binds preferentially to reticulocytes and may mediate an alternative P. vivax invasion pathway. To gain insight into the natural genetic diversity of the DBL domain of EBP2 (region II; EBP2-II), we analyzed ebp2-II gene sequences of 71 P. vivax isolates collected in different endemic settings of the Brazilian Amazon rainforest, where P. vivax is the predominant malaria-associated species. Although most of the substitutions in the ebp2-II gene were non-synonymous and suggested positive selection, the results showed that the DBL domain of the EBP2 was much less polymorphic than that of DBPII. The predominant EBP2 haplotype in the Amazon region corresponded to the C127 reference sequence first described in Cambodia (25% C127-like haplotype). An overview of ebp2-II gene sequences available at GenBank (n = 352) from seven countries (Cambodia, Madagascar, Myanmar, PNG, South Korea, Thailand, Vietnam) confirmed the C127-like haplotype as highly prevalent worldwide. Two out of 43 haplotypes (5 to 20 inferred per country) showed a global frequency of 60%. The results presented here open new avenues of research pursuit while suggesting that a vaccine based on the DBL domain of EBP2 should target a few haplotypes for broad coverage.

Evaluation of transmission-blocking potential of PvPSOP25 using transgenic murine malaria parasite and clinical isolates.

Malaria transmission-blocking vaccines (TBVs) aim to inhibit malaria parasite development in mosquitoes and prevent further transmission to the human host. The putative-secreted ookinete protein 25 (PSOP25), highly conserved in Plasmodium spp., is a promising TBV target. Here, we investigated PvPSOP25 from P. vivax as a TBV candidate using transgenic murine parasite P. berghei and clinical P. vivax isolates. A transgenic P. berghei line expressing PvPSOP25 (TrPvPSOP25Pb) was generated. Full-length PvPSOP25 was expressed in the yeast Pichia pastoris and used to immunize mice to obtain anti-rPvPSOP25 sera. The transmission-blocking activity of the anti-rPvPSOP25 sera was evaluated through in vitro assays and mosquito-feeding experiments. The antisera generated by immunization with rPvPSOP25 specifically recognized the native PvPSOP25 antigen expressed in TrPvPSOP25Pb ookinetes. In vitro assays showed that the immune sera significantly inhibited exflagellation and ookinete formation of the TrPvPSOP25Pb parasite. Mosquitoes feeding on mice infected with the transgenic parasite and passively transferred with the anti-rPvPSOP25 sera showed a 70.7% reduction in oocyst density compared to the control group. In a direct membrane feeding assay conducted with five clinical P. vivax isolates, the mouse anti-rPvPSOP25 antibodies significantly reduced the oocyst density while showing a negligible influence on mosquito infection prevalence. This study supported the feasibility of transgenic murine malaria parasites expressing P. vivax antigens as a useful tool for evaluating P. vivax TBV candidates. Meanwhile, the moderate transmission-reducing activity of the generated anti-rPvPSOP25 sera necessitates further research to optimize its efficacy.

Genetic diversity of the PvMSP-3α gene in Plasmodium vivax isolates circulating in the National Capital Region (NCR) of India

Malaria is still a public health problem in tropical countries like India; major malaria parasite species are Plasmodium falciparum and P. vivax. Of which, P. vivax is responsible for ∼40% of the malaria burden at least in the Indian scenario. Unfortunately, there is limited data on the population structure and genetic diversity of P. vivax parasites in India. In this study, we investigated the genetic diversity of P. vivax strains in the South-west district, Delhi and, Nuh district, Haryana [National Capital Region (NCR)], using a polymorphic marker- P. vivax merozoite surface protein-3α (PvMSP-3α) gene. Dried blood spots from microscopically confirmed P. vivax patients were used for investigation of the PvMSP-3α gene. PCR-RFLP was performed on the PvMSP-3α gene to investigate the genotypes and allelic variability with HhaI and AluI restriction enzymes. In total, 40 successfully PCR amplified PvMSP-3α gene segments were subjected to RFLP analysis. Amplified products showed three different base pair size variations viz. genotype A in 31(77.5%), genotype B in 4(10%) and genotype C in 5(12.5%) P. vivax specimens. RFLP with HhaI and AluI revealed 17 (H1-H17) and 25 (A1-A25) allelic variants, respectively. Interestingly, two similar sub-allelic variants, ie. H8 (with HhaI), and A4 (with AluI) clustered within the rural area of Nuh district, Haryana in two samples. With this study, we propose to commission such type of genetic diversity analysis of P. vivax to investigate the circulating genotypes of the parasites from distinct geographical locations across India, that can have significant implications in understanding the population structures of P. vivax.

