Absence of association between pyronaridine in vitro responses and polymorphisms in genes involved in quinoline resistance in Plasmodium falciparum

<p>Abstract</p> <p>Background</p> <p>The aim of the present work was to assess the <it>in vitro </it>cross-resistance of pyronaridine with other quinoline drugs, artesunate and several other commonly used anti-malarials and to evaluate whether decreased susceptibility to pyronaridine could be associated with genetic polymorphisms in genes involved in reduced quinoline susceptibility, such as <it>pfcrt</it>, <it>pfmdr1</it>, <it>pfmrp </it>and <it>pfnhe</it>.</p> <p>Methods</p> <p>The <it>in vitro </it>chemosusceptibility profiles of 23 strains of <it>Plasmodium falciparum </it>were analysed by the standard 42-hour ³H-hypoxanthine uptake inhibition method for pyronaridine, artesunate, chloroquine, monodesethylamodiaquine, quinine, mefloquine, lumefantrine, atovaquone, pyrimethamine and doxycycline. Genotypes were assessed for <it>pfcrt</it>, <it>pfmdr1</it>, <it>pfnhe-1 </it>and <it>pfmrp </it>genes.</p> <p>Results</p> <p>The IC₅₀values for pyronaridine ranged from 15 to 49 nM (geometric mean = 23.1 nM). A significant positive correlation was found between responses to pyronaridine and responses to artesunate (<it>r²</it>= 0.20; <it>P </it>= 0.0317) but too low to suggest cross-resistance. No significant correlation was found between pyronaridine IC₅₀and responses to other anti-malarials. Significant associations were not found between pyronaridine IC₅₀and polymorphisms in <it>pfcrt</it>, <it>pfmdr1</it>, <it>pfmrp </it>or <it>pfnhe-1</it>.</p> <p>Conclusion</p> <p>There was an absence of cross-resistance between pyronaridine and quinolines, and the IC₅₀values for pyronaridine were found to be unrelated to mutations in the transport protein genes <it>pfcrt</it>, <it>pfmdr1</it>, <it>pfmrp </it>or <it>pfnhe-1</it>, known to be involved in quinoline resistance. These results confirm the interest and the efficacy of the use of a combination of pyronaridine and artesunate in areas in which parasites are resistant to quinolines.</p

Sequence analysis of coding DNA fragments of pfcrt and pfmdr-1 genes in Plasmodium falciparum isolates from Odisha, India

The global emergence and spread of malaria parasites resistant to antimalarial drugs is the major problem in malaria control. The genetic basis of the parasite's resistance to the antimalarial drug chloroquine (CQ) is well-documented, allowing for the analysis of field isolates of malaria parasites to address evolutionary questions concerning the origin and spread of CQ-resistance. Here, we present DNA sequence analyses of both the second exon of the Plasmodium falciparum CQ-resistance transporter (pfcrt) gene and the 5' end of the P. falciparum multidrug-resistance 1 (pfmdr-1) gene in 40 P. falciparum field isolates collected from eight different localities of Odisha, India. First, we genotyped the samples for the pfcrt K76T and pfmdr-1 N86Y mutations in these two genes, which are the mutations primarily implicated in CQ-resistance. We further analyzed amino acid changes in codons 72-76 of the pfcrt haplotypes. Interestingly, both the K76T and N86Y mutations were found to co-exist in 32 out of the total 40 isolates, which were of either the CVIET or SVMNT haplotype, while the remaining eight isolates were of the CVMNK haplotype. In total, eight nonsynonymous single nucleotide polymorphisms (SNPs) were observed, six in the pfcrt gene and two in the pfmdr-1 gene. One poorly studied SNP in the pfcrt gene (A97T) was found at a high frequency in many P. falciparum samples. Using population genetics to analyze these two gene fragments, we revealed comparatively higher nucleotide diversity in the pfcrt gene than in the pfmdr-1 gene. Furthermore, linkage disequilibrium was found to be tight between closely spaced SNPs of the pfcrt gene. Finally, both the pfcrt and the pfmdr-1 genes were found to evolve under the standard neutral model of molecular evolution