Plasmodium vivax spleen-dependent protein 1 and its role in extracellular vesicles-mediated intrasplenic infections.

Recent studies indicate that human spleen contains over 95% of the total parasite biomass during chronic asymptomatic infections caused by Plasmodium vivax. Previous studies have demonstrated that extracellular vesicles (EVs) secreted from infected reticulocytes facilitate binding to human spleen fibroblasts (hSFs) and identified parasite genes whose expression was dependent on an intact spleen. Here, we characterize the P. vivax spleen-dependent hypothetical gene (PVX_114580). Using CRISPR/Cas9, PVX_114580 was integrated into P. falciparum 3D7 genome and expressed during asexual stages. Immunofluorescence analysis demonstrated that the protein, which we named P. vivax Spleen-Dependent Protein 1 (PvSDP1), was located at the surface of infected red blood cells in the transgenic line and this localization was later confirmed in natural infections. Plasma-derived EVs from P. vivax-infected individuals (PvEVs) significantly increased cytoadherence of 3D7_PvSDP1 transgenic line to hSFs and this binding was inhibited by anti-PvSDP1 antibodies. Single-cell RNAseq of PvEVs-treated hSFs revealed increased expression of adhesion-related genes. These findings demonstrate the importance of parasite spleen-dependent genes and EVs from natural infections in the formation of intrasplenic niches in P. vivax, a major challenge for malaria elimination.

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

BACKGROUND: 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. METHODS AND FINDINGS: 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. CONCLUSIONS: 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.

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.

Plasmodium vivax tryptophan-rich antigen reduces type I collagen secretion via the NF-κBp65 pathway in splenic fibroblasts.

BACKGROUND: The spleen plays a critical role in the immune response against malaria parasite infection, where splenic fibroblasts (SFs) are abundantly present and contribute to immune function by secreting type I collagen (collagen I). The protein family is characterized by Plasmodium vivax tryptophan-rich antigens (PvTRAgs), comprising 40 members. PvTRAg23 has been reported to bind to human SFs (HSFs) and affect collagen I levels. Given the role of type I collagen in splenic immune function, it is important to investigate the functions of the other members within the PvTRAg protein family. METHODS: Protein structural prediction was conducted utilizing bioinformatics analysis tools and software. A total of 23 PvTRAgs were successfully expressed and purified using an Escherichia coli prokaryotic expression system, and the purified proteins were used for co-culture with HSFs. The collagen I levels and collagen-related signaling pathway protein levels were detected by immunoblotting, and the relative expression levels of inflammatory factors were determined by quantitative real-time PCR. RESULTS: In silico analysis showed that P. vivax has 40 genes encoding the TRAg family. The C-terminal region of all PvTRAgs is characterized by the presence of a domain rich in tryptophan residues. A total of 23 recombinant PvTRAgs were successfully expressed and purified. Only five PvTRAgs (PvTRAg5, PvTRAg16, PvTRAg23, PvTRAg30, and PvTRAg32) mediated the activation of the NF-κBp65 signaling pathway, which resulted in the production of inflammatory molecules and ultimately a significant reduction in collagen I levels in HSFs. CONCLUSIONS: Our research contributes to the expansion of knowledge regarding the functional role of PvTRAgs, while it also enhances our understanding of the immune evasion mechanisms utilized by parasites.

Evidence of Plasmodium vivax circulation in western and eastern regions of Senegal: implications for malaria control.