Tracing the origins of Plasmodium vivax resurgence after malaria elimination on Aneityum Island in Vanuatu

BackgroundFive years after successful malaria elimination, Aneityum Island in Vanuatu experienced an outbreak of Plasmodium vivax of unknown origin in 2002. Epidemiological investigations revealed several potential sources of P. vivax. We aimed to identify the genetic origin of P. vivax responsible for the resurgence.MethodsFive P. vivax microsatellite markers were genotyped using DNA extracted from archived blood samples. A total of 69 samples from four P. vivax populations was included: 29 from the outbreak in 2002, seven from Aneityum in 1999 and 2000, 18 from visitors to Aneityum in 2000, and 15 from nearby Tanna Island in 2002. A neighbour-joining phylogenetic tree was constructed to elucidate the relationships among P. vivax isolates. STRUCTURE and principal component analysis were used to assess patterns of genetic structure.ResultsHere we show distinct genetic origins of P. vivax during the outbreak on Aneityum. While the origin of most P. vivax lineages found during the outbreak remains unidentified, limited genetic diversity among these lineages is consistent with a rapid expansion from a recent common ancestor. Contemporaneous P. vivax from neighboring Tanna and potential relapse of P. vivax acquired from other islands in 1999 and 2000 are also identified as minor contributors to the outbreak.ConclusionsMultiple reintroductions of P. vivax after elimination highlight the high receptivity and vulnerability to malaria resurgence in island settings of Vanuatu, despite robust surveillance and high community compliance to control measures.

A two-dose viral-vectored Plasmodium vivax multistage vaccine confers durable protection and transmission-blockade in a pre-clinical study.

Among Plasmodium spp. responsible for human malaria, Plasmodium vivax ranks as the second most prevalent and has the widest geographical range; however, vaccine development has lagged behind that of Plasmodium falciparum, the deadliest Plasmodium species. Recently, we developed a multistage vaccine for P. falciparum based on a heterologous prime-boost immunization regimen utilizing the attenuated vaccinia virus strain LC16m8Δ (m8Δ)-prime and adeno-associated virus type 1 (AAV1)-boost, and demonstrated 100% protection and more than 95% transmission-blocking (TB) activity in the mouse model. In this study, we report the feasibility and versatility of this vaccine platform as a P. vivax multistage vaccine, which can provide 100% sterile protection against sporozoite challenge and >95% TB efficacy in the mouse model. Our vaccine comprises m8Δ and AAV1 viral vectors, both harboring the gene encoding two P. vivax circumsporozoite (PvCSP) protein alleles (VK210; PvCSP-Sal and VK247; -PNG) and P25 (Pvs25) expressed as a Pvs25-PvCSP fusion protein. For protective efficacy, the heterologous m8Δ-prime/AAV1-boost immunization regimen showed 100% (short-term; Day 28) and 60% (long-term; Day 242) protection against PvCSP VK210 transgenic Plasmodium berghei sporozoites. For TB efficacy, mouse sera immunized with the vaccine formulation showed >75% TB activity and >95% transmission reduction activity by a direct membrane feeding assay using P. vivax isolates in blood from an infected patient from the Brazilian Amazon region. These findings provide proof-of-concept that the m8Δ/AAV1 vaccine platform is sufficiently versatile for P. vivax vaccine development. Future studies are needed to evaluate the safety, immunogenicity, vaccine efficacy, and synergistic effects on protection and transmission blockade in a non-human primate model for Phase I trials.

Low Genetic Diversity of Plasmodium vivax Circumsporozoite Surface Protein in Clinical Isolates from Southern Thailand.

