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Plasmodium vivax is increasingly the dominant species of malaria in the Greater Mekong Subregion (GMS), which is pursuing regional malaria elimination. P. vivax lineages in the GMS are poorly characterized. Currently, P. vivax reference genomes are scarce due to difficulties in culturing the parasite and lack of high-quality samples. In addition, P. vivax is incredibly diverse, necessitating the procurement of reference genomes from different geographical regions. Here we present four new P. vivax draft genomes assembled de novo from clinical samples collected in the China-Myanmar border area. We demonstrate comparable length and content to existing genomes, with the majority of structural variation occurring around subtelomeric regions and exported proteins, which we corroborated with detection of copy number variations in these regions. We predicted peptides from all PIR gene subfamilies, except for PIR D. We confirmed that proteins classically labeled as PIR D family members are not identifiable by PIR motifs, and actually bear stronger resemblance to DUF (domain of unknown function) family DUF3671, potentially pointing to a new, closely related gene family. Further, phylogenetic analyses of MSP7 genes showed high variability within the MSP7-B family compared to MSP7-A and -C families, and the result was comparable to that from whole genome analyses. The new genome assemblies serve as a resource for studying P. vivax within the GMS.

Transmission-blocking vaccine (TBV) is a promising strategy to interfere with the transmission of malaria. To date, only limited TBV candidate antigens have been identified for Plasmodium vivax. HAP2 is a gamete membrane fusion protein, with homology to the class II viral fusion proteins. Herein we reported the characterization of the PvHAP2 for its potential as a TBV candidate for P. vivax. The HAP2/GCS1 domain of PvHAP2 was expressed in the baculovirus expression system and the recombinant protein was used to raise antibodies in rabbits. Indirect immunofluorescence assays showed that anti-PvHAP2 antibodies reacted only with the male gametocytes on blood smears. Direct membrane feeding assays were conducted using four field P. vivax isolates in Anopheles dirus. At a mean infection intensity of 72.4, 70.7, 51.3, and 15.6 oocysts/midgut with the control antibodies, anti-PvHAP2 antibodies significantly reduced the midgut oocyst intensity by 40.3, 44.4, 61.9, and 89.7%. Whereas the anti-PvHAP2 antibodies were not effective in reducing the infection prevalence at higher parasite exposure (51.3-72.4 oocysts/midgut in the control group), the anti-PvHAP2 antibodies reduced infection prevalence by 50% at a low challenge (15.6 oocysts/midgut). Multiple sequence alignment showed 100% identity among these Thai P. vivax isolates, suggesting that polymorphism may not be an impediment for the utilization of PvHAP2 as a TBV antigen. In conclusion, our results suggest that PvHAP2 could serve as a TBV candidate for P. vivax, and further optimization and evaluation are warranted.

Background Border malaria, a shared phenomenon in the Greater Mekong Sub-region of Southeast Asia, is a major obstacle for regional malaria elimination. Along the China–Myanmar border, an additional problem arose as a result of the settlement of internally displaced people (IDP) in the border region. Since asymptomatic malaria significantly impacts transmission dynamics, assessment of the prevalence, dynamics and risk factors of asymptomatic malaria infections is necessary. Methods Cross-sectional surveys were carried out in 3 seasons (March and April, July and November) and 2 sites (villages and IDP camps) in 2015. A total of 1680 finger-prick blood samples were collected and used for parasite detection by microscopy and nested RT-PCR (nRT-PCR). Logistic regression models were used to explore the risk factors associated with asymptomatic malaria at individual and household levels. Results The prevalence of asymptomatic Plasmodium infections was 23.3% by nRT-PCR, significantly higher than that detected by microscopy (1.5%). The proportions of Plasmodium vivax, Plasmodium falciparum and mixed-species infections were 89.6, 8.1 and 2.3%, respectively. Asymptomatic infections showed obvious seasonality with higher prevalence in the rainy season. Logistic regression analysis identified males and school children (≤ 15 years) as the high-risk populations. Vector-based interventions, including bed net and indoor residual spray, were found to have significant impacts on asymptomatic Plasmodium infections, with non-users of these measures carrying much higher risks of infection. In addition, individuals living in poorly constructed households or farther away from clinics were more prone to asymptomatic infections. Conclusions Sub-microscopic Plasmodium infections were highly prevalent in the border human populations from IDP camps and surrounding villages. Both individual- and household-level risk factors were identified, which provides useful information for identifying the high-priority populations to implement targeted malaria control.

