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.

Prevalence of pvmrp1 Polymorphisms and Its Contribution to Antimalarial Response.

As more sporadic cases of chloroquine resistance occur (CQR) in Plasmodium vivax (P. vivax) malaria, molecular markers have become an important tool to monitor the introduction and spread of drug resistance. P. vivax multidrug resistance-associated protein 1 (PvMRP1), as one of the members of the ATP-binding cassette (ABC) transporters, may modulate this phenotype. In this study, we investigated the gene mutations and copy number variations (CNVs) in the pvmrp1 in 102 P. vivax isolates from China, the Republic of Korea (ROK), Myanmar, Papua New Guinea (PNG), Pakistan, the Democratic People's Republic of Korea (PRK), and Cambodia. And we also obtained 72 available global pvmrp1 sequences deposited in the PlasmoDB database to investigate the genetic diversity, haplotype diversity, natural selection, and population structure of pvmrp1. In total, 29 single nucleotide polymorphisms reflected in 23 non-synonymous, five synonymous mutations and one gene deletion were identified, and CNVs were found in 2.9% of the isolates. Combined with the antimalarial drug susceptibility observed in the previous in vitro assays, except the prevalence of S354N between the two CQ sensitivity categories revealed a significant difference, no genetic mutations or CNVs associated with drug sensitivity were found. The genetic polymorphism analysis of 166 isolates worldwide found that the overall nucleotide diversity (π) of pvmrp1 was 0.0011, with 46 haplotypes identified (Hd = 0.9290). The ratio of non-synonymous to synonymous mutations (dn/ds = 0.5536) and the neutrality tests statistic Fu and Li's D* test (Fu and Li's D* = −3.9871, p < 0.02) suggests that pvmrp1 had evolved under a purifying selection. Due to geographical differences, genetic differentiation levels of pvmrp1 in different regions were different to some extent. Overall, this study provides a new idea for finding CQR molecular monitoring of P. vivax and provides more sequences of pvmrp1 in Asia for subsequent research. However, further validation is still needed through laboratory and epidemiological field studies of P. vivax samples from more regions.

Geographical distribution and genetic diversity of Plasmodium vivax reticulocyte binding protein 1a correlates with patient antigenicity.

Plasmodium vivax is the most widespread cause of human malaria. Recent reports of drug resistant vivax malaria and the challenge of eradicating the dormant liver forms increase the importance of vaccine development against this relapsing disease. P. vivax reticulocyte binding protein 1a (PvRBP1a) is a potential vaccine candidate, which is involved in red cell tropism, a crucial step in the merozoite invasion of host reticulocytes. As part of the initial evaluation of the PvRBP1a vaccine candidate, we investigated its genetic diversity and antigenicity using geographically diverse clinical isolates. We analysed pvrbp1a genetic polymorphisms using 202 vivax clinical isolates from six countries. Pvrbp1a was separated into six regions based on specific domain features, sequence conserved/polymorphic regions, and the reticulocyte binding like (RBL) domains. In the fragmented gene sequence analysis, PvRBP1a region II (RII) and RIII (head and tail structure homolog, 152-625 aa.) showed extensive polymorphism caused by random point mutations. The haplotype network of these polymorphic regions was classified into three clusters that converged to independent populations. Antigenicity screening was performed using recombinant proteins PvRBP1a-N (157-560 aa.) and PvRBP1a-C (606-962 aa.), which contained head and tail structure region and sequence conserved region, respectively. Sensitivity against PvRBP1a-N (46.7%) was higher than PvRBP1a-C (17.8%). PvRBP1a-N was reported as a reticulocyte binding domain and this study identified a linear epitope with moderate antigenicity, thus an attractive domain for merozoite invasion-blocking vaccine development. However, our study highlights that a global PvRBP1a-based vaccine design needs to overcome several difficulties due to three distinct genotypes and low antigenicity levels.

Therapeutic efficacy of chloroquine for uncomplicated Plasmodium vivax malaria in southeastern and western border areas of Myanmar.