BACKGROUND: Malaria elimination in Senegal requires accurate diagnosis of all Plasmodium species. Plasmodium falciparum is the most prevalent species in Senegal, although Plasmodium malariae, Plasmodium ovale, and recently Plasmodium vivax have also been reported. Nonetheless, most malaria control tools, such as Histidine Rich Protein 2 rapid diagnosis test (PfHRP2-RDT,) can only diagnose P. falciparum. Thus, PfHRP2-RDT misses non-falciparum species and P. falciparum infections that fall below the limit of detection. These limitations can be addressed using highly sensitive Next Generation Sequencing (NGS). This study assesses the burden of the four different Plasmodium species in western and eastern regions of Senegal using targeted PCR amplicon sequencing. METHODS: Three thousand samples from symptomatic and asymptomatic individuals in 2021 from three sites in Senegal (Sessene, Diourbel region; Parcelles Assainies, Kaolack region; Gabou, Tambacounda region) were collected. All samples were tested using PfHRP2-RDT and photoinduced electron transfer polymerase chain reaction (PET-PCR), which detects all Plasmodium species. Targeted sequencing of the nuclear 18S rRNA and the mitochondrial cytochrome B genes was performed on PET-PCR positive samples. RESULTS: Malaria prevalence by PfHRP2-RDT showed 9.4% (94/1000) and 0.2% (2/1000) in Diourbel (DBL) and Kaolack (KL), respectively. In Tambacounda (TAM) patients who had malaria symptoms and had a negative PfHRP2-RDT were enrolled. The PET-PCR had a positivity rate of 23.5% (295/1255) overall. The PET-PCR positivity rate was 37.6%, 12.3%, and 22.8% in Diourbel, Kaolack, and Tambacounda, respectively. Successful sequencing of 121/295 positive samples detected P. falciparum (93%), P. vivax (2.6%), P. malariae (4.4%), and P. ovale wallikeri (0.9%). Plasmodium vivax was co-identified with P. falciparum in thirteen samples. Sequencing also detected two PfHRP2-RDT-negative mono-infections of P. vivax in Tambacounda and Kaolack. CONCLUSION: The findings demonstrate the circulation of P. vivax in western and eastern Senegal, highlighting the need for improved malaria control strategies and accurate diagnostic tools to better understand the prevalence of non-falciparum species countrywide.

Prevalence and Characteristics of Plasmodium vivax Gametocytes in Duffy-Positive and Duffy-Negative Populations across Ethiopia.

Plasmodium parasites replicate asexually in human hosts. The proportion of infections that carries gametocytes is a proxy for human-to-mosquito transmissibility. It is unclear which proportion of Plasmodium vivax infections in Duffy-negative populations carries gametocytes. We determined the prevalence and characteristics of P. vivax gametocytes in Duffy-positive and -negative populations across broad regions of Ethiopia. Finger-prick blood samples were collected for microscopic and molecular screening of Plasmodium parasites and Duffy status of individuals. Molecular screening of Plasmodium species and Duffy blood group genotyping was done using SYBR green and the Taqman quantitative polymerase chain reaction method. Of the 447 febrile patients who were shown to be P. vivax smear positive, 414 (92.6%) were confirmed by molecular screening as P. vivax and 16 (3.9%) of them were from Duffy-negative individuals. Of these, 5 of 16 (31.3%) Duffy-negative P. vivax-infected samples were detected with gametocytes. Of the 398 Duffy-positive P. vivax-infected samples, 150 (37.7%) were detected with gametocytes, slightly greater than that in Duffy-negative samples. This study highlights the presence of P. vivax gametocytes in Duffy-negative infections, suggestive of human-to-mosquito transmissibility. Although P. vivax infections in Duffy-negative individuals were commonly associated with low parasitemia, some of these infections were shown to have relatively high parasitemia and may represent a prominent erythrocyte invasion capability of P. vivax, and hidden reservoirs that can contribute to transmission. A better understanding of P. vivax transmission biology and gametocyte function particularly in Duffy-negative populations would aid future treatment and management of P. vivax malaria in Africa.

Exploring the genetic diversity pattern of PvEBP/DBP2: A promising candidate for an effective Plasmodium vivax vaccine.