Malaria was concentrated in a few townships along the China–Myanmar border., To identify township-level high-risk foci of malaria transmission in Yunnan Province, China, along the international border, we retrospectively reviewed data collected in hospitals and clinics of 58 townships in 4 counties during 2011–2013. We analyzed spatiotemporal distribution, especially hot spots of confirmed malaria, using geographic information systems and Getis-Ord Gi*(d) cluster analysis. Malaria incidence, transmission seasonality, and Plasmodium vivax:P. falciparum ratio remained almost unchanged from 2011 to 2013, but heterogeneity in distribution increased. The number of townships with confirmed malaria decreased significantly during the 3 years; incidence became increasingly concentrated within a few townships. High-/low-incidence clusters of P. falciparum shifted in location and size every year, whereas the locations of high-incidence P. vivax townships remained unchanged. All high-incidence clusters were located along the China–Myanmar border. Because of increasing heterogeneity in malaria distribution, microgeographic analysis of malaria transmission hot spots provided useful information for designing targeted malaria intervention during the elimination phase.

Plasmodium vivax has become the predominant malaria parasite and a major challenge for malaria elimination in the Greater Mekong Subregion (GMS). Yet, our knowledge about the evolution of P. vivax populations in the GMS is fragmental. We performed whole genome sequencing on 23 P. vivax samples from the China-Myanmar border (CMB) and used 21 high-coverage samples to compare to over 200 samples from the rest of the GMS. Using genome-wide single nucleotide polymorphisms (SNPs), we analyzed population differentiation, genetic structure, migration and potential selection using an array of methods. The CMB parasites displayed a higher proportion of monoclonal infections, and 52% shared over 90% of their genomes in identity-by-descent segments with at least one other sample from the CMB, suggesting preferential expansion of certain parasite strains in this region, likely resulting from the P. vivax outbreaks occurring during this study period. Principal component, admixture, fixation index and phylogenetic analyses all identified that parasites from the CMB were genetically distinct from parasites from eastern parts of the GMS (Cambodia, Laos, Vietnam, and Thailand), whereas the eastern GMS parasite populations were largely undifferentiated. Such a genetic differentiation pattern of the P. vivax populations from the GMS parasite was largely explainable through geographic distance. Using the genome-wide SNPs, we narrowed down to a set of 36 SNPs for differentiating parasites from different areas of the GMS. Genome-wide scans to determine selection in the genome with two statistical methods identified genes potentially under drug selection, including genes associated with antifolate resistance and genes linked to chloroquine resistance in Plasmodium falciparum., Author summary Plasmodium vivax is an understudied malaria parasite compared to P. falciparum despite that it is the most common Plasmodium species outside of Africa. In the Greater Mekong Subregion (GMS), the increased proportion of P. vivax proves its resilience to conventional malaria control measures. Within the GMS malaria incidence is highly heterogeneous, typified by more intensive malaria transmission along international borders. Understanding the transmission between countries and tracking parasite introduction are therefore essential to eliminating malaria within this region. The China-Myanmar border (CMB) presents such an example wherein China has eliminated autochthonous malaria cases, while Myanmar has high malaria incidence. Malaria on the CMB is nearly entirely due to P. vivax, yet few studies investigated the genetics and evolution of the P. vivax populations in the area. Here we used whole-genome sequencing for a holistic analysis of P. vivax from the CMB and compared them to those from other sites of the GMS. Parasites on the CMB had a significantly higher proportion (75%) of monoclonal infection than parasites from other regions. Many of the CMB parasites showed significant genetic sharing that is consistent with the result of clonal expansion, consistent with the malaria outbreak occurring during the study period. While P. vivax parasites from the entire GMS were substantially mixed with no evidence of significant gene flow barriers, those from the CMB were more genetically distinct from other populations. Genome-wide scans for selection identified genes potentially under selection, and especially notable are genes associated with sulfadoxine/pyrimethamine resistance. Genes also under selection include those potentially encoding membrane channels and transporters, which were associated with drug resistance in P. falciparum. Moreover, this population genomic study also identified a set of 36 single nucleotide polymorphisms, which could serve as a barcode for differentiating parasites from various regions of the GMS, a task that is important for the final phase of regional malaria elimination.