BACKGROUND: In the Greater Mekong Subregion of Southeast Asia, Plasmodium vivax malaria is endemic and causes significant morbidity. In this study, the efficacy of chloroquine for treating uncomplicated P. vivax malaria at the eastern and western borders of Myanmar was investigated. METHODS: A total of 197 participants with microscopically confirmed P. vivax infection were enrolled from three townships of the southeastern (Thanbyuzayat and Kawthoung) and western (Kyauktaw) borders of Myanmar. Patients were treated with chloroquine according to the national malaria treatment guidelines and followed for 28 days. RESULTS: Among the 197 enrollments, 172 completed the 28-day follow-up. Twelve recurrent P. vivax infections, all occurring in the third and fourth week, were detected, resulting in an overall cumulative rate of recurrence of 4.7% [95% confidence interval (CI) 1.5-7.8]. The incidence rate of recurrence varied among the three sites. In Thanbyuzayat township, no patients had recurrent parasitemia between days 7 and 28. In contrast, Kyauktaw township had a day 28 cumulative incidence rate of recurrence of 7.2% (95% CI 0.6-13.9%) compared to 6.9% (95% CI 0.6-13.2) in Kawthoung township. CONCLUSION: While this study confirmed the relatively high clinical efficacy of chloroquine for treating P. vivax in Myanmar with modest rates of recurrent infections within 28 days of the treatment, it also revealed considerable geographical heterogeneity of chloroquine efficacy, which warrants continuous surveillance efforts.

Identification of Reticulocyte Binding Domain of Plasmodium ovale curtisi Duffy Binding Protein (PocDBP) Involved in Reticulocyte Invasion.

The Plasmodium ovale curtisi (Poc) prevalence has increased substantially in sub-Saharan African countries as well as regions of Southeast Asia. Poc parasite biology has not been explored much to date; in particular, the invasion mechanism of this malaria parasite remains unclear. In this study, the binding domain of the Duffy binding protein of P. ovale curtisi (PocDBP) was characterized as an important ligand for reticulocyte invasion. The homologous region of the P. vivax Duffy binding protein in PocDBP, named PocDBP-RII herein, was selected, and the recombinant PocDBP-RII protein was expressed in an Escherichia coli system. This was used to analyze reticulocyte binding activity using fluorescence-activated cell sorting and immune serum production in rabbits. The binding specificity was proven by treating reticulocytes with trypsin, chymotrypsin and neuraminidase. The amino acid sequence homology in the N-terminal Cys-rich region was found to be ~ 44% between PvDBP and PocDBP. The reticulocyte binding activity of PocDBP-RII was significantly higher than the erythrocyte binding activity and was concentration dependent. Erythrocyte binding was reduced significantly by chymotrypsin treatment and inhibited by an anti-PocDBP-RII antibody. This finding suggests that PocDBP may be an important ligand in the reticulocyte invasion process of P. ovale curtisi.

Residual malaria among migrant workers in Myanmar: why still persistent and how to eliminate it?

BACKGROUND: Residual malaria is probably an important source for the re-emergence of malaria infection in the elimination era. Assessment to identify the factors influencing residual malaria in high-risk groups is needed to develop evidence-based decisions by stakeholders and policymakers. METHODS: This study was conducted to explore the factors influencing the residual malaria infection among migrant workers in two sentinel sites (endemic vs. pre-elimination areas) in Myanmar using the mixed-model method. RESULTS: A total of 102 migrant respondents (65 in Bamauk and 37 in Shwegyin) were included for the quantitative assessment using pretested questionnaires during household visits. Although 87.3% of them had insecticidal bed nets (ITNs/LLINs), only 68.3% of the migrants in Bamauk and 57.9% in Shwegyin used it regularly. The use of any bed net was high (79.9% in Bamauk vs. 91.0% in Shwegyin). The mean LLINs in their families were 1.64 (95%CI: 1.48-1.81) in Bamauk and 2.89 (95%CI: 2.67-3.11) in Shwegyin. Most of them received no health information for malaria prevention within the last year and their knowledge about malaria was low. Their working nature was a challenge for control measures against malaria in migrants. CONCLUSION: The strategy for distributing LLINs and health promotion activities for mobile/migrant populations should be reviewed, and an appropriate action plan should be developed for the specific migrant group. Moreover, health promotion activities for behavior change communication should be strengthened in the migrant population in Myanmar.