Malaria remains a public health challenge. Since many control strategies have proven ineffective in eradicating this disease, new strategies are required, among which the design of a multivalent vaccine stands out. However, the effectiveness of this strategy has been hindered, among other reasons, by the genetic diversity observed in parasite antigens. In Plasmodium vivax, the Erythrocyte Binding Protein (PvEBP, also known as DBP2) is an alternate ligand to Duffy Binding Protein (DBP); given its structural resemblance to DBP, EBP/DBP2 is proposed as a promising antigen for inclusion in vaccine design. However, the extent of genetic diversity within the locus encoding this protein has not been comprehensively assessed. Thus, this study aimed to characterize the genetic diversity of the locus encoding the P. vivax EBP/DBP2 protein and to determine the evolutionary mechanisms modulating this diversity. Several intrapopulation genetic variation parameters were estimated using 36 gene sequences of PvEBP/DBP2 from Colombian P. vivax clinical isolates and 186 sequences available in databases. The study then evaluated the worldwide genetic structure and the evolutionary forces that may influence the observed patterns of genetic variation. It was found that the PvEBP/DBP2 gene exhibits one of the lowest levels of genetic diversity compared to other vaccine-candidate antigens. Four major haplotypes were shared worldwide. Analysis of the protein's 3D structure and epitope prediction identified five regions with potential antigenic properties. The results suggest that the PvEBP/DBP2 protein possesses ideal characteristics to be considered when designing a multivalent effective antimalarial vaccine against P. vivax.

Autochthonous Plasmodium vivax Infections, Florida, USA, 2023.

During May-July 2023, a cluster of 7 patients at local hospitals in Florida, USA, received a diagnosis of Plasmodium vivax malaria. Whole-genome sequencing of the organism from 4 patients and phylogenetic analysis with worldwide representative P. vivax genomes indicated probable single parasite introduction from Central/South America.

The Plasmodium vivax MSP1P-19 is involved in binding of reticulocytes through interactions with the membrane proteins band3 and CD71.

The parasite Plasmodium vivax preferentially invades human reticulocytes. Its merozoite surface protein 1 paralog (PvMSP1P), particularly the 19-kDa C-terminal region (PvMSP1P-19), has been shown to bind to reticulocytes, and this binding can be inhibited by antisera obtained by PvMSP1P-19 immunization. The molecular mechanism of interactions between PvMSP1P-19 and reticulocytes during P. vivax invasion, however, remains unclear. In this study, we analyzed the ability of MSP1P-19 to bind to different concentrations of reticulocytes and confirmed its reticulocyte preference. LC-MS analysis was used to identify two potential reticulocyte receptors, band3 and CD71, that interact with MSP1P-19. Both PvMSP1P-19 and its sister taxon Plasmodium cynomolgi MSP1P-19 were found to bind to the extracellular loop (loop 5) of band3, where the interaction of MSP1P-19 with band3 was chymotrypsin sensitive. Antibodies against band3-P5, CD71, and MSP1P-19 reduced the binding activity of PvMSP1P-19 and Plasmodium cynomolgi MSP1P-19 to reticulocytes, while MSP1P-19 proteins inhibited Plasmodium falciparum invasion in vitro in a concentration-dependent manner. To sum up, identification and characterization of the reticulocyte receptor is important for understanding the binding of reticulocytes by MSP1P-19.

Characterization of drug resistance genes in Indian Plasmodium falciparum and Plasmodium vivax field isolates.

One of the major challenges for malaria control and elimination is the spread and emergence of antimalarial drug resistance. Mutations in Plasmodium falciparum (Pf) and Plasmodium vivax (Pv) field isolates for five drug resistance genes viz. crt, mdr1, dhps, dhfr and kelch known to confer resistance to choloroquine (CQ), sulfadoxine-pyrimethamine (SP) and artemisinin (ART) and its derivatives were analyzed. A total of 342 symptomatic isolates of P. falciparum (Pf) and P. vivax (Pv) from 1993 to 2014 were retrieved from malaria parasite repository at National Institute of Malaria Research (NIMR). Sample DNA was extracted from dried blood spots and various targeted single nucleotide polymorphisms (SNPs) associated with antimalarial drug resistance were analysed for these isolates. 72S (67.7%) and 76T (83.8%) mutations along with SVMNT haplotype (67.7%) predominated the study population for Pfcrt. The most prevalent SNPs were 108N (73.2%) and 437G (24.8%) and the most prevalent haplotypes were ACNRNI (51.9%) and SAKAA (74.5%) in Pfdhfr and Pfdhps respectively. Only two mutations in Pfmdr1, 86Y (26.31%) and 184F (56.26%), were seen frequently in our study population. No mutations associated with Pfk13 were observed. For Pv, all the studied isolates showed two Pvdhps mutations, 383G and 553G, and two Pfdhfr mutations, 58R and 117N. Similarly, three mutations, viz. 958M, 908L and 1076L were found in Pvmdr1. No variations were observed in Pvcrt-o and Pvk12 genes. Overall, our study demonstrates an increase in mutations associated with SP resistance in both Pf and Pv, however, no single nucleotide polymorphisms (SNPs) associated with ART resistance have been observed for either species. Various SNPs associated with CQ resistance were seen in Pf; whereas only Pvmdr1 associated resistant SNPs were observed in Pv. Therefore, molecular characterization of drug resistance genes is essential for timely monitoring and prevention of malaria by identifying the circulating drug resistant parasites in the country.