Malaria transmission is heterogeneous in the Greater Mekong Subregion with most of the cases occurring along international borders. Knowledge of transmission hotspots is essential for targeted malaria control and elimination in this region. This study aimed to determine the dynamics of malaria transmission and possible existence of transmission hotspots on a microgeographical scale along the China-Myanmar border. Microscopically confirmed clinical malaria cases were recorded in five border villages through a recently established surveillance system between January 2011 and December 2014. A total of 424 clinical cases with confirmed spatial and temporal information were analyzed, of which 330 (77.8%) were Plasmodium vivax and 88 (20.8%) were Plasmodium falciparum, respectively. The P. vivax and P. falciparum case ratio increased dramatically from 2.2 in 2011 to 4.7 in 2014, demonstrating that P. vivax malaria has become the predominant parasite species. Clinical infections showed a strong bimodal seasonality. There were significant differences in monthly average incidence rates among the study villages with rates in a village in China being 3-8 folds lower than those in nearby villages in Myanmar. Spatial analysis revealed the presence of clinical malaria hotspots in four villages. This information on malaria seasonal dynamics and transmission hotspots should be harnessed for planning targeted control.

Background Sensitive methods for detecting asymptomatic malaria infections are essential for identifying potential transmission reservoirs and obtaining an accurate assessment of malaria epidemiology in low-endemicity areas aiming to eliminate malaria. PCR techniques to detect parasite nucleic acids (DNA or RNA) are among the most commonly used molecular methods. However, most of these methods are of low throughput and cannot be used for large-scale molecular epidemiological studies. A recently developed capture and ligation probe-PCR (CLIP-PCR) is claimed to have the sensitivity of molecular techniques and the high throughput capacity needed for screening purposes. This study aimed to compare several molecular methods for detecting asymptomatic and submicroscopic Plasmodium infections in healthy residents of a malaria-hypoendemic region in Southeast Asia, where malaria elimination is in sight. Method This study compared three molecular detection methods side-by-side, namely nested PCR targeting the rRNA genes, nested RT-PCR to detect parasite rRNA, and CLIP-PCR to detect parasite rRNA in 1005 healthy individuals in northeastern Myanmar. For nested PCR and RT-PCR, parasite DNA and total RNA were extracted from ~100 µL of blood, whereas RNA used for CLIP-PCR was from a 3 mm disk of dried blood filter paper. The sensitivity and specificity of these methods were compared with those of conventional light microscopy. In addition, RT-PCR and quantitative RT-PCR (qRT-PCR) targeting the Pvs25 gene in Plasmodium vivax were used to assess gametocyte prevalence in the samples. Results Light microscopy detected Plasmodium infections in only 1.19% of the residents harbouring the parasites. CLIP-PCR had slightly better performance and detected Plasmodium infections in 1.89% of the population. Further improvement was achieved by nested PCR to detect parasite DNA, which detected P. vivax and Plasmodium falciparum infections in 2.39% of the residents. The nested RT-PCR targeting rRNA, however, detected as many as 187 (18.61%) individuals having Plasmodium infections with P. vivax being the predominant species (176 P. vivax, 5 P. falciparum and 6 P. falciparum/P. vivax mixed infections). Of the 210 Plasmodium-positive samples detected by all molecular methods, 115 were Pvs25-positive by qRT-PCR, indicating that a large proportion of asymptomatic individuals were gametocyte carriers. Conclusion Nested RT-PCR based on the detection of asexual-stage parasite rRNA was the most sensitive, with a more than sixfold higher sensitivity than the other two molecular methods of parasite detection. CLIP-PCR has an increased throughput, but its sensitivity in this study was much lower than those of other molecular methods, which may be partially due to the smaller amount of RNA input used. Electronic supplementary material The online version of this article (doi:10.1186/s12936-017-1813-0) contains supplementary material, which is available to authorized users.

