Sensitivity to Antifolates and Genetic Analysis of Plasmodium Vivax Isolates From Thailand

Abstract

We investigated the association between the Plasmodium vivax dihydrofolate reductase (Pvdhfrtas) and the P. vivax dihydropteroate synthase (Pvdhps) genotype and in vitro sensitivity to the antifolates pyrimethamine, WR99210, chlorcycloguanil, sulfadoxine, and dapsone. Drug responses of 32 P. vivax isolates were assessed in two in vitro systems: schizont maturation inhibition and a yeast expression system. The geometric mean of 50% inhibition concentration (IC(50)) values for pyrimethamine, chlorcycloguanil, WR99210, sulfadoxine, and dapsone were 85 +/- 88, 784 +/- 662, 95 +/- 87, 2,424 +/- 2,784, and 1,625 +/- 1,801 nM, respectively, for the schizont maturation assay. Five different Pvdhfr alleles and four Pvdhps alleles were observed: 26 of 32 quadruple mutant alleles of Pvdhfr (F57I,L/S58R/T61M/S117T), four triple mutants (S58R/T61M/S117T, K49C/S58R/S117N), and two double mutant isolates (S58R/S117N). All isolates carried Pvdhps 585V. Twenty four isolates carried double mutant Pvdhps (A383G/A553G), six an additional mutation, S382A,C/A383G/A553G, and two a single mutation, A383G. Increasing geometric mean IC(50) values were observed with increased number of Pvdhfr mutations from double to quadruple. Results suggest that quadruple mutant alleles confer decreased sensitivity to pyrimethamine but retain sensitivity to WR99210.

Assessment of in vitro sensitivity of Plasmodium vivax fresh isolates

“…The drug shows activity against the most pyrimethamine-resistant P. falciparum strains and is the extremely effective inhibitor of the P. vivax DHFR including mutations that confer high-level resistance to pyrimethamine [14] . Median (95% CI) IC 50 of WR99210 in P. vivax isolates collected in the present study was similar to our previous observation in the same area [15] . The relatively poor in vitro susceptibility of P. vivax to WR99210 could be explained by the slow action of this drug and/or the innate resistance as well as the presence of p-aminobenzoic acid and folate in the media used which acted as competitive antagonists of antifolate activity [16] .…”

Prevalence of Polymorphisms in Antifolate Drug Resistance Molecular Marker Genes pvdhfr and pvdhps in Clinical Isolates of Plasmodium vivax from Kolkata, India

“…Several reports on in vitro drug assays and clinical therapeutic assessments generally agree with this hypothesis (27)(28)(29), suggesting that genotyping of molecular markers may provide valuable information about the trends of SP resistance in P. vivax. Mutant 117N alone could increase the 50% inhibitory concentration (IC 50 ) of pyrimethamine by more than 80 times, and a combination of S58R and S117N increases resistance to this drug 400 times more than the wild type (28).…”

Different Allele Prevalence in the Dihydrofolate Reductase and Dihydropteroate Synthase Genes in Plasmodium vivax Populations from China

“…Results from in vitro drug assays with limited numbers of field samples and tests using a yeast expression system are generally agreeable with this assumption. 18,20,38 Furthermore, limited clinical assessments of the efficacy of SP have associated pvdhfr quadruple mutations and increased risks of clinical resistance to SP, 19,22,23,25 suggesting that molecular genotyping data for pvdhfr and pvdhps should provide useful information about SP resistance in P. vivax .…”