The genetic diversity within the circumsporozoite surface protein (PvCSP) of Plasmodium vivax, the predominant malaria species in Thailand, is primarily observed in the northwestern region along the Thailand-Myanmar border. However, as P. vivax cases shift to southern provinces, particularly Yala Province near the Thailand-Malaysia border, PvCSP diversity remains understudied. Between 2018 and 2020, 89 P. vivax isolates were collected in Yala Province, a significant malaria hotspot. Employing polymerase chain reaction amplification, restriction fragment length polymorphism (PCR-RFLP), and DNA sequencing, the gene encoding PvCSP (Pvcsp) was analyzed. All Yala P. vivax isolates belonged to the VK210 type, distinct from strains in the western region near the Myanmar border. The central repeat region of Pvcsp revealed two common peptide repeat motifs-GDRADGQPA and GDRAAGQPA-across all southern isolates. Sequence analysis identified two subtypes, with S1 more prevalent (92%) than S2 (8%). This study underscores the limited diversity of VK210 variants of P. vivax populations in southern Thailand. These baseline findings facilitate monitoring for potential new parasite variants, aiding in the future control and management of P. vivax in the region.

Alterations in histopathology and stress-associated gene expression induced by infection with Prohemistomum vivax encysted metacercariae in Nile tilapia

Due to limited data on the pathogenicity of Prohemistomum vivax (P. vivax) and its impacts on fish health, this study aimed to determine the morphological, molecular characteristics, pathogenicity, and histopathological alterations in fish infected with P. vivax. Eight hundred (800) Nile tilapia (Oreochromis niloticus) were collected from various farms in Kafr El Sheikh Governorate. The fish were examined for encysted metacercariae (EMC) in different organs. Tissue specimens were collected and underwent histopathological analysis, expression of stress-related genes, and genetic characterization by sequencing of the internal transcribed spacer 2 (ITS2). P. vivax metacercariae were oval to round in shape and were collected from various organs including the muscle, skin, eyes, intestine, liver, kidney, and gills of infected O. niloticus. Sequencing and phylogenetic analysis of the ITS2 region revealed a 507-bp fragment, confirming parasite identity and matching within the same clade as other P. vivax isolates. Infected fish displayed abdominal hydropsy, skin darkening, and emaciation. P. vivax encysted metacercariae were detected during the study period in 620/800 fish, with an overall prevalence of 77.5%. The seasonal prevalence was 95% in summer, 85% in spring, 55% in autumn, and 75% in winter. The intensity of infection was 1–40 cysts per microscopic field. Histopathological examination of muscles revealed parasitic cysts embedded within muscle fibers, causing severe degeneration and necrosis. Upregulation of cytochrome P450 (cpy1a1), heat shock protein 70 (hsp-70), and tumor suppressor p53 (p53) was recorded in both liver and muscle samples of infected tilapia compared to controls. This indicates activation of detoxification, cellular stress, and apoptotic pathways in response to P. vivax infection. There is limited data available on the pathogenicity of P. vivax and its impacts on fish health; thus, this study provides key insights into the morphology, pathogenicity, and histopathological impacts of P. vivax in Nile tilapia.

Genetic differentiation of Plasmodium vivax duffy binding protein in Ethiopia and comparison with other geographical isolates