Background With the premise of diminishing parasite genetic diversity following the reduction of malaria incidence, the analysis of polymorphic antigenic markers may provide important information about the impact of malaria control on local parasite populations. Here we evaluated the genetic diversity of Plasmodium vivax apical membrane antigen 1 (Pvama1) gene in a parasite population from the China-Myanmar border and compared it with global P. vivax populations. Methods We performed evolutionary analysis to examine the genetic diversity, natural selection, and population differentiation of 73 Pvama1 sequences acquired from the China-Myanmar border as well as 615 publically available Pvama1 sequences from seven global P. vivax populations. Results A total of 308 Pvama1 haplotypes were identified among the global P. vivax isolates. The overall nucleotide diversity of Pvama1 gene among the 73 China-Myanmar border parasite isolates was 0.008 with 41 haplotypes being identified (Hd = 0.958). Domain I (DI) harbored the majority (26/33) of the polymorphic sites. The McDonald Kreitman test showed a significant positive selection across the ectodomain and the DI of Pvama1. The fixation index (F ST) estimation between the China-Myanmar border, Thailand (0.01) and Myanmar (0.10) showed only slight geographical genetic differentiation. Notably, the Sal-I haplotype was not detected in any of the analyzed global isolates, whereas the Belem strain was restricted to the Thai population. The detected mutations are mapped outside the overlapped region of the predicted B-cell epitopes and intrinsically unstructured/disordered regions. Conclusions This study revealed high levels of genetic diversity of Pvama1 in the P. vivax parasite population from the China-Myanmar border with DI displaying stronger diversifying selection than other domains. There were low levels of population subdivision among parasite populations from the Greater Mekong Subregion. Electronic supplementary material The online version of this article (doi:10.1186/s13071-016-1899-1) contains supplementary material, which is available to authorized users.

Background The Greater Mekong Subregion is aiming to achieve regional malaria elimination by 2030. Though a shift in malaria parasite species predominance by Plasmodium vivax has been recently documented, the transmission of the two minor Plasmodium species, Plasmodium malariae and Plasmodium ovale spp., is poorly characterized in the region. This study aims to determine the prevalence of these minor species in the China–Myanmar border area and their genetic diversity. Methods Epidemiology study was conducted during passive case detection in hospitals and clinics in Myanmar and four counties in China along the China–Myanmar border. Cross-sectional surveys were conducted in villages and camps for internally displaced persons to determine the prevalence of malaria infections. Malaria infections were diagnosed initially by microscopy and later in the laboratory using nested PCR for the SSU rRNA genes. Plasmodium malariae and P. ovale infections were confirmed by sequencing the PCR products. The P. ovale subtypes were determined by sequencing the Pocytb, Pocox1 and Pog3p genes. Parasite populations were evaluated by PCR amplification and sequencing of the MSP-1 genes. Antifolate sensitivity was assessed by sequencing the dhfr-ts and dhps genes from the P. malariae and P. ovale isolates. Results Analysis of 2701 blood samples collected from the China–Myanmar border by nested PCR targeting the parasite SSU rRNA genes identified 561 malaria cases, including 161 Plasmodium falciparum, 327 P. vivax, 66 P. falciparum/P. vivax mixed infections, 4 P. malariae and 3 P. ovale spp. P. vivax and P. falciparum accounted for >60 and ~30% of all malaria cases, respectively. In comparison, the prevalence of P. malariae and P. ovale spp. was very low and only made up ~1% of all PCR-positive cases. Nevertheless, these two species were often misidentified as P. vivax infections or completely missed by microscopy even among symptomatic patients. Phylogenetic analysis of the SSU rRNA, Pocytb, Pocox1 and Pog3p genes confirmed that the three P. ovale spp. isolates belonged to the subtype P. ovale curtisi. Low-level genetic diversity was detected in the MSP-1, dhfr and dhps genes of these minor parasite species, potentially stemming from the low prevalence of these parasites preventing their mixing. Whereas most of the dhfr and dhps positions equivalent to those conferring antifolate resistance in P. falciparum and P. vivax were wild type, a new mutation S113C corresponding to the S108 position in pfdhfr was identified in two P. ovale curtisi isolates. Conclusions The four human malaria parasite species all occurred sympatrically at the China–Myanmar border. While P. vivax has become the predominant species, the two minor parasite species also occurred at very low prevalence but were often misidentified or missed by conventional microscopy. These minor parasite species displayed low levels of polymorphisms in the msp-1, dhfr and dhps genes. Electronic supplementary material The online version of this article (doi:10.1186/s12936-016-1605-y) contains supplementary material, which is available to authorized users.