Antibody signatures of asymptomatic Plasmodium falciparum malaria infections measured from dried blood spots.

BACKGROUND: Screening malaria-specific antibody responses on protein microarrays can help identify immune factors that mediate protection against malaria infection, disease, and transmission, as well as markers of past exposure to both malaria parasites and mosquito vectors. Most malaria protein microarray work has used serum as the sample matrix, requiring prompt laboratory processing and a continuous cold chain, thus limiting applications in remote locations. Dried blood spots (DBS) pose minimal biohazard, do not require immediate laboratory processing, and are stable at room temperature for transport, making them potentially superior alternatives to serum. The goals of this study were to assess the viability of DBS as a source for antibody profiling and to use DBS to identify serological signatures of low-density Plasmodium falciparum infections in malaria-endemic regions of Myanmar. METHODS: Matched DBS and serum samples from a cross-sectional study in Ingapu Township, Myanmar were probed on protein microarrays populated with P. falciparum antigen fragments. Signal and trends in both sample matrices were compared. A case-control study was then performed using banked DBS samples from malaria-endemic regions of Myanmar, and a regularized logistic regression model was used to identify antibody signatures of ultrasensitive PCR-positive P. falciparum infections. RESULTS: Approximately 30% of serum IgG activity was recovered from DBS. Despite this loss of antibody activity, antigen and population trends were well-matched between the two sample matrices. Responses to 18 protein fragments were associated with the odds of asymptomatic P. falciparum infection, albeit with modest diagnostic characteristics (sensitivity 58%, specificity 85%, negative predictive value 88%, and positive predictive value 52%). CONCLUSIONS: Malaria-specific antibody responses can be reliably detected, quantified, and analysed from DBS, opening the door to serological studies in populations where serum collection, transport, and storage would otherwise be impossible. While test characteristics of antibody signatures were insufficient for individual diagnosis, serological testing may be useful for identifying exposure to asymptomatic, low-density malaria infections, particularly if sero-surveillance strategies target individuals with low previous exposure as sentinels for population exposure.

Surge of severe acute respiratory syndrome coronavirus 2 infections linked to single introduction of a virus strain in Myanmar, 2020.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection is a major health concern globally. Genomic epidemiology is an important tool to assess the pandemic of coronavirus disease 2019 (COVID-19). Several mutations have been reported by genome analysis of the SARS-CoV-2. In the present study, we investigated the mutational and phylogenetic analysis of 30 whole-genome sequences for the virus's genomic characteristics in the specimens collected in the early phase of the pandemic (March-June, 2020) and the sudden surge of local transmission (August-September, 2020). The four samples in the early phase of infection were B.6 lineage and located within a clade of the samples collected at the same time in Singapore and Malaysia, while five returnees by rescue flights showed the lineage B. 1.36.1 (three from India), B.1.1 (one from India) and B.1.80 (one from China). However, there was no evidence of local spread from these returnees. Further, all 19 whole-genome sequences collected in the sudden surge of local transmission showed lineage B.1.36. The surge of the second wave on SARS-CoV-2 infection was linked to the single-introduction of a variant (B.1.36) that may result from the strict restriction of international travel and containment efforts. These genomic data provides the useful information to disease control and prevention strategy.

Seroprevalence of toxoplasmosis among reproductive-aged women in Myanmar and evaluation of luciferase immunoprecipitation system assay.

BACKGROUNDS: Primary infection with Toxoplasma gondii during pregnancy can pose serious health problems for the fetus. However, the epidemiological status of toxoplasmosis among reproductive-aged population in Myanmar is largely unknown. Although luciferase immunoprecipitation system (LIPS) assays for serodiagnosis of toxoplasmosis was developed mostly using mouse infection model, had not been tested by using field-derived human samples. METHODS: A total of 251 serum samples were collected from reproductive-aged women, residing in Shwegyin township, Bago region, Myanmar and analyzed with a commercial ELISA kit, as well as in-house LIPS assays. RESULTS: The overall seroprevalence for Toxoplasma gondii infection by the commercial ELISA was 11.5%. No clear risk factor was identified except for being in the younger age group (15-30 years old). Overall, LIPS assays showed low sensitivity when the commercial ELSA was used as a reference test. CONCLUSION: We identified the epidemiological situation of toxoplasmosis in some rural communities in Myanmar. The data obtained here will serve as a primary information for the effort to reduce toxoplasmosis in this region. Although looked promising in the previous experiments with mouse infection model, we found that the reported LIPS procedures need further improvements to increase the sensitivities.