BackgroundPlasmodium vivax Duffy binding protein (PvDBP) is a merozoite surface protein located in the micronemes of P. vivax. The invasion of human reticulocytes by P. vivax merozoites depends on the parasite DBP binding domain engaging Duffy Antigen Receptor for Chemokine (DARC) on these red blood cells (RBCs). PvDBPII shows high genetic diversity which is a major challenge to its use in the development of a vaccine against vivax malaria.MethodsA cross-sectional study was conducted from February 2021 to September 2022 in five study sites across Ethiopia. A total of 58 blood samples confirmed positive for P. vivax by polymerase chain reaction (PCR) were included in the study to determine PvDBPII genetic diversity. PvDBPII were amplified using primers designed from reference sequence of P. vivax Sal I strain. Assembling of sequences was done using Geneious Prime version 2023.2.1. Alignment and phylogenetic tree constructions using MEGA version 10.1.1. Nucleotide diversity and haplotype diversity were analysed using DnaSP version 6.12.03, and haplotype network was generated with PopART version 1.7.ResultsThe mean age of the participants was 25 years, 5 (8.6%) participants were Duffy negatives. From the 58 PvDBPII sequences, seven haplotypes based on nucleotide differences at 8 positions were identified. Nucleotide diversity and haplotype diversity were 0.00267 ± 0.00023 and 0.731 ± 0.036, respectively. Among the five study sites, the highest numbers of haplotypes were identified in Arbaminch with six different haplotypes while only two haplotypes were identified in Gambella. The phylogenetic tree based on PvDBPII revealed that parasites of different study sites shared similar genetic clusters with few exceptions. Globally, a total of 39 haplotypes were identified from 223 PvDBPII sequences representing different geographical isolates obtained from NCBI archive. The nucleotide and haplotype diversity were 0.00373 and 0.845 ± 0.015, respectively. The haplotype prevalence ranged from 0.45% to 27.3%. Two haplotypes were shared among isolates from all geographical areas of the globe.ConclusionsPvDBPII of the Ethiopian P. vivax isolates showed low nucleotide but high haplotype diversity, this pattern of genetic variability suggests that the population may have undergone a recent expansion. Among the Ethiopian P. vivax isolates, almost half of the sequences were identical to the Sal-I reference sequence. However, there were unique haplotypes observed in the Ethiopian isolates, which does not share with isolates from other geographical areas. There were two haplotypes that were common among populations across the globe. Categorizing population haplotype frequency can help to determine common haplotypes for designing an effective blood-stage vaccine which will have a significant role for the control and elimination of P. vivax.

Genomic insights into Plasmodium vivax population structure and diversity in central Africa

BackgroundThough Plasmodium vivax is the second most common malaria species to infect humans, it has not traditionally been considered a major human health concern in central Africa given the high prevalence of the human Duffy-negative phenotype that is believed to prevent infection. Increasing reports of asymptomatic and symptomatic infections in Duffy-negative individuals throughout Africa raise the possibility that P. vivax is evolving to evade host resistance, but there are few parasite samples with genomic data available from this part of the world.MethodsWhole genome sequencing of one new P. vivax isolate from the Democratic Republic of the Congo (DRC) was performed and used in population genomics analyses to assess how this central African isolate fits into the global context of this species.ResultsPlasmodium vivax from DRC is similar to other African populations and is not closely related to the non-human primate parasite P. vivax-like. Evidence is found for a duplication of the gene PvDBP and a single copy of PvDBP2.ConclusionThese results suggest an endemic P. vivax population is present in central Africa. Intentional sampling of P. vivax across Africa would further contextualize this sample within African P. vivax diversity and shed light on the mechanisms of infection in Duffy negative individuals. These results are limited by the uncertainty of how representative this single sample is of the larger population of P. vivax in central Africa.

PvDBPII elicits multiple antibody-mediated mechanisms that reduce growth in a Plasmodium vivax challenge trial

The receptor-binding domain, region II, of the Plasmodium vivax Duffy binding protein (PvDBPII) binds the Duffy antigen on the reticulocyte surface to mediate invasion. A heterologous vaccine challenge trial recently showed that a delayed dosing regimen with recombinant PvDBPII SalI variant formulated with adjuvant Matrix-MTM reduced the in vivo parasite multiplication rate (PMR) in immunized volunteers challenged with the Thai P. vivax isolate PvW1. Here, we describe extensive analysis of the polyfunctional antibody responses elicited by PvDBPII immunization and identify immune correlates for PMR reduction. A classification algorithm identified antibody features that significantly contribute to PMR reduction. These included antibody titre, receptor-binding inhibitory titre, dissociation constant of the PvDBPII-antibody interaction, complement C1q and Fc gamma receptor binding and specific IgG subclasses. These data suggest that multiple immune mechanisms elicited by PvDBPII immunization are likely to be associated with protection and the immune correlates identified could guide the development of an effective vaccine for P. vivax malaria. Importantly, all the polyfunctional antibody features that correlated with protection cross-reacted with both PvDBPII SalI and PvW1 variants, suggesting that immunization with PvDBPII should protect against diverse P. vivax isolates.

Drug resistance markers in Plasmodium vivax isolates from a Kanchanaburi province, Thailand between January to May 2023.

