Prevalence and genetic diversity of polymorphisms in pfcrt, pfdhfr-ts and pfk13 propeller genes of Plasmodium falciparum in southern Côte d'Ivoire.

Abstract

Background: Plasmodium falciparum has developed resistance to almost all the antimalarial drugs currently in use. This resistance has been and remains one of the greatest threats to the control and elimination of malaria. The use of molecular markers of resistance to monitor the emergence and spread of antimalarial drug-resistant parasite strains has proved highly effective. The aim of this study was to analyse the polymorphism of the pfcrt, pfdhfr-ts and pfK13 propeller genes for resistance in P. falciparum to chloroquine (CQ), pyrimethamine and artemisinin-based combination therapies (ACTs) in three sites in southern Côte d'Ivoire.

Methodology: Blood samples were collected in Anonkoua-kouté, Port-Bouët, and Ayamé from 94 patients with microscopically confirmed uncomplicated P. falciparum malaria. These patients, aged over 2 years, gave their informed consent prior to blood sampling. P. falciparum genomic DNA extracted from these samples was amplified by nested PCR using primers specific to the pfcrt, pfdhfr-ts and Pfk13 propeller genes. The amplification products were sequenced using the Sanger method. After sequencing, the prevalence of pfcrt (M74I, N75E, K76T), pfdhfr (N51I, C59R, S108N) and pfk13 propeller (Y493H, R539T, I543T, C580Y, M476I and R561H) mutations confirmed to be involved in P. falciparum resistance to CQ, pyrimethamine and ACTs, respectively was determined. Data were analysed using R statistical software, version 3.2.2.

Results: For all three study sites, 93 (93/94, i.e. 98.94%), 86 (86/94, i.e. 94.49%) and 74 (74/94, i.e. 78.72%) DNA fragments from patient isolates were successfully amplified for the Pfk13 propeller, pfdhfr-ts and pfcrt genes, respectively. Of the successfully amplified fragments, 93 (93/93, i.e. 100%), 81 (81/86, i.e. 94.18%) and 64 (64/74, i.e. 86.48%) were successfully sequenced for the pfk13 propeller, pfdhfr-ts and pfcrt genes, respectively. Sequence analysis indicated that S108N mutations in the pfdhfr gene and K76T mutations in the pfcrt gene were observed in 74.07% (60/81) and 15.62% (10/64) respectively. Analysis of the k13 propeller gene also showed a predominance of the YRICMR allelic form representing the sensitive haplotype (72/93, i.e. 78.49%).

Conclusions: More than a decade after the abandonment of the use of CQ and the adoption of sulfadoxinepyrimethamine (SP) as intermittent preventive treatment (IPT) for pregnant women, the prevalence of alleles associated with CQ chemoresistance, represented by the K76T mutation in the pfcrt gene, fell, while that of alleles associated with pyrimethamine chemoresistance, represented by the S108N mutation in the pfdhfr-ts gene, increased in Anonkoua-Kouté, Port-Bouët and Ayamé. No mutations in mutant alleles of the K13 propeller gene conferring resistance to artemisinin derivatives were observed at any of the study sites. The study thus showed that the ACTs used for first-line treatment of malaria in Côte d'Ivoire are still effective.