Molecular surveillance for drug resistance markers in Plasmodium vivax isolates from symptomatic and asymptomatic infections at the China-Myanmar border.

BACKGROUND: In the Greater Mekong sub-region, Plasmodium vivax has become the predominant species and imposes a major challenge for regional malaria elimination. This study aimed to investigate the variations in genes potentially related to drug resistance in P. vivax populations from the China-Myanmar border area. In addition, this study also wanted to determine whether divergence existed between parasite populations associated with asymptomatic and acute infections. METHODS: A total of 66 P. vivax isolates were obtained from patients with acute malaria who attended clinics at the Laiza area, Kachin State, Myanmar in 2015. In addition, 102 P. vivax isolates associated with asymptomatic infections were identified by screening of volunteers without signs or symptoms from surrounding villages. Slide-positive samples were verified with nested PCR detecting the 18S rRNA gene. Multiclonal infections were further excluded by genotyping at msp-3α and msp-3ß genes. Parasite DNA from 60 symptomatic cases and 81 asymptomatic infections was used to amplify and sequence genes potentially associated with drug resistance, including pvmdr1, pvcrt-o, pvdhfr, pvdhps, and pvk12. RESULTS: The pvmdr1 Y976F and F1076L mutations were present in 3/113 (2.7%) and 97/113 (85.5%) P. vivax isolates, respectively. The K10 insertion in pvcrt-o gene was found in 28.2% of the parasites. Four mutations in the two antifolate resistance genes reached relatively high levels of prevalence: pvdhfr S58R (53.4%), S117N/T (50.8%), pvdhps A383G (75.0%), and A553G (36.3%). Haplotypes with wild-type pvmdr1 (976Y/997K/1076F) and quadruple mutations in pvdhfr (13I/57L/58R/61M/99H/117T/173I) were significantly more prevalent in symptomatic than asymptomatic infections, whereas the pvmdr1 mutant haplotype 976Y/997K/1076L was significantly more prevalent in asymptomatic than symptomatic infections. In addition, quadruple mutations at codons 57, 58, 61 and 117 of pvdhfr and double mutations at codons 383 and 553 of pvdhps were found both in asymptomatic and symptomatic infections with similar frequencies. No mutations were found in the pvk12 gene. CONCLUSIONS: Mutations in pvdhfr and pvdhps were prevalent in both symptomatic and asymptomatic P. vivax infections, suggestive of resistance to antifolate drugs. Asymptomatic carriers may act as a silent reservoir sustaining drug-resistant parasite transmission necessitating a rational strategy for malaria elimination in this region.

Genetic diversity and neutral selection in Plasmodium vivax erythrocyte binding protein correlates with patient antigenicity.

Plasmodium vivax is the most widespread and difficult to treat cause of human malaria. The development of vaccines against the blood stages of P. vivax remains a key objective for the control and elimination of vivax malaria. Erythrocyte binding-like (EBL) protein family members such as Duffy binding protein (PvDBP) are of critical importance to erythrocyte invasion and have been the major target for vivax malaria vaccine development. In this study, we focus on another member of EBL protein family, P. vivax erythrocyte binding protein (PvEBP). PvEBP was first identified in Cambodian (C127) field isolates and has subsequently been showed its preferences for binding reticulocytes which is directly inhibited by antibodies. We analysed PvEBP sequence from 316 vivax clinical isolates from eight countries including China (n = 4), Ethiopia (n = 24), Malaysia (n = 53), Myanmar (n = 10), Papua New Guinea (n = 16), Republic of Korea (n = 10), Thailand (n = 174), and Vietnam (n = 25). PvEBP gene exhibited four different phenotypic clusters based on the insertion/deletion (indels) variation. PvEBP-RII (179-479 aa.) showed highest polymorphism similar to other EBL family proteins in various Plasmodium species. Whereas even though PvEBP-RIII-V (480-690 aa.) was the most conserved domain, that showed strong neutral selection pressure for gene purifying with significant population expansion. Antigenicity of both of PvEBP-RII (16.1%) and PvEBP-RIII-V (21.5%) domains were comparatively lower than other P. vivax antigen which expected antigens associated with merozoite invasion. Total IgG recognition level of PvEBP-RII was stronger than PvEBP-RIII-V domain, whereas total IgG inducing level was stronger in PvEBP-RIII-V domain. These results suggest that PvEBP-RII is mainly recognized by natural IgG for innate protection, whereas PvEBP-RIII-V stimulates IgG production activity by B-cell for acquired immunity. Overall, the low antigenicity of both regions in patients with vivax malaria likely reflects genetic polymorphism for strong positive selection in PvEBP-RII and purifying selection in PvEBP-RIII-V domain. These observations pose challenging questions to the selection of EBP and point out the importance of immune pressure and polymorphism required for inclusion of PvEBP as a vaccine candidate.