Exploring mosquito abundance and Plasmodium infection through nested-PCR: implications for disease surveillance and control.

The Plasmodium is responsible for malaria which poses a major health threat, globally. This study is based on the estimation of the relative abundance of mosquitoes, and finding out the correlations of meteorological parameters (temperature, humidity and rainfall) with the abundance of mosquitoes. In addition, this study also focused on the use of nested PCR (species-specific nucleotide sequences of 18S rRNA genes) to explore the Plasmodium spp. in female Anopheles. In the current study, the percentage relative abundance of Culex mosquitoes was 57.65% and Anopheles 42.34% among the study areas. In addition, the highest number of mosquitoes was found in March in district Mandi Bahauddin at 21 °C (Tmax = 27, Tmin = 15) average temperature, 69% average relative humidity and 131 mm rainfall, and these climatic factors were found to affect the abundance of the mosquitoes, directly or indirectly. Molecular analysis showed that overall, 41.3% of the female Anopheles pools were positive for genus Plasmodium. Among species, the prevalence of Plasmodium (P.) vivax (78.1%) was significantly higher than P. falciparum (21.9%). This study will be helpful in the estimation of future risk of mosquito-borne diseases along with population dynamic of mosquitoes to enhance the effectiveness of vector surveillance and control programs.

Profile of metacaspase gene expression in Plasmodium vivax field isolates from the Brazilian Amazon.

BACKGROUND: Metacaspases comprise a family of cysteine proteases implicated in both cell death and cell differentiation of protists that has been considered a potential drug target for protozoan parasites. However, the biology of metacaspases in Plasmodium vivax - the second most prevalent and most widespread human malaria parasite worldwide, whose occurrence of chemoresistance has been reported in many endemic countries, remains largely unexplored. Therefore, the present study aimed to address, for the first time, the expression pattern of metacaspases in P. vivax parasites. METHODS AND RESULTS: P. vivax blood-stage parasites were obtained from malaria patients in the Brazilian Amazon and the expression of the three putative P. vivax metacaspases (PvMCA1-3) was detected in all isolates by quantitative PCR assay. Of note, the expression levels of each PvMCA varied noticeably across isolates, which presented different frequencies of parasite forms, supporting that PvMCAs may be expressed in a stage-specific manner as previously shown in P. falciparum. CONCLUSION: The detection of metacaspases in P. vivax blood-stage parasites reported herein, allows the inclusion of these proteases as a potential candidate drug target for vivax malaria, while further investigations are still required to evaluate the activity, role and essentiality of metacaspases in P. vivax biology.

Epidemiology of Plasmodium vivax in Duffy negatives and Duffy positives from community and health centre collections in Ethiopia.

