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Background: Currently, artemisinin-based combination therapy (ACT) is the first-line anti-malarial treatment in malaria-endemic areas. However, resistance in Plasmodium falciparum to artemisinin-based combinations emerging in the Greater Mekong Sub-region is a major problem hindering malaria elimination. To continuously monitor the potential spread of ACT-resistant parasites, this study assessed the efficacy of artemether-lumefantrine (AL) for falciparum malaria in western Myanmar., Methods: Ninety-five patients with malaria symptoms from Paletwa Township, Chin State, Myanmar were screened for P. falciparum infections in 2015. After excluding six patients with a parasite density below 100 or over 150,000/µL, 41 P. falciparum patients were treated with AL and followed for 28 days. Molecular markers associated with resistance to 4-amino-quinoline drugs (pfcrt and pfmdr1), antifolate drugs (pfdhps and pfdhfr) and artemisinin (pfk13) were genotyped to determine the prevalence of mutations associated with anti-malarial drug resistance., Results: For the 41 P. falciparum patients (27 children and 14 adults), the 28-day AL therapeutic efficacy was 100%, but five cases (12.2%) were parasite positive on day 3 by microscopy. For the pfk13 gene, the frequency of NN insert after the position 136 was 100% in the day-3 parasite-positive group as compared to 50.0% in the day-3 parasite-negative group, albeit the difference was not statistically significant (P = 0.113). The pfk13 K189T mutation (10.0%) was found in Myanmar for the first time. The pfcrt K76T and A220S mutations were all fixed in the parasite population. In pfmdr1, the Y184F mutation was present in 23.3% of the parasite population, and found in both day-3 parasite-positive and -negative parasites. The G968A mutation of pfmdr1 gene was first reported in Myanmar. Prevalence of all the mutations in pfdhfr and pfdhps genes assessed was over 70%, with the exception of the pfdhps A581G mutation, which was 3.3%., Conclusions: AL remained highly efficacious in western Myanmar. Pfk13 mutations associated with artemisinin resistance were not found. The high prevalence of mutations in pfcrt, pfdhfr and pfdhps suggests high-degree resistance to chloroquine and antifolate drugs. The pfmdr1 N86/184F/D1246 haplotype associated with selection by AL in Africa reached > 20% in this study. The detection of > 10% patients who were day-3 parasite-positive after AL treatment emphasizes the necessity of continuously monitoring ACT efficacy in western Myanmar.

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-3β 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.

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.