Real-time PCR for dihydrofolate reductase gene single-nucleotide polymorphisms in Plasmodium vivax isolates

Abstract

Mutations in the dhfr gene of Plasmodium vivax (pvdhfr) are associated with resistance to the antifolate antimalarial drugs. Polymorphisms in the pvdhfr gene were assessed by hybridization probe technology on the LightCycler instrument with 134 P. vivax-infected blood samples from Turkey (n = 24), Azerbaijan (n = 39), Thailand (n = 16), Indonesia (n = 53), and travelers (n = 19). Double mutations (S58R and S117N) or quadruple mutations (F57L/I, S58R, T61M, and S117N) in the pvdhfr genes were found in all Thai samples (100%). pvdhfr mutant-type alleles were significantly more common in samples from travelers (42%) than in those from patients from Indonesia (5%). Surprisingly, the pvdhfr single-mutation allele (S117N) was identified at a high frequency in parasites from Turkey and Azerbaijan (71 and 36%, respectively), where sulfadoxine-pyrimethamine is not recommended for the treatment of P. vivax malaria by the World Health Organization and the Malaria National Programs.

FIG. 1.

Nucleotide sequence of the P. vivax DHFR-TS gene (GenBank accession no. X98123).

FIG. 2.

Genotyping of pvdhfr by melting curve analysis of 134 P. vivax-infected samples. (A) Melting temperature peaks of the alleles obtained for the F57I/L, S58R, and T61M mutations: ^a, wild-type alleles F57, S58, and T61; ^b, mutant type 1 alleles F57, S58R, and T61; ^c, mutant type 2 alleles F57L, S58R, and T61M; ^d, mutant type 3 alleles I57L, S58R, and T61M. (B) Melting peaks of the alleles for S117N/T. ^e, wild-type allele S117; ^f, mutant type 1 allele S117N; ^g, mutant type 2 allele S117T. (C) Melting peaks obtained for mutations I172V and I173L. ^h, wild-type alleles I172 and I173; ⁱ, mutant type alleles I172 and I173L. (D) Double peaks indicating the presence of both wild-type and mutant alleles simultaneously in the same sample.

FIG. 3.

Comparison of the amino acid sequences of P. vivax DHFR enzymes deduced from the nucleotide sequences of the pvdhfr genes amplified from P. vivax isolates. Alignment of the sequences is based on the ARI-Pakistan sequence (GenBank accession no. X98123), considered to represent the wild-type sequence, from residues 39 to 185. Dashes represent deletions, and polymorphisms are in boldface with gray shading.