Cross-species reactivity of antibodies against Plasmodium vivax blood-stage antigens to Plasmodium knowlesi.

Malaria is caused by multiple different species of protozoan parasites, and interventions in the pre-elimination phase can lead to drastic changes in the proportion of each species causing malaria. In endemic areas, cross-reactivity may play an important role in the protection and blocking transmission. Thus, successful control of one species could lead to an increase in other parasite species. A few studies have reported cross-reactivity producing cross-immunity, but the extent of cross-reactive, particularly between closely related species, is poorly understood. P. vivax and P. knowlesi are particularly closely related species causing malaria infections in SE Asia, and whilst P. vivax cases are in decline, zoonotic P. knowlesi infections are rising in some areas. In this study, the cross-species reactivity and growth inhibition activity of P. vivax blood-stage antigen-specific antibodies against P. knowlesi parasites were investigated. Bioinformatics analysis, immunofluorescence assay, western blotting, protein microarray, and growth inhibition assay were performed to investigate the cross-reactivity. P. vivax blood-stage antigen-specific antibodies recognized the molecules located on the surface or released from apical organelles of P. knowlesi merozoites. Recombinant P. vivax and P. knowlesi proteins were also recognized by P. knowlesi- and P. vivax-infected patient antibodies, respectively. Immunoglobulin G against P. vivax antigens from both immune animals and human malaria patients inhibited the erythrocyte invasion by P. knowlesi. This study demonstrates that there is extensive cross-reactivity between antibodies against P. vivax to P. knowlesi in the blood stage, and these antibodies can potently inhibit in vitro invasion, highlighting the potential cross-protective immunity in endemic areas.

Inhibition of parasite invasion by monoclonal antibody against epidermal growth factor-like domain of Plasmodium vivax merozoite surface protein 1 paralog.

The Plasmodium vivax merozoite surface protein 1 paralog (PvMSP1P), which has epidermal growth factor (EGF)-like domains, was identified as a novel erythrocyte adhesive molecule. This EGF-like domain (PvMSP1P-19) elicited high level of acquired immune response in patients. Antibodies against PvMSP1P significantly reduced erythrocyte adhesion activity to its unknown receptor. To determine PvMSP1P-19-specific antibody function and B-cell epitopes in vivax patients, five monoclonal antibodies (mAbs) and 18-mer peptides were generated. The mAb functions were determined by erythrocyte-binding inhibition assay and invasion inhibition assay with P. knowlesi. B-cell epitopes of PvMSP1P-19 domains were evaluated by peptide microarray. The pvmsp1p-19 sequences showed limited polymorphism in P. vivax worldwide isolates. The 1BH9-A10 showed erythrocyte binding inhibitory by interaction with the N-terminus of PvMSP1P-19, while this mAb failed to recognize PkMSP1P-19 suggesting the species-specific for P. vivax. Other mAbs showed cross-reactivity with PkMSP1P-19. Among them, the 2AF4-A2 and 2AF4-A6 mAb significantly reduced parasite invasion through C-terminal recognition. The linear B-cell epitope in naturally exposed P. vivax patient was identified at three linear epitopes. In this study, PvMSP1P-19 N-terminal-specific 1BH9-A10 and C-terminal-specific 2AF4 mAbs showed functional activity for epitope recognition suggesting that PvMSP1P may be useful for vaccine development strategy for specific single epitope to prevent P. vivax invasion.