Plasmodium vivax has become the predominant species in the border regions of Thailand. The emergence and spread of antimalarial drug resistance in P. vivax is one of the significant challenges for malaria control. Continuous surveillance of drug resistance is therefore necessary for monitoring the development of drug resistance in the region. This study aims to investigate the prevalence of the mutation in the P. vivax multidrug resistant 1 (Pvmdr1), dihydrofolate reductase (Pvdhfr), and dihydropteroate synthetase (Pvdhps) genes conferred resistance to chloroquine (CQ), pyrimethamine (P) and sulfadoxine (S), respectively. 100 P. vivax isolates were obtained between January to May 2023 from a Kanchanaburi province, western Thailand. Nucleotide sequences of Pvmdr1, Pvdhfr, and Pvdhps genes were amplified and sequenced. The frequency of single nucleotide polymorphisms (SNPs)-haplotypes of drug-resistant alleles was assessed. The linkage disequilibrium (LD) tests were also analyzed. In Pvmdr1, T958M, Y976F, and F1076L, mutations were detected in 100%, 21%, and 23% of the isolates, respectively. In Pvdhfr, the quadruple mutant allele (I57R58M61T117) prevailed in 84% of the samples, followed by (L57R58M61T117) in 11%. For Pvdhps, the double mutant allele (G383G553) was detected (48%), followed by the triple mutant allele (G383M512G553) (47%) of the isolates. The most prevalent combination of Pvdhfr (I57R58M61T117) and Pvdhps (G383G553) alleles was sextuple mutated haplotypes (48%). For LD analysis, the association in the SNPs pairs was found between the intragenic and intergenic regions of the Pvdhfr and Pvdhps genes. The study has recently updated the high prevalence of three gene mutations associated with CQ and SP resistance. Genetic monitoring is therefore important to intensify in the regions to further assess the spread of drug resistant. Our data also provide evidence on the distribution of drug resistance for the early warning system, thereby threatening P. vivax malaria treatment policy decisions at the national level.

Genomic analysis of Plasmodium vivax describes patterns of connectivity and putative drivers of adaptation in Ethiopia

Ethiopia has the greatest burden of Plasmodium vivax in Africa, but little is known about the epidemiological landscape of parasites across the country. We analysed the genomic diversity of 137 P. vivax isolates collected nine Ethiopian districts from 2012 to 2016. Signatures of selection were detected by cross-country comparisons with isolates from Thailand (n = 104) and Indonesia (n = 111), representing regions with low and high chloroquine resistance respectively. 26% (35/137) of Ethiopian infections were polyclonal, and 48.5% (17/35) of these comprised highly related clones (within-host identity-by-descent > 25%), indicating frequent co-transmission and superinfection. Parasite gene flow between districts could not be explained entirely by geographic distance, with economic and cultural factors hypothesised to have an impact on connectivity. Amplification of the duffy binding protein gene (pvdbp1) was prevalent across all districts (16–75%). Cross-population haplotype homozygosity revealed positive selection in a region proximal to the putative chloroquine resistance transporter gene (pvcrt-o). An S25P variant in amino acid transporter 1 (pvaat1), whose homologue has recently been implicated in P. falciparum chloroquine resistance evolution, was prevalent in Ethiopia (96%) but not Thailand or Indonesia (35–53%). The genomic architecture in Ethiopia highlights circulating variants of potential public health concern in an endemic setting with evidence of stable transmission.

Genetic diversity of merozoite surface protein-5 (MSP-5) of Plasmodium vivax isolates from Malaria patients in Iran

Malaria has not yet been eradicated in Iran, and Plasmodium vivax (P. vivax) is the main cause of malaria in the country. This study aimed to investigate and analyze the amount of genetic diversity of Plasmodium vivax merozoite surface protein-5 (PvMSP-5) exon 1 gene in the southeast of Iran.Thirty-five patients with clinical symptoms of P. vivax malaria participated. The exon 1 of PvMSP-5 was amplified by PCR, and the PCR product of all isolates was sequenced, and genetic polymorphisms were determined using various genetic software.The analysis showed that studied isolates are different from one another in the DnaSP software version. Out of the 612 sites, 477 were monomorphic and 135 were segregated. The total number of mutations was 143. The singleton variable and the parsimony informative sites were 23 and 112, respectively. There were 17 specific haplotypes with haplotype diversity equal to 0.943. Nucleotide diversity was equal to 0.06766 in the isolates. The ratio of nonsynonymous (0.06446) to synonymous (0.07909) mutations was 0.815020. Tajima’s D, which expressed coding, and non-coding regions, was 0.72403, which was not deemed significant (P > 0.10).The analysis of intrapopulation diversity revealed nucleotide and haplotype diversity in the msp-5 gene of Iranian P. vivax isolates. In addition to balancing or purifying selection, intragenic recombination also contributed to the variation observed in exon 1 of PvMSP-5, according to the findings.