BACKGROUND: Malaria remains a significant cause of morbidity and mortality in Ethiopia with an estimated 3.8 million cases in 2021 and 61% of the population living in areas at risk of malaria transmission. Throughout the country Plasmodium vivax and Plasmodium falciparum are co-endemic, and Duffy expression is highly heterogeneous. The public health significance of Duffy negativity in relation to P. vivax malaria in Ethiopia, however, remains unclear. This study seeks to explore the prevalence and rates of P. vivax malaria infection across Duffy phenotypes in clinical and community settings. METHODS: A total of 9580 and 4667 subjects from community and health facilities from a malaria endemic site and an epidemic-prone site in western Ethiopia were enrolled and examined for P. vivax infection and Duffy expression from February 2018 to April 2021. Association between Duffy expression, P. vivax and P. falciparum infections were examined for samples collected from asymptomatic community volunteers and symptomatic subjects from health centres. RESULTS: Infection rate of P. vivax among Duffy positives was 2-22 fold higher than Duffy negatives in asymptomatic volunteers from the community. Parasite positivity rate was 10-50 fold higher in Duffy positives than Duffy negatives among samples collected from febrile patients attending health centres and mixed P. vivax and P. falciparum infections were significantly more common than P. vivax mono infections among Duffy negative individuals. Plasmodium vivax parasitaemia measured by 18sRNA parasite gene copy number was similar between Duffy positives and Duffy negatives. CONCLUSIONS: Duffy negativity does not offer complete protection against infection by P. vivax, and cases of P. vivax in Duffy negatives are widespread in Ethiopia, being found in asymptomatic volunteers from communities and in febrile patients from health centres. These findings offer evidence for consideration when developing control and intervention strategies in areas of endemic P. vivax and Duffy heterogeneity.

Characterization of merozoite-specific thrombospondin-related anonymous protein (MTRAP) in Plasmodium vivax and P. knowlesi parasites.

Plasmodium vivax, the most widespread human malaria parasite, and P. knowlesi, an emerging Plasmodium that infects humans, are the phylogenetically closest malarial species that infect humans, which may induce cross-species reactivity across most co-endemic areas in Southeast Asia. The thrombospondin-related anonymous protein (TRAP) family is indispensable for motility and host cell invasion in the growth and development of Plasmodium parasites. The merozoite-specific TRAP (MTRAP), expressed in blood-stage merozoites, is supposed to be essential for human erythrocyte invasion. We aimed to characterize MTRAPs in blood-stage P. vivax and P. knowlesi parasites and ascertain their cross-species immunoreactivity. Recombinant P. vivax and P. knowlesi MTRAPs of full-length ectodomains were expressed in a mammalian expression system. The MTRAP-specific immunoglobulin G, obtained from immune animals, was used in an immunofluorescence assay for subcellular localization and invasion inhibitory activity in blood-stage parasites was determined. The cross-species humoral immune responses were analyzed in the sera of patients with P. vivax or P. knowlesi infections. The MTRAPs of P. vivax (PvMTRAP) and P. knowlesi (PkMTRAP) were localized on the rhoptry body of merozoites in blood-stage parasites. Both anti-PvMTRAP and anti-PkMTRAP antibodies inhibited erythrocyte invasion of blood-stage P. knowlesi parasites. The humoral immune response to PvMTRAP showed high immunogenicity, longevity, and cross-species immunoreactivity with P. knowlesi. MTRAPs are promising candidates for development of vaccines and therapeutics against vivax and knowlesi malaria.

A Novel Ex Vivo Assay to Evaluate Functional Effectiveness of Plasmodium vivax Transmission-Blocking Vaccine Using Pvs25 Transgenic Plasmodium berghei.

BACKGROUND: Plasmodium falciparum and Plasmodium vivax account for >90% global malaria burden. Transmission intervention strategies encompassing transmission-blocking vaccines (TBV) and drugs represent ideal public health tools to eliminate malaria at the population level. The availability of mature P. falciparum gametocytes through in vitro culture has facilitated development of a standard membrane feeding assay to assess efficacy of transmission interventions against P. falciparum. The lack of in vitro culture for P. vivax has significantly hampered similar progress on P. vivax and limited studies have been possible using blood from infected patients in endemic areas. The ethical and logistical limitations of on-time access to blood from patients have impeded the development of P. vivax TBVs. METHODS: Transgenic murine malaria parasites (Plasmodium berghei) expressing TBV candidates offer a promising alternative for evaluation of P. vivax TBVs through in vivo studies in mice, and ex vivo membrane feeding assay (MFA). RESULTS: We describe the development of transmission-competent transgenic TgPbvs25 parasites and optimization of parameters to establish an ex vivo MFA to evaluate P. vivax TBV based on Pvs25 antigen. CONCLUSIONS: The MFA is expected to expedite Pvs25-based TBV development without dependence on blood from P. vivax-infected patients in endemic areas for evaluation.