Background Artemisinin resistance in Plasmodium falciparum has emerged as a major threat for malaria control and elimination worldwide. Mutations in the Kelch propeller domain of PfK13 are the only known molecular markers for artemisinin resistance in this parasite. Over 100 non-synonymous mutations have been identified in PfK13 from various malaria endemic regions. This study aimed to investigate the genetic diversity of PvK12, the Plasmodium vivax ortholog of PfK13, in parasite populations from Southeast Asia, where artemisinin resistance in P. falciparum has emerged. Methods The PvK12 sequences in 120 P. vivax isolates collected from Thailand (22), Myanmar (32) and China (66) between 2004 and 2008 were obtained and 353 PvK12 sequences from worldwide populations were retrieved for further analysis. Results These PvK12 sequences revealed a very low level of genetic diversity (π = 0.00003) with only three single nucleotide polymorphisms (SNPs). Of these three SNPs, only G581R is nonsynonymous. The synonymous mutation S88S is present in 3% (1/32) of the Myanmar samples, while G704G and G581R are present in 1.5% (1/66) and 3% (2/66) of the samples from China, respectively. None of the mutations observed in the P. vivax samples were associated with artemisinin resistance in P. falciparum. Furthermore, analysis of 473 PvK12 sequences from twelve worldwide P. vivax populations confirmed the very limited polymorphism in this gene and detected only five distinct haplotypes. Conclusions The PvK12 sequences from global P. vivax populations displayed very limited genetic diversity indicating low levels of baseline polymorphisms of PvK12 in these areas. Electronic supplementary material The online version of this article (doi:10.1186/s12936-016-1583-0) contains supplementary material, which is available to authorized users.

Plasmodium vivax is a major public health burden, responsible for the majority of malaria infections outside Africa. We explored the impact of demographic history and selective pressures on the P. vivax genome by sequencing 182 clinical isolates sampled from 11 countries across the globe, using hybrid selection to overcome human DNA contamination. We confirmed previous reports of high genomic diversity in P. vivax relative to the more virulent Plasmodium falciparum species; regional populations of P. vivax exhibited greater diversity than the global P. falciparum population, indicating a large and/or stable population. Signals of natural selection suggest that P. vivax is evolving in response to antimalarial drugs and is adapting to regional differences in the human host and the mosquito vector. These findings underline the variable epidemiology of this parasite species and highlight the breadth of approaches that may be required to eliminate P. vivax globally.

Background The male gamete fertilization factor P48/45 in malaria parasites is a prime transmission-blocking vaccine (TBV) candidate. Efforts to develop antimalarial vaccines are often thwarted by genetic diversity of the target antigens. Here we evaluated the genetic diversity of Pvs48/45 gene in global Plasmodium vivax populations. Methods We determined 200 Pvs48/45 sequences collected from temperate and subtropical parasite populations in China. Population genetic and evolutionary analyses were performed to determine the levels of genetic diversity, potential signature of selection, and population differentiation. Results Analysis of the Pvs48/45 sequences from 200 P. vivax parasites collected in a temperate and a tropical region revealed a low level of genetic diversity (π = 0.0012) with 14 single nucleotide polymorphisms, of which 11 were nonsynonymous. Analysis of 344 Pvs48/45 sequences from nine worldwide P. vivax populations detected a total of 38 haplotypes, of which 13 haplotypes were present only once. Multiple tests for selection confirmed a signature of positive selection on Pvs48/45 with selection skewed to the second cysteine domain. Haplotype network analysis and Wright’s fixation index showed large geographical differentiation with the presence of continent-or region-specific mutations in this gene. Conclusions Pvs48/45 displays low levels of genetic diversity with the presence of region-specific mutations. Some of the mutations may be potential epitope targets based on their positions in the predicted structure, highlighting the need for future evaluation of these mutations in designing Pvs48/45-based TBV. Electronic supplementary material The online version of this article (doi:10.1186/s13071-015-1232-4) contains supplementary material, which is available to authorized users.

