Prevalence of molecular markers of sulfadoxine-pyrimethamine and artemisinin resistance in Plasmodium falciparum from Pakistan

Abstract

Background: In Pakistan, artesunate (AS) in combination with sulfadoxine-pyrimethamine (SP) is the recommended treatment for uncomplicated Plasmodium falciparum malaria. Monitoring molecular markers of anti-malarial drug resistance is crucial for early detection and containment of parasite resistance to treatment. Currently, no data are available on molecular markers of artemisinin resistance (K13 mutations) in P. falciparum isolates from Pakistan. In this study, the prevalence of mutations associated with SP and artemisinin resistance was estimated in different regions of Pakistan.

Methods: A total of 845 blood samples that were positive for malaria parasites by microscopy or rapid diagnostic test were collected from January 2016 to February 2017 from 16 different sites in Pakistan. Of these samples, 300 were positive for P. falciparum by PCR. Polymorphisms in the P. falciparum dihydrofolate reductase (pfdhfr) and dihydropteroate synthase (pfdhps) genes were identified by pyrosequencing while polymorphisms in the propeller domain of the pfk13 gene were identified by Sanger sequencing.

Results: The prevalence of the PfDHFR 108N and 59R mutations was 100% and 98.8%, respectively, while the prevalence of PfDHFR 50R and 51I mutations was 8.6%. No mutation was observed at PfDHFR position 164. In PfDHPS, the prevalence of mutations at positions 436, 437, and 613 was 9.9%, 45.2%, and 0.4%, respectively. No mutations were found at PfDHPS positions 540 and 581. The prevalence of double PfDHFR mutants (59R + 108N) ranged from 93.8% to 100%, while the prevalence of parasites having the PfDHFR 59R + 108N mutations in addition to the PfDHPS 437G mutation ranged from 9.5% to 83.3% across different regions of Pakistan. Nine non-synonymous and four synonymous mutations were observed in the PfK13 propeller domain, none of which correspond to mutations validated to contribute to artemisinin resistance.

Conclusion: The absence of the highly resistant PfDHFR/PfDHPS quintuple mutant parasites and the lack of PfK13 mutations associated with artemisinin resistance is consistent with AS + SP being effective in Pakistan.

Keywords: Drug resistance; Malaria; Pakistan; Plasmodium falciparum.

Fig. 1

Geographic location of sample collection sites in 16 different areas of Pakistan. Red dots indicate the areas from where samples were collected

Fig. 2

Prevalence of PfDHFR and PfDHPS mutations across all study sites in Pakistan. Amino acid positions are shown on x-axis, while the proportion of infections containing a mutation at a given position is shown on the y-axis. Error bars indicate the standard error

Fig. 3

Distribution of PfDHFR and PfDHPS mutant alleles in different regions of Pakistan. Amino acid positions and geographic regions are shown on x-axis, while the proportion of infections containing a mutation at a given position is shown on the y-axis. Error bars indicate the standard error

Fig. 4

Amino acid sequence alignment of PfK13 haplotypes. Haplotypes observed in this study are highlighted in grey. Numbers in the top row are amino acid positions. Positions highlighted in yellow have been associated with delayed parasite clearance in previous studies, with mutations validated for their role in artemisinin resistance marked with an asterisk. Positions highlighted in green represent non-synonymous mutations identified in this study