Genetic Variations Associated with Drug Resistance Markers in Asymptomatic Plasmodium falciparum Infections in Myanmar

Abstract

The emergence and spread of drug resistance is a problem hindering malaria elimination in Southeast Asia. In this study, genetic variations in drug resistance markers of Plasmodium falciparum were determined in parasites from asymptomatic populations located in three geographically dispersed townships of Myanmar by PCR and sequencing. Mutations in dihydrofolate reductase (pfdhfr), dihydropteroate synthase (pfdhps), chloroquine resistance transporter (pfcrt), multidrug resistance protein 1 (pfmdr1), multidrug resistance-associated protein 1 (pfmrp1), and Kelch protein 13 (k13) were present in 92.3%, 97.6%, 84.0%, 98.8%, and 68.3% of the parasites, respectively. The pfcrt K76T, pfmdr1 N86Y, pfmdr1 I185K, and pfmrp1 I876V mutations were present in 82.7%, 2.5%, 87.5%, and 59.8% isolates, respectively. The most prevalent haplotypes for pfdhfr, pfdhps, pfcrt and pfmdr1 were 51I/59R/108N/164L, 436A/437G/540E/581A, 74I/75E/76T/220S/271E/326N/356T/371I, and 86N/130E/184Y/185K/1225V, respectively. In addition, 57 isolates had three different point mutations (K191T, F446I, and P574L) and three types of N-terminal insertions (N, NN, NNN) in the k13 gene. In total, 43 distinct haplotypes potentially associated with multidrug resistance were identified. These findings demonstrate a high prevalence of multidrug-resistant P. falciparum in asymptomatic infections from diverse townships in Myanmar, emphasizing the importance of targeting asymptomatic infections to prevent the spread of drug-resistant P.falciparum.

Keywords: Plasmodium falciparum; asymptomatic infection; drug resistance genes; haplotypes; multidrug resistance.

Figure 1

Map of study areas and flow chart of resistance markers detection. (A) Three study townships are indicated as black dots in the maps. (B) The sequences of the samples in the final black coiled frame were analyzed for single nucleotide polymorphism (SNP) and haplotypes.

Figure 2

Prevalence of dihydrofolate reductase (pfdhfr), dihydropteroate synthase (pfdhps), chloroquine resistance transporter (pfcrt), multidrug resistance protein 1 (pfmdr1), multidrug resistance-associated protein 1 (pfmrp1), and Kelch protein 13 (k13) haplotypes in P. falciparum isolates from asymptomatic carriers. (A) Prevalence of pfdhfr haplotypes, mutations at codons 51, 59, 108, and 164. including wild-type NCSI, double mutant ICNI and NRNI, triple mutant IRNI and NRNL, and quadruple mutant IRNL; (B) Prevalence of pfdhps haplotypes, mutations at codons 436, 437, 540, and 581, including wild-type SGKA, single mutant AGKA, SAKA, SGEA, and SGKG, double mutant AGEA, AGNA, SAEA, SGEG, and SGEG; (C) Prevalence of pfcrt haplotypes, mutations at codons 74, 75, 76, 220, 271, 326, 356, and 371, including wild-type MNKAQNIR, single mutant MNKAQNII, sextuple mutant IETSENII, septuple mutant IETSENTI, and octuple mutant IETSESTI; (D) Prevalence of pfmdr1 haplotypes, mutations at codons 86, 130, 184, 185, and 1225, including wild-type NEYIV, single mutant NEFIV and NEYKV, double mutant YEYKV, NKYKV, NEFKV, and NEFNV, triple mutant NEFKL; (E) Prevalence of pfmrp1 haplotypes, mutations at codons 191, 325, 437, 572, 785, 876, 1007, 1339, and 1390, including wild-type HNSFHITAF, single mutant HNSFHVTAF, double mutant YNAFHITAF, quadruple mutant YSAFHVTAF, YNAFHVMAF, and YNAFHVTAI, quintuple mutant YNAFNVMAF, YNAFHVMAI, and YNAFHVTSI, sextuple mutant YNALNVMAF; (F) Prevalence of k13 haplotypes, mutations at codons 191, 446, 574 and N, NN, NNN insertion, including wild-type, N insertion, NN insertion, NNN insertion, NN insertion with 446I, NN insertion with 574L, and single mutant 191T.

Figure 3

Median-joining haplotype network of asymptomatic P. falciparum isolates harboring mutations in six drug resistance-associated genes. The haplotype network was constructed for asymptomatic P. falciparum isolates using 43 haplotypes obtained from amino acid changes observed in pfdhfr, pfdhps, pfcrt, pfmdr1, pfmrp1, and k13. The size of each circle shows the same haplotype prevalence, with a color corresponding to a population of origin: Laiza (red), Banmauk (blue), or Paletwa (yellow). The length of an edge is proportional to the number of variations between two haplotypes, and enlarged is the torso of the tree. K13 individual mutations are labeled by amino acid position.