Understanding naturally acquired immunity to infections caused by Plasmodia in different malaria endemicity settings is needed for better vaccine designs and for exploring antibody responses as a proxy marker of malaria transmission intensity. This study investigated the sero-epidemiology of malaria along the international border between China and Myanmar, where malaria elimination action plans are in place. This study recruited 233 P. vivax and 156 P. falciparum infected subjects with acute malaria at the malaria clinics and hospitals. In addition, 93 and 67 healthy individuals from the same endemic region or from non-endemic region, respectively, were used as controls. Acute malaria infections were identified by microscopy. Anti-recombinant PfMSP119 and PvMSP119 antibody levels were measured by ELISA. Antibody responses to respective MSP119 were detected in 50.9% and 78.2% patients with acute P. vivax and P. falciparum infections, respectively. There were cross-reacting antibodies in Plasmodium patients against these two recombinant proteins, though we could not exclude the possibility of submicroscopic mixed-species infections. IgG1, IgG3 and IgG4 were the major subclasses. Interestingly, 43.2% of the healthy endemic population also had antibodies against PfMSP119, whereas only 3.9% of this population had antibodies against PvMSP119. Higher antibody levels were correlated with age and parasite density, but not with season, gender or malaria history. Both total IgG and individual IgG subclasses underwent substantial declines during the convalescent period in three months. This study demonstrated that individuals in a hypoendemic area with coexistence of P. vivax and P. falciparum can mount rapid antibody responses against both PfMSP119 and PvMSP119. The significantly higher proportion of responders to PfMSP119 in the healthy endemic population indicates higher prevalence of P. falciparum in the recent past. Specific antibodies against PvMSP119 could serve as a marker of recent exposure to P. vivax in epidemiological studies.

© 2015 Parker et al.; licensee BioMed Central. Background: Endemic malaria in Thailand continues to only exist along international borders. This pattern is frequently attributed to importation of malaria from surrounding nations. A microgeographical approach was used to investigate malaria cases in a study village along the Thailand-Myanmar border. Methods: Three mass blood surveys were conducted during the study period (July and December 2011, and May 2012) and were matched to a cohort-based demographic surveillance system. Blood slides and filter papers were taken from each participant. Slides were cross-verified by an expert microscopist and filter papers were analysed using nested PCR. Cases were then mapped to households and analysed using spatial statistics. A risk factor analysis was done using mixed effects logistic regression. Results: In total, 55 Plasmodium vivax and 20 Plasmodium falciparum cases (out of 547 participants) were detected through PCR, compared to six and two (respectively) cases detected by field microscopy. The single largest risk factor for infection was citizenship. Many study participants were ethnic Karen people with no citizenship in either Thailand or Myanmar. This subpopulation had over eight times the odds of malaria infection when compared to Thai citizens. Cases also appeared to cluster near a major drainage system and year-round water source within the study village. Conclusion: This research indicates that many cases of malaria remain undiagnosed in the region. The spatial and demographic clustering of cases in a sub-group of the population indicates either transmission within the Thai village or shared exposure to malaria vectors outside of the village. While it is possible that malaria is imported to Thailand from Myanmar, the existence of undetected infections, coupled with an ecological setting that is conducive to malaria transmission, means that indigenous transmission could also occur on the Thai side of the border. Improved, timely, and active case detection is warranted.