Burden of Submicroscopic Plasmodium Infections and Detection of kelch13 Mutant Parasites in Military and Civilian Populations in Papua New Guinea.

Malaria remains a major public health problem in Papua New Guinea (PNG) and an important force health protection issue for both PNG and Australian Defence Forces. To investigate the malaria burden in the military and civilians residing on military bases, a cross-sectional survey was conducted in April 2019 at three military bases in Wewak, Manus Island, and Vanimo, PNG. A total of 1,041 participants were enrolled; 235 military personnel from three bases and 806 civilians from Wewak and Vanimo. Polymerase chain reaction (PCR) revealed an overall high prevalence of Plasmodium infection in both the military and civilians. Among the military, the infection prevalence was significantly higher in Wewak (35.5%) and Vanimo (33.3%) bases than on Manus Island (11.8%). Among civilians, children (<16 years old) had significantly higher odds of being PCR positive than adults (≥16 years old). At Wewak and Vanimo, Plasmodium vivax accounted for 85.4%, 78.2%, and 66.2% of infections in military, children, and adult populations. Overall, 87.3%, 41.3%, and 61.3% of Plasmodium infections in the military, children, and adults, respectively, were detected only by PCR, not by microscopy (submicroscopic [SM] infections). Children had a significantly lower proportion of SM infections than adults and Papua New Guinea Defence Force personnel. Infection status was not associated with hemoglobin levels in these populations at the time of the survey. Mutant kelch13 (C580Y) parasites were identified in 5/68 Plasmodium falciparum-infected individuals. The survey results indicate extensive malaria transmission on these bases, especially in Wewak and Vanimo. More intensified interventions are required to reduce malaria transmission on PNG military bases.

The Genome of Plasmodium gonderi: Insights into the Evolution of Human Malaria Parasites.

Plasmodium species causing malaria in humans are not monophyletic, sharing common ancestors with nonhuman primate parasites. Plasmodium gonderi is one of the few known Plasmodium species infecting African old-world monkeys that are not found in apes. This study reports a de novo assembled P. gonderi genome with complete chromosomes. The P. gonderi genome shares codon usage, syntenic blocks, and other characteristics with the human parasites Plasmodium ovale s.l. and Plasmodium malariae, also of African origin, and the human parasite Plasmodium vivax and species found in nonhuman primates from Southeast Asia. Using phylogenetically aware methods, newly identified syntenic blocks were found enriched with conserved metabolic genes. Regions outside those blocks harbored genes encoding proteins involved in the vertebrate host-Plasmodium relationship undergoing faster evolution. Such genome architecture may have facilitated colonizing vertebrate hosts. Phylogenomic analyses estimated the common ancestor between P. vivax and an African ape parasite P. vivax-like, within the Asian nonhuman primates parasites clade. Time estimates incorporating P. gonderi placed the P. vivax and P. vivax-like common ancestor in the late Pleistocene, a time of active migration of hominids between Africa and Asia. Thus, phylogenomic and time-tree analyses are consistent with an Asian origin for P. vivax and an introduction of P. vivax-like into Africa. Unlike other studies, time estimates for the clade with Plasmodium falciparum, the most lethal human malaria parasite, coincide with their host species radiation, African hominids. Overall, the newly assembled genome presented here has the quality to support comparative genomic investigations in Plasmodium.

Evaluation of the Performance of Rapid Diagnostic Tests for Malaria Diagnosis and Mapping of Different Plasmodium Species in Mali.

