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

Abstract

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.

Keywords: Artemisinin; Drug resistance; Falciparum; Kelch 13; Malaria; Myanmar.

Fig. 1

Summary of samples involved for clinical and molecular analysis. Year of the study, anti-malarial, and number of the samples collected are shown. DP dihydroartemisinin–piperaquine, AL artemether–lumefantrine, AM artesunate–mefloquine, LFU lost to follow-up, TF treatment failure, ACPR adequate clinical and parasitological response, D3 pos. day 3 parasite positivity

Fig. 2

Distribution of day 3 positivity after treatment with artemisinin-based combination therapy (ACT) and molecular markers (k13, pfarps10, pffd and pfmdr2) in six sentinel sites. Day 3 prevalence and high mutant rate of molecular markers were observed in southern Myanmar sites, Myanmar Artemisinin Resistance Containment (MARC) Tier I areas

Fig. 3

Frequency of K13 propeller alleles in 550 samples isolates in six sentinel sites in Myanmar. All mutant type isolates carry a single non-synonymous mutation. Significant decreased of wile type alleles and increasing of C580Y alleles were noted in Kawthaung, southern Myanmar site

Fig. 4

Correlation between frequencies of wile type of target genes. K13 propeller (a), pfarps10 (b), pffd (c), and pfmdr2 (d), and prevalence of day 3 parasite positivity after ACT treatment in six sentinel sites in Myanmar. The frequency of day 3 parasite positivity is plotted against the frequency of wild type alleles of target genes. Spearman’s coefficient of rank correlation: K13 propeller (r = −0.9590, 95% confidence interval −0.665 to −0.995); pfarps10 (r = −0.8840, 95% confidence interval −0.257 to 0.987); pffd (r = −0.6704, 95% confidence interval −0.309 to −0.959) and pfmdr2 (r = −0.7679, 95% confidence interval −0.115 to −0.970