Chloroquine-primaquine (CQ-PQ) continues to be the frontline therapy for radical cure of Plasmodium vivax malaria. Emergence of CQ-resistant (CQR) P. vivax parasites requires a shift to artemisinin combination therapies (ACTs), which imposes a significant financial, logistical, and safety burden. Monitoring the therapeutic efficacy of CQ is thus important. Here, we evaluated the therapeutic efficacy of CQ-PQ for P. vivax malaria in northeast Myanmar. We recruited 587 patients with P. vivax monoinfection attending local malaria clinics during 2012 to 2013. These patients received three daily doses of CQ at a total dose of 24 mg of base/kg of body weight and an 8-day PQ treatment (0.375 mg/kg/day) commencing at the same time as the first CQ dose. Of the 401 patients who finished the 28-day follow-up, the cumulative incidence of recurrent parasitemia was 5.20% (95% confidence interval [CI], 3.04% to 7.36%). Among 361 (61%) patients finishing a 42-day follow-up, the cumulative incidence of recurrent blood-stage infection reached 7.98% (95% CI, 5.20% to 10.76%). The cumulative risk of gametocyte carriage at days 28 and 42 was 2.21% (95% CI, 0.78% to 3.64%) and 3.93% (95% CI, 1.94% to 5.92%), respectively. Interestingly, for all 15 patients with recurrent gametocytemia, this was associated with concurrent asexual stages. Genotyping of recurrent parasites at the merozoite surface protein 3α gene locus from 12 patients with recurrent parasitemia within 28 days revealed that 10 of these were the same genotype as at day 0, suggesting recrudescence or relapse. Similar studies in 70 patients in the same area in 2007 showed no recurrent parasitemias within 28 days. The sensitivity to chloroquine of P. vivax in northeastern Myanmar may be deteriorating.

Background Malaria within the Greater Mekong sub-region is extremely heterogeneous. While China and Thailand have been relatively successful in controlling malaria, Myanmar continues to see high prevalence. Coupled with the recent emergence of artemisinin-resistant malaria along the Thai-Myanmar border, this makes Myanmar an important focus of malaria within the overall region. However, accurate epidemiological data from Myanmar have been lacking, in part because of ongoing and emerging conflicts between the government and various ethnic groups. Here the results are reported from a risk analysis of malaria slide positivity in a conflict zone along the China-Myanmar border. Methods Surveys were conducted in 13 clinics and hospitals around Laiza City, Myanmar between April 2011 and October 2012. Demographic, occupational and educational information, as well as malaria infection history, were collected. Logistic models were used to assess risk factors for slide positivity. Results Age patterns in Plasmodium vivax infections were younger than those with Plasmodium falciparum. Furthermore, males were more likely than females to have falciparum infections. Patients who reported having been infected with malaria during the previous year were much more likely to have a current vivax infection. During the second year of the study, falciparum infections among soldiers increased signficiantly. Conclusions These results fill some knowledge gaps with regard to risk factors associated with malaria slide positivity in this conflict region of north-eastern Myanmar. Since epidemiological studies in this region have been rare or non-existent, studies such as the current are crucial for understanding the dynamic nature of malaria in this extremely heterogeneous epidemiological landscape.

Background Rapid diagnostic tests (RDTs) have become an essential tool in the contemporary malaria control and management programmes in the world. This study aims to evaluate the performance of two commonly used RDTs for malaria diagnosis in the China-Myanmar border area. Methods A total 606 febrile patients in the China-Myanmar border were recruited to this study and were diagnosed for malaria infections by microscopy, two RDTs tests (Pf/Pan device, and Pv/Pf device) and nested PCR. Results Malaria parasites were found in 143 patients by microscopy, of which 51, 73, and 19 were Plasmodium falciparum, Plasmodium vivax and P. falciparum/P. vivax mixed infections, respectively. Compared to microscopy, the sensitivity of the Pf/Pan device was 88.6% for P. falciparum and 69.9% for P. vivax with the specificity of 90.4%. For a subset of 350 patients, the sensitivity of the Pf/Pan device and Pv/Pf device for detection of P. falciparum was 87.5% and 91.7%, respectively; and for detection of P. vivax was 72.0% and 73.8%, respectively. The specificity of the Pf/Pan device and Pv/Pf device was 94.3% and 96.5%, respectively. Nested PCR detected malaria parasites in 174 of 606 samples, of which 67, 79, two and 26 were P. falciparum, P. vivax, P. ovale and P. falciparum/P. vivax mixed infections, respectively. Compared to nested PCR, all other methods had sensitivity below 80%, suggesting that a significant number of cases were missed. Conclusions Compared to PCR, both microscopy and RDTs had lower sensitivities. RDTs had similar performance to microscopy for P. falciparum diagnosis, but performed worse for P. vivax diagnosis. Other RDT products should be selected with higher sensitivity (and good specificity) for both P. falciparum and P. vivax diagnosis.

