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.

FIG. 1.

Map of locations from which samples were derived. All locations had numerous samples, except the southernmost Kalimantan site which had only a single patient sample. That location is indicated by a dot only, and the sample was included with the West Kalimantan samples for analysis. The pie charts show the proportions of alleles that showed a particular number of point mutations. The darkest color indicates 4 or more nonsynonymous mutations, and the progressively lighter shades show 3, 2, or 1 mutation; white indicates the wild type.

FIG. 2.

Pyrimethamine sensitivity of yeast strains dependent upon P. vivax dhfr alleles. Yeast strains were grown in liquid culture with the indicated concentrations of pyrimethamine, and growth relative to the same strain without pyrimethamine was measured after 24 h as described in Materials and Methods.