In vitro sensitivity of Plasmodium falciparum clinical isolates from the China-Myanmar border area to quinine and association with polymorphism in the Na+/H+ exchanger

Abstract

Quinine resistance (QNR) in Plasmodium falciparum has been detected in many regions of the world where malaria is endemic. Genetic polymorphisms in at least four genes are implicated in QN susceptibility, and their significance often depends on the genetic background of the parasites. In this study, we have culture-adapted 60 P. falciparum clinical isolates from the China-Myanmar border and assessed their in vitro responses to QN. Our results showed that >50% of the parasite isolates displayed reduced sensitivity to QN, with a half-maximal inhibitory concentration (IC(50)) above 500 nM. Genotyping of pfcrt found that an overwhelming proportion of the parasite population had the chloroquine-resistant genotype, whereas pfmdr1 mutation genotypes and gene amplification were rare. Genotyping of the P. falciparum Na(+)/H(+) exchanger gene (pfnhe1) at the minisatellite ms4760 locus identified 10 haplotypes. Haplotype 7, which harbors three copies of the DNNND repeat, was the most predominant, accounting for nearly half of the parasite isolates. Correlation studies did not reveal significant associations of the polymorphisms in pfcrt and pfmdr1 genes with QN response. However, the ms4760 haplotypes were highly associated with in vitro QN responses. In particular, parasite isolates with an increased DNNND copy number tended to have significantly reduced QN susceptibility, whereas parasite isolates with a higher NHNDNHNNDDD copy number had increased QN susceptibility. This study provided further support for the importance of pfnhe1 polymorphisms in influencing QNR in P. falciparum.

FIG. 1.

Alignment of the 35 pfnhe1 ms4760 haplotypes identified to date. Two types of repeats (Rep 1, DNNND, and Rep 2, NHNDNHNNDDD) are highlighted. Bold letters indicate mutated amino acids.

FIG. 2.

Scatter plot of mean IC₅₀ values (± SD) of the parasite isolates for QN, categorized by the number of copies Rep 1 (1, 2, 3, or 4) or Rep 2 (1 or 2). For Rep 1 and Rep 2, the P values were determined by using a nonparametric Kruskal-Wallis test with Bonferroni correction.