The Greater Mekong Subregion (GMS), comprised of six countries including Cambodia, China's Yunnan Province, Lao PDR, Myanmar (Burma), Thailand and Vietnam, is one of the most threatening foci of malaria. Since the initiation of the WHO's Mekong Malaria Program a decade ago, malaria situation in the GMS has greatly improved, reflected in the continuous decline in annual malaria incidence and deaths. However, as many nations are moving towards malaria elimination, the GMS nations still face great challenges. Malaria epidemiology in this region exhibits enormous geographical heterogeneity with Myanmar and Cambodia remaining high-burden countries. Within each country, malaria distribution is also patchy, exemplified by 'border malaria' and 'forest malaria' with high transmission occurring along international borders and in forests or forest fringes, respectively. 'Border malaria' is extremely difficult to monitor, and frequent malaria introductions by migratory human populations constitute a major threat to neighboring, malaria-eliminating countries. Therefore, coordination between neighboring countries is essential for malaria elimination from the entire region. In addition to these operational difficulties, malaria control in the GMS also encounters several technological challenges. Contemporary malaria control measures rely heavily on effective chemotherapy and insecticide control of vector mosquitoes. However, the spread of multidrug resistance and potential emergence of artemisinin resistance in Plasmodium falciparum make resistance management a high priority in the GMS. This situation is further worsened by the circulation of counterfeit and substandard artemisinin-related drugs. In most endemic areas of the GMS, P. falciparum and Plasmodium vivax coexist, and in recent malaria control history, P. vivax has demonstrated remarkable resilience to control measures. Deployment of the only registered drug (primaquine) for the radical cure of vivax malaria is severely undermined due to high prevalence of glucose-6-phosphate dehydrogenase deficiency in target human populations. In the GMS, the dramatically different ecologies, diverse vector systems, and insecticide resistance render traditional mosquito control less efficient. Here we attempt to review the changing malaria epidemiology in the GMS, analyze the vector systems and patterns of malaria transmission, and identify the major challenges the malaria control community faces on its way to malaria elimination.

Using two polymorphic genetic markers, the merozoite surface protein-3alpha (MSP-3alpha) and the circumsporozoite protein (CSP), we investigated the population diversity of Plasmodium vivax in Mae Sod, Thailand from April 2000 through June 2001. Genotyping the parasites isolated from 90 malaria patients attending two local clinics for the dimorphic CSP gene revealed that the majority of the parasites (77%) were the VK210 type. Genotyping the MSP3-alpha gene indicated that P. vivax populations exhibited an equally high level of polymorphism as those from Papua New Guinea, a hyperendemic region. Based on the length of polymerase chain reaction products, three major types of the MSP-3alpha locus were distinguished, with frequencies of 74.8%, 18.7%, and 6.5%, respectively. The 13 alleles distinguished by restriction fragment length polymorphism analysis did not show a significant seasonal variation in frequency. Genotyping the MSP-3alpha and CSP genes showed that 19.3% and 25.6% of the patients had multiple infections, respectively, and the combined rate was 35.6%. Comparisons of MSP-3alpha sequences from nine clones further confirmed the high level of genetic diversity of the parasite and also suggested that geographic isolation may exist. These results strongly indicate that P. vivax populations are highly diverse and multiple clonal infections are common in this malaria-hypoendemic region of Thailand.