Novel Plasmodium vivax dhfr Alleles from the Indonesian Archipelago and Papua New Guinea: Association with Pyrimethamine Resistance Determined by a Saccharomyces cerevisiae Expression System

Abstract

In plasmodia, the dihydrofolate reductase (DHFR) enzyme is the target of the pyrimethamine component of sulfadoxine-pyrimethamine (S/P). Plasmodium vivax infections are not treated intentionally with antifolates. However, outside Africa, coinfections with Plasmodium falciparum and P. vivax are common, and P. vivax infections are often exposed to S/P. Cloning of the P. vivax dhfr gene has allowed molecular comparisons of dhfr alleles from different regions. Examination of the dhfr locus from a few locations has identified a very diverse set of alleles and showed that mutant alleles of the vivax dhfr gene are prevalent in Southeast Asia where S/P has been used extensively. We have surveyed patient isolates from six locations in Indonesia and two locations in Papua New Guinea. We sequenced P. vivax dhfr alleles from 114 patient samples and identified 24 different alleles that differed from the wild type by synonymous and nonsynonymous point mutations, insertions, or deletions. Most importantly, five alleles that carried four or more nonsynonymous mutations were identified. Only one of these highly mutant alleles had been previously observed, and all carried the 57L and 117T mutations. P. vivax cannot be cultured continuously, so we used a yeast assay system to determine in vitro sensitivity to pyrimethamine for a subset of the alleles. Alleles with four nonsynonymous mutations conferred very high levels of resistance to pyrimethamine. This study expands significantly the total number of novel dhfr alleles now identified from P. vivax and provides a foundation for understanding how antifolate resistance arises and spreads in natural P. vivax populations.Plasmodium vivax causes a severe and debilitating febrile illness. This mosquito-borne parasite infects an estimated 80 million people each year and is a prevalent cause of malaria outside sub-Saharan Africa (39). Unfortunately, P. vivax cannot be maintained in continuous culture, so despite its prevalence, the study of vivax has lagged markedly behind that of Plasmodium falciparum. This difference is particularly true with respect to the genetics and epidemiology of drug resistance in P. vivax. Chloroquine has been the first-line antimalarial treatment in most areas of endemicity, but resistance to this drug has virtually eliminated its usefulness against P. falciparum (61, 63, 65) and chloroquine resistance has now been observed with P. vivax as well (1,21,34,45,52,55,58,59). Fansidar, a fixed combination of sulfadoxine and pyrimethamine (S/P), is most often the alternative chosen when chloroquine-resistant P. falciparum parasites render chloroquine ineffective (4). However, S/P has not been recommended for primary therapy of vivax malaria due to the poor clinical efficacy reported when the drug was introduced in the 1950s (13,14,26,49,64,68).In both P. falciparum and P. vivax, the dihydrofolate reductase (DHFR, E.C. 1.5.1.3) enzyme is the therapeutic target of the pyrimethamine component of S/P (12,18,23,26,27,43,44,60). A combination of in vitro analysis of cultu...

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 .…”

“…23,39 Similar pvdhfr quadruple mutations are also prevalent in certain areas of Indonesia and Papua New Guinea. 18 In comparison, in southern Asia areas such as India, Pakistan, and Sri Lanka, parasites harboring multiple mutations in pvdhfr and pvdhps are relatively rare. 28,30,40 In such regions where P. falciparum and P. vivax coexist, mutations in pvdhfr and pvdhps have been attributed to selective pressure of antifolate drugs specific for controlling P. falciparum .…”

“…16 To date, some 20 mutations have been detected in pvdhfr ; among them mutations at codons 57, 58, 61, 117, and 173, corresponding to positions 50, 51, 59, 108 and 164 in pfdhfr , respectively, are associated with pyrimethamine resistance. [17][18][19][20] A structural study has confirmed that the most common mutations (S58R and S117N) cause structural changes in P . vivax DHFR and lead to decreased binding to pyrimethamine.…”

Resistance to Therapies for Infection by Plasmodium vivax

“…Hawkins et al (101) contrasted that with data from a report by Young and Burgess (241) emphasizing the intrinsic susceptibility to induced resistance to pyrimethamine. Those investigators also linked the clinical failure of SP against P. vivax to the accumulation of multiple mutations in DHFR, with wildtype DHFR isolates showing in vivo sensitivity to SP therapy (97,109), along with in vitro evidence of a similar process in transfected Saccharomyces cerevisiae systems (97,98).…”

Assessment of in vitro sensitivity of Plasmodium vivax fresh isolates

“…In vitro sensitivity of this compound was assessed in our study in view of previous reports showing its promise as a possible treatment of P. vivax malaria. 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] .…”