Persistence of Long-lived Memory B Cells specific to Duffy Binding Protein in individuals exposed to Plasmodium vivax.

The major challenge in designing a protective Duffy binding protein region II (DBPII)-based vaccine against blood-stage vivax malaria is the high number of polymorphisms in critical residues targeted by binding-inhibitory antibodies. Here, longevity of antibody and memory B cell response (MBCs) to DBL-TH variants, DBL-TH2, -TH4, -TH5, -TH6 and -TH9 were analyzed in P. vivax-exposed individuals living in a low malaria transmission area of southern Thailand. Antibody to DBL-TH variants were significantly detected during P. vivax infection and it was persisted for up to 9 months post-infection. However, DBL-TH-specific MBC responses were stably maintained longer than antibody response, at least 3 years post-infection in the absence of re-infection. Phenotyping of B cell subsets showed the expansion of activated and atypical MBCs during acute and recovery phase of infection. While the persistence of DBL-TH-specific MBCs was found in individuals who had activated and atypical MBC expansion, anti-DBL-TH antibody responses was rapidly declined in plasma. The data suggested that these two MBCs were triggered by P. vivax infection, its expansion and stability may have impact on antibody responses. Our results provided evidence for ability of DBPII variant antigens in induction of long-lasting MBCs among individuals who were living in low malaria endemicity.

Cross-species analysis of apical asparagine-rich protein of Plasmodium vivax and Plasmodium knowlesi.

The Plasmodium falciparum apical asparagine (Asn)-rich protein (AARP) is one of malarial proteins, and it has been studied as a candidate of malaria subunit vaccine. Basic characterization of PvAARP has been performed with a focus on its immunogenicity and localization. In this study, we further analyzed the immunogenicity of PvAARP, focusing on the longevity of the antibody response, cross-species immunity and invasion inhibitory activity by using the primate malaria parasite Plasmodium knowlesi. We found that vivax malaria patient sera retained anti-PvAARP antibodies for at least one year without re-infection. Recombinant PvAARP protein was strongly recognized by knowlesi malaria patients. Antibody raised against the P. vivax and P. knowlesi AARP N-termini reacted with the apical side of the P. knowlesi merozoites and inhibited erythrocyte invasion by P. knowlesi in a concentration-dependent manner, thereby suggesting a cross-species nature of anti-PvAARP antibody against PkAARP. These results can be explained by B cell epitopes predicted in conserved surface-exposed regions of the AARP N-terminus in both species. The long-lived anti-PvAARP antibody response, cross-reactivity, and invasion inhibitory activity of anti-PvAARP support a critical role of AARP during the erythrocyte invasion and suggest that PvAARP induces long-lived cross-species protective immunity against P. vivax and P. knowlesi.

Estimation on local transmission of malaria by serological approach under low transmission setting in Myanmar.

BACKGROUND: As the prevalence of the malaria has been decreasing in many endemic countries including Myanmar, malaria elimination in Greater Mekong Region was targeted not later than 2030. The relevance of molecular and serological tools to identify residual transmission remains to be established in this setting. METHODS: One-year cohort study was conducted and sera samples were collected in every 3 months with active and passive case detection for clinical malaria episodes by RDT, microscopy and molecular method. The sera were used to detect the malaria antibody against PfMSP1-19, PvAMA1, PvDBPII and PvMSP1-19 by protein microarray. RESULTS: Among the recruited 1182 participants, there was no RDT positive case for malaria infection although two vivax infections were detected by microscopy in initial collection. Molecular methods detected the asymptomatic cases of 28/1182 (2.37%) in first, 5/894 (0.42%) in second, 12/944 (1.02%) in third, 6/889 (0.51%) in fourth collection, respectively. Seropositivity rates against the PfMSP1-19, PvMSP1-19, PvAMA1 and PvDBPII were 73/270 (27.0%), 85/270 (31.5%), 65/270 (24.1%) and 160/270 (59.3%), respectively. PfMSP1-19 and PvMSP1-19 showed high and stable antigenicity in acute and subacute samples but declining in 1-year history samples. No cross reactivity of PfMSP1-19 and PvMSP1-19 between the two species and higher seropositivity among the asymptomatic carriers were observed. Mapping data indicated serological surveillance can detect the geographical pattern of malaria infection under low transmission setting. CONCLUSIONS: These findings support that PfMSP1-19 and PvMSP1-19 are suggested for serosurveillance of the malaria especially in low transmission setting for further necessary actions have to be carried out to eliminate the malaria.

