Background Vivax malaria is an important public health problem in the Greater Mekong Subregion (GMS), including the China-Myanmar border. Previous studies have found that Plasmodium vivax has decreased sensitivity to antimalarial drugs in some areas of the GMS, but the sensitivity of P. vivax to antimalarial drugs is unclear in the China-Myanmar border. Here, we investigate the drug sensitivity profile and genetic variations for two drug resistance related genes in P. vivax isolates to provide baseline information for future drug studies in the China-Myanmar border. Methodology/Principal findings A total of 64 P. vivax clinical isolates collected from the China-Myanmar border area were assessed for ex vivo susceptibility to eight antimalarial drugs by the schizont maturation assay. The medians of IC 50 (half-maximum inhibitory concentrations) for chloroquine, mefloquine, pyronaridine, piperaquine, quinine, artesunate, artemether, dihydroartemisinin were 84.2 nM, 34.9 nM, 4.0 nM, 22.3 nM, 41.4 nM, 2.8 nM, 2.1 nM and 2.0 nM, respectively. Twelve P. vivax clinical isolates were found over the cutoff IC 50 value (220 nM) for chloroquine resistance. In addition, sequence polymorphisms in pvmdr1 (P. vivax multidrug resistance-1), pvcrt-o (P. vivax chloroquine resistance transporter-o), and difference in pvmdr1 copy number were studied. Sequencing of the pvmdr1 gene in 52 samples identified 12 amino acid substitutions, among which two (G698S and T958M) were fixed, M908L were present in 98.1% of the isolates, while Y976F and F1076L were present in 3.8% and 78.8% of the isolates, respectively. Amplification of the pvmdr1 gene was only detected in 4.8% of PLOS NEGLECTED TROPICAL DISEASES

Multidrug-resistant Plasmodium falciparum in the Greater Mekong Subregion of Southeast Asia is a major threat to malaria elimination and requires close surveillance. In this study, we collected 107 longitudinal clinical samples of P. falciparum in 2007–2012 from the malaria hypoendemic region of the China-Myanmar border and measured their in vitro susceptibilities to 10 antimalarial drugs. Overall, parasites had significantly different IC50 values to all the drugs tested as compared to the reference 3D7 strain. Parasites were also genotyped in seven genes that were associated with drug resistance including pfcrt, pfmdr1, pfmrp1, pfdhfr, pfdhps, pfnhe1, and PfK13 genes. Despite withdrawal of chloroquine and antifolates from treating P. falciparum, parasites remained highly resistant to these drugs and mutations in pfcrt, pfdhfr, and pfdhps genes were highly prevalent and almost reached fixation in the study parasite population. Except for pyronaridine, quinine and lumefantrine, all other tested drugs exhibited significant temporal variations at least between some years, but only chloroquine and piperaquine had a clear temporal trend of continuous increase of IC50s. For the pfmrp1 gene, several mutations were associated with altered sensitivity to a number of drugs tested including chloroquine, piperaquine, lumefantrine and dihydroartemisinin. The association of PfK13 mutations with resistance to multiple drugs suggests potential evolution of PfK13 mutations amid multidrug resistance genetic background. Furthermore, network analysis of drug resistance genes indicated that certain haplotypes associated multidrug resistance persisted in these years, albeit there were year-to-year fluctuations of the predominant haplotypes.

Background: Chloroquine (CQ) and primaquine (PQ) remain the frontline drugs for radical cure of uncomplicated P. vivax malaria in the Greater Mekong Sub-region (GMS). Recent reports of decreased susceptibility of P. vivax to CQ in many parts of the GMS raise concerns.Methods: From April 2014 to September 2016, 281 patients with uncomplicated P. vivax infection attending clinics in border settlements for internally displaced people in northeast Myanmar were recruited into this study. Patients were treated with standard regimen of 3-day CQ and concurrent 14-day PQ (3.5 mg/kg total dose) as directly observed therapy, and followed for recurrent parasitemia within 28 days post-patency.Results: Within the 28-day follow-up period, seven patients developed recurrent parasitemia, resulting in a cumulative rate of parasite recurrence of 2.6%. Five of the seven parasitemias recurred within two weeks, and two of those failed to clear within seven days, indicating high-grade resistance. Conclusion:Although failure of CQ/PQ treatment of P. vivax was relatively infrequent in northeast Myanmar, this study nonetheless confirms that CQ/PQ-resistant strains do circulate in this area, some of them of a highly resistant phenotype. It is thus recommended that patients who acquire vivax malaria in Myanmar be treated an artemisinin-combination therapy along with hypnozoitocidal primaquine therapy to achieve radical cure.