Molecular detection of antimalarial resistance in Plasmodium vivax isolates from a tertiary care setting in Puducherry

PurposeThe study was aimed at detecting the mutation patterns in the drug targets in Plasmodium vivax that confer resistance to the common antimalarial agents used in India. MethodsA total of 27 Plasmodium vivax isolates collected from whole blood samples over a three year period were subjected to PCR amplification followed by sequencing of the genes pvmdr1, pvdhfr, pvdhps and pvk12, which serve as the molecular targets to detect resistance to chloroquine, pyrimethamine, sulfadoxine and artemisinin respectively. ResultsThe study found T958 M F1076L double mutants of pvmdr1 in 52 %(14/27) isolates, S58R S117 N double mutants of pvdhfr in 67 % (18/27) isolates, A383G A553G double mutant pvdhps in 59 % (16/27) isolates and wild type of pvk12 gene in all the isolates. ConclusionsThere was a rise in the proportion of double mutants of pvmdr1 and pvdhfr over time. Those cases with double mutant pvmdr1 gene in their isolates were found to have a prolonged hospital stay compared to those without, indicating reduced clinical response to chloroquine.

Molecular surveillance of chloroquine resistance in Plasmodium vivax isolates from malaria cases in Yunnan Province of China using pvcrt-o gene polymorphisms

BackgroundThe efficacy of chloroquine treatment for vivax malaria has been rarely evaluated due to a lack of an appropriate testing method. The objective of this study was to conduct molecular monitoring of chloroquine resistance in Plasmodium vivax strains from vivax malaria patients in Yunnan Province, focusing on the analysis of polymorphism in the P. vivax chloroquine resistance transporter protein orthologous gene (pvcrt-o).MethodsIn accordance with the principles of a cohort study, blood samples were collected from malaria cases diagnosed with a P. vivax mono-infection in Yunnan Province from 2020 to 2022. Segmental PCR was used to amplify the whole pvcrt-o gene in the blood samples and their products were subsequently sequenced. The sequencing data were arranged to obtain the full coding DNA sequence (CDS) as well as the gene’s promoter region sequences. The CDSs were aligned with the reference sequence (XM_001613407.1) of the P. vivax SalI isolate to identify the mutant loci.ResultsFrom a total of 375 blood samples taken from vivax malaria cases, 272 both whole gene CDSs (1272–1275 bp) and promoter DNA sequences (707 bp) of pvcrt-o gene were obtained. Among the whole CDSs, there were 7 single nucleotide polymorphic sites in which c.7 A>G was the minor allele frequency (MAF) site with 4.4% (12/272) detection rate. The mutation detection rate showed a significant decrease from 9.8% (10/102) in 2020 to 1.1% (1/92) in 2021 and 1.3% (1/78) in 2022, indicating statistical significance (χ2 = 11.256, P < 0.05). Among the identified 12 haplotypes, the majority of which were wild type (75.7%; 206/272). These four mutant haplotypes (Hap_3, Hap_5, Hap_9, and Hap_10) were classified as “K10 insertion type” and accounted for 12.1% (33/272). The detection rate of Hap_3 increased from 1.0% (1/102) in 2020 to 13.0% (12/92) in 2021 and 14.1% (11/78) in 2022, indicating statistical significance. A total of 23.8% (65/272) of the samples exhibited 14 bp (bp) deletions in the promoter region, occurring most frequently in the wild type haplotype (Hap_1) samples at a rate of 28.6% (59/206).ConclusionsIn recent years in Yunnan Province, a notable proportion of vivax malaria patients are infected by P. vivax strains with a “K10 insertion” and partial sequence deletions in the promoter region of the pvcrt-o gene, necessitating vigilance.