Clinical and molecular surveillance of artemisinin resistant falciparum malaria in Myanmar (2009-2013).

BACKGROUND: Emergence of artemisinin-resistant malaria in Southeast Asian countries threatens the global control of malaria. Although K13 kelch propeller has been assessed for artemisinin resistance molecular marker, most of the mutations need to be validated. In this study, artemisinin resistance was assessed by clinical and molecular analysis, including k13 and recently reported markers, pfarps10, pffd and pfmdr2. METHODS: A prospective cohort study in 1160 uncomplicated falciparum patients was conducted after treatment with artemisinin-based combination therapy (ACT), in 6 sentinel sites in Myanmar from 2009 to 2013. Therapeutic efficacy of ACT was assessed by longitudinal follow ups. Molecular markers analysis was done on all available day 0 samples. RESULTS: True recrudescence treatment failures cases and day 3 parasite positivity were detected at only the southern Myanmar sites. Day 3 positive and k13 mutants with higher prevalence of underlying genetic foci predisposing to become k13 mutant were detected only in southern Myanmar since 2009 and comparatively fewer mutations of pfarps10, pffd, and pfmdr2 were observed in western Myanmar. K13 mutations, V127M of pfarps10, D193Y of pffd, and T448I of pfmdr2 were significantly associated with day 3 positivity (OR: 6.48, 3.88, 2.88, and 2.52, respectively). CONCLUSIONS: Apart from k13, pfarps10, pffd and pfmdr2 are also useful for molecular surveillance of artemisinin resistance especially where k13 mutation has not been reported. Appropriate action to eliminate the resistant parasites and surveillance on artemisinin resistance should be strengthened in Myanmar. Trial registration This study was registered with ClinicalTrials.gov, identifier NCT02792816.

Genetic diversity of Plasmodium falciparum populations in southeast and western Myanmar.

BACKGROUND: The genetic diversity of malaria parasites reflects the complexity and size of the parasite populations. This study was designed to explore the genetic diversity of Plasmodium falciparum populations collected from two southeastern areas (Shwekyin and Myawaddy bordering Thailand) and one western area (Kyauktaw bordering Bangladesh) of Myanmar. METHODS: A total of 267 blood samples collected from patients with acute P. falciparum infections during 2009 and 2010 were used for genotyping at the merozoite surface protein 1 (Msp1), Msp2 and glutamate-rich protein (Glurp) loci. RESULTS: One hundred and eighty four samples were successfully genotyped at three genes. The allelic distributions of the three genes were all significantly different among three areas. MAD20 and 3D7 were the most prevalent alleles in three areas for Msp1 and Msp2, respectively. The Glurp allele with a bin size of 700-750 bp was the most prevalent both in Shwekyin and Myawaddy, whereas two alleles with bin sizes of 800-850 bp and 900-1000 bp were the most prevalent in the western site Kyauktaw. Overall, 73.91% of samples contained multiclonal infections, resulting in a mean multiplicity of infection (MOI) of 1.94. Interestingly, the MOI level presented a rising trend with the order of Myawaddy, Kyauktaw and Shwekyin, which also paralleled with the increasing frequencies of Msp1 RO33 and Msp2 FC27 200-250 bp alleles. Msp1 and Msp2 genes displayed higher levels of diversity and higher MOI rates than Glurp. PCR revealed four samples (two from Shwekyin and two from Myawaddy) with mixed infections of P. falciparum and P. vivax. CONCLUSIONS: This study genotyped parasite clinical samples from two southeast regions and one western state of Myanmar at the Msp1, Msp2 and Glurp loci, which revealed high levels of genetic diversity and mixed-strain infections of P. falciparum populations at these sites. The results indicated that malaria transmission intensity in these regions remained high and more strengthened control efforts are needed. The genotypic data provided baseline information for monitoring the impacts of malaria elimination efforts in the region.