BackgroundImported cases of infectious disease provide invaluable information about epidemiological conditions abroad, and should guide treatment decisions at home and abroad. Here, we examined cases of malaria imported from Africa to China for mutations eroding the efficacy of sulfadoxine-pyrimethamine (SP), sometimes used as an intermittent preventive treatment during for pregnant women and infants.MethodsA total of 208 blood samples were collected from P. falciparum-infected workers who had returned from Western and Central Africa to Guangxi Province Frequency distribution. Samples were analyzed for the mutations in dhfr and dhps genes by PCR -sequencing. The prevalence of dhfr and dhps polymorphisms was analyzed. Among the isolates, polymorphisms were detected in mutants N51I, C59R, S108N and I164L of Pfdhfr and I431V, S436 A/F, A437G, K540 E/N, A581G and A613T of pfdhps.ResultsMutations promoting drug resistance were widespread in this cohort. For pfdhfr and pfdhps, wild types were equally rare among patients returned from Western Africa and Central Africa. A triple-mutant dhfr haplotype was most prevalent (>70%). We report for the first time mutation I164L-dhfr and I431V-dhps in Ghana, and for the first time we found A581G to exceed a clinically-relevant threshold that may counter-indicate current clinical practices. For Pfdhps, the double-mutant IAGKAA was high prevalent haplotype in Ghana, Western Africa. The single-mutant ISGKAA was a majority haplotype in Cameroon. Alarmingly, a “super resistance” quintuple mutant was detected, for the first time, in parasites of West African origin (defined by IAGKAA/IRNI in combination with pfdhps 581G and dhfr I164L). This may limit the efficacy of this drug combination for even intermittent clinical applications.ConclusionsThese data are cause for great concern and call for continued surveillance of the efficacy of SP in source and recipient populations, and should be considered when developing treatment policy for imported malaria cases in China and elsewhere.

Malaria parasites in different areas where malaria is endemic display different levels of resistance to antimalarial drugs as the result of varied drug use histories. To provide updated knowledge of drug sensitivities during the malaria elimination phase in Southeast Asia, an epicenter of multidrug resistance, we determined in vitro susceptibilities of culture-adapted Plasmodium falciparum isolates from two eastern border regions (Wa and Kachin) of Myanmar to 10 drugs. Despite their close proximity, the Kachin parasites displayed higher 50% inhibitory concentrations than the Wa parasites to chloroquine, piperaquine, naphthoquine, mefloquine, quinine, pyrimethamine, pyronaridine, lumefantrine, and dihydroartemisinin. Genotyping of genes associated with drug resistance also showed significant differences in the prevalence rates of mutant alleles between the two regions. Particularly, major pfdhfr mutations mediating pyrimethamine resistance and the pfdhps A437G mutation had significantly higher frequencies in the Kachin parasites (P Ͻ 0.005). Moreover, when pfdhfr and pfdhps were considered together, the wild-type allele was found only in the Wa samples (22.6%). In addition, the pfmdr1 Y184F mutation reached 38.7% in the Kachin parasites, compared to 9.7% in the Wa parasites, whereas N86Y was only detected in the Wa parasites, at 22.6%. Furthermore, the F446I mutation and all mutations in the propeller domain of the PfK13 gene were significantly more frequent in the Kachin parasites. Collectively, this work demonstrates that even in spatially closely separated regions, parasites can exhibit drastic differences in drug sensitivities and genetic makeups underlying drug resistance, which may reflect regionally different drug histories and genetic drift of these isolated parasite populations.