In vitro susceptibility to pyrimethamine of DHFR I164L single mutant Plasmodium falciparum

Abstract

BackgroundRecently, Plasmodium falciparum parasites bearing Pfdhfr I164L single mutation were found in Madagascar. These new mutants may challenge the use of antifolates for the intermittent preventive treatment of malaria during pregnancy (IPTp). Assays with transgenic bacteria suggested that I164L parasites have a wild-type phenotype for pyrimethamine but it had to be confirmed by testing the parasites themselves.MethodsThirty Plasmodium falciparum clinical isolates were collected in 2008 in the south-east of Madagascar. A part of Pfdhfr gene encompassing codons 6 to 206 was amplified by PCR and the determination of the presence of single nucleotide polymorphisms was performed by DNA sequencing. The multiplicity of infection was estimated by using an allelic family-specific nested PCR. Isolates that appeared monoclonal were submitted to culture adaptation. Determination of IC50s to pyrimethamine was performed on adapted isolates.ResultsFour different Pfdhfr alleles were found: the 164L single mutant-type (N = 13), the wild-type (N = 7), the triple mutant-type 51I/59R/108N (N = 9) and the double mutant-type 108N/164L (N = 1). Eleven out 30 (36.7%) of P. falciparum isolates were considered as monoclonal infection. Among them, five isolates were successfully adapted in culture and tested for pyrimethamine in vitro susceptibility. The wild-type allele was the most susceptible with a 50% inhibitory concentration (IC50) < 10 nM. The geometric mean of IC50 of the three I164L mutant isolates was 6-fold higher than the wild-type with 61.3 nM (SD = 3.2 nM, CI95%: 53.9-69.7 nM). These values remained largely below the IC50 of the triple mutant parasite (13,804 nM).ConclusionThe IC50s of the I164L mutant isolates were significantly higher than those of the wild-type (6-fold higher) and close from those usually reported for simple mutants S108N (roughly10-fold higher than wild type). Given the observed values, the determination of IC50s directly on parasites did not confirm what has been found on transgenic bacteria. The prevalence increase of the Pfdhfr I164L single mutant parasite since 2006 could be explained by the selective advantage of this allele under sulphadoxine-pyrimethamine pressure. The emergence of highly resistant alleles should be considered in the future, in particular because an unexpected double mutant-type allele S108N/I164L has been already detected.

Polymorphism in dhfr/dhps genes, parasite density and ex vivo response to pyrimethamine in Plasmodium falciparum malaria parasites in Thies, Senegal

“…We found a correlation between the dhfr S108N single mutation and pyrimethamine resistance, and a correlation between the dhfr N51I/C59R/S108N triple mutation, as well as the dhfr N51I/C59R/S108N and dhps A437G quadruple mutation, and pyrimethamine resistance. Overall, we found a significant difference in the geometric mean IC 50 values for pyrimethamine ( p = 0.0009) between parasites possessing wild-type and resistant alleles in dhfr , as has been reported by others ( Andriantsoanirina et al, 2011 ). We confirmed the existence of an association between the dhfr genotype and chemosensitivity to pyrimethamine in P. falciparum isolates from Thies, as the increase in the number of mutations was associated with an increase in ex vivo resistance to pyrimethamine, similar to what has been observed in Gabon ( Aubouy et al, 2003 ), Central African Republic ( Menard et al, 2006 ), and Cote D’Ivoire ( Djaman et al, 2007 ).…”

MRP1 mediates folate transport and antifolate sensitivity in Plasmodium falciparum

“…The IC 50 concentrations that were obtained in this study are within the range of previously detected in vitro sensitivity profiles. For pyrimethamine, the IC 50 value of 12 n m lies within the range of reported inhibitory concentrations (< 10 to 480 n m ) in different assay types . Also for WR99210, the IC 50 of 0.039 n m was very near to those previously reported for different parasite lines (0.075–0.21 n m ) .…”

A clinical and molecular study of artesunate + sulphadoxine-pyrimethamine in three districts of central and eastern India

“…The results of this study showed a similar pattern with the exception of an absence of mutation in codon N51I. It was observed the I164L mutation associated with high levels of pyrimethamine resistance [21]. Previous studies in Assam, in north-east India and Odisha in eastern India, documented the dhfr triple mutant Asn108 + Ile51 + Arg59 associated with pyrimethamine resistance [17].…”