BACKGROUND: The first-line diagnosis of malaria in Mali is based on the use of rapid diagnostic tests (RDT) that detect the Histidin Rich Protein 2 (HRP2) antigen specific to Plasmodium falciparum. Our study, based on a real-time polymerase chain reaction (qPCR) gold standard, aimed to describe the distribution of the Plasmodium species in each administrative region of Mali and to assess the performance of RDTs. METHODS: We randomly selected 150 malaria-negative and up to 30 malaria-positive RDTs in 41 sites distributed in 9 regions of Mali. DNA extracted from the RDT nitrocellulose strip was assayed with a pan-Plasmodium qPCR. Positive samples were then analyzed with P. falciparum-, P. malariae-, P. vivax-, or P. ovale-specific qPCRs. RESULTS: Of the 1496 RDTs, 258 (18.6%) were positive for Plasmodium spp., of which 96.9% were P. falciparum. The P. vivax prevalence reached 21.1% in the north. RDT displayed acceptable diagnostic indices; the lower CI95% bounds of Youden indices were all ≥0.50, except in the north (Youden index 0.66 (95% CI [0.44-0.82]) and 0.63 (95% CI [0.33-0.83]. CONCLUSIONS: Overall, RDT diagnostic indices are adequate for the biological diagnosis of malaria in Mali. We recommend the use of RDTs detecting P. vivax-specific antigens in the north.

Delayed gametocyte clearance in Plasmodium vivax malaria is associated with polymorphisms in the cytochrome P450 reductase (CPR).

Primaquine (PQ) is the main drug used to eliminate dormant liver stages and prevent relapses in Plasmodium vivax malaria. It also has an effect on the gametocytes of Plasmodium falciparum; however, it is unclear to what extent PQ affects P. vivax gametocytes. PQ metabolism involves multiple enzymes, including the highly polymorphic CYP2D6 and the cytochrome P450 reductase (CPR). Since genetic variability can impact drug metabolism, we conducted an evaluation of the effect of CYP2D6 and CPR variants on PQ gametocytocidal activity in 100 subjects with P. vivax malaria. To determine gametocyte density, we measured the levels of pvs25 transcripts in samples taken before treatment (D0) and 72 hours after treatment (D3). Generalized estimating equations (GEEs) were used to examine the effects of enzyme variants on gametocyte densities, adjusting for potential confounding factors. Linear regression models were adjusted to explore the predictors of PQ blood levels measured on D3. Individuals with the CPR mutation showed a smaller decrease in gametocyte transcript levels on D3 compared to those without the mutation (P = 0.02, by GEE). Consistent with this, higher PQ blood levels on D3 were associated with a lower reduction in pvs25 transcripts. Based on our findings, the CPR variant plays a role in the persistence of gametocyte density in P. vivax malaria. Conceptually, our work points to pharmacogenetics as a non-negligible factor to define potential host reservoirs with the propensity to contribute to transmission in the first days of CQ-PQ treatment, particularly in settings and seasons of high Anopheles human-biting rates.

Genetic polymorphisms of Plasmodium vivax ookinete (sexual stage) surface proteins (Pvs25 and Pvs28) from Thailand.

Plasmodium vivax is the most geographically widespread malaria parasite in human presently. The ookinete surface proteins of sexual stage of malaria parasites, Pvs25 and Pvs28, are candidates for the transmission blocking vaccine. The antigenic variation in population might be barrier for vaccine development. The objective of this study was to investigate the genetic diversity of Pvs25 and Pvs28 in endemic areas of Thailand. P. vivax clinical isolates collected from Thai-neighboring border areas were analyzed using polymerase chain reaction and sequencing method. Three and 14 amino acid substitutions were observed in 43 Pvs25 and 48 Pvs28 sequences, respectively. Three haplotypes in Pvs25 and 14 haplotypes with 5-7 GSGGE/D tandem repeats in Pvs28 were identified. The nucleotide diversity of pvs25 (π = 0.00059) had lower level than pvs28 (π = 0.00517). Tajima's D value for both pvs25 and pvs28 genes were negative while no significant difference was found (P > 0.10). Low genetic diversity was found in pvs25 and pvs28 genes in Thailand. The finding of the most frequent amino acid substitutions was consistent with global isolates. Therefore, the data could be helpful in developing of effective transmission blocking vaccine in malaria endemic areas.