Polymorphisms of the pfmdr1 but not the pfnhe-1 gene is associated with in vitro quinine sensitivity in Thai isolates of Plasmodium falciparum

Abstract

BackgroundThe emergence of Plasmodium falciparum resistance to most currently used anti-malarial drugs is a major problem in malaria control along the Thai-Myanmar and Thai-Cambodia borders. Quinine (QN) with tetracycline/doxycycline has been used as the second-line treatment for uncomplicated falciparum malaria. In addition, QN monotherapy has been the first-line treatment for falciparum malaria in pregnant women. However, reduced in vitro and in vivo responses to QN have been reported. To date, a few genetic markers for QN resistance have been proposed including Plasmodium falciparum chloroquine resistance transporter (pfcrt), P. falciparum multidrug resistance 1 (pfmdr1), and P. falciparum Na+/H+ exchanger (pfnhe-1). This study was to investigate the role of the pfmdr1 and pfnhe-1 gene on in vitro QN sensitivity in Thai isolates of P. falciparum.MethodsEighty-five Thai isolates of P. falciparum from the Thai-Myanmar and Thai-Cambodia borders from 2003-2008 were determined for in vitro QN sensitivity using radioisotopic assay. Polymorphisms of the pfmdr1 and pfnhe-1 gene were determined by PCR-RFLP and sequence analysis. Associations between the in vitro QN sensitivity and the polymorphisms of the pfmdr1 and pfnhe-1 gene were evaluated.ResultsThe mean QN IC50 was 202.8 nM (range 25.7-654.4 nM). Only four isolates were QN resistant when the IC50 of >500 nM was used as the cut-off point. Significant associations were found between the pfmdr1 mutations at codons N86Y and N1042D and in vitro QN sensitivity. However, no associations with the number of DNNND, DDNNNDNHNDD, and NHNDNHNNDDD repeats in the microsatellite ms4760 of the pfnhe-1 gene were identified.ConclusionData from the present study put doubt regarding the pfnhe-1 gene as to whether it could be used as the suitable marker for QN resistance in Thailand. In contrast, it confirms the influence of the pfmdr1 gene on in vitro QN sensitivity.

Mutation in the Plasmodium falciparum CRT Protein Determines the Stereospecific Activity of Antimalarial Cinchona Alkaloids

“…To further characterize the relationship between PfCRT and the three-dimensional structure of CA, we measured the in vitro potency of four diastereomer alkaloid pairs against a set of pfcrt mutant parasite clones derived from the 106/1 K76 strain (Table 1). The resulting IC 50 s showed that, among the pharmacologically active erythro alkaloids, the dextrorotatory (ϩ) QD, HQD, and CN (8R, 9S) were 1.7-to 7.7-fold more potent than the levorotatory (Ϫ) QN, HQN, and CD (8S, 9R) against all control parasite lines and (Table 2). Among the CQR pfcrt mutants, trends emerged that (ϩ)/(Ϫ) IC 50 ratios increased (76N and 76T) or decreased (76I) relative to the original 106/1 K76 parent line.…”

“…Based on the weak activity of the 9-epimers against the other parasite lines, it was unlikely that the large increase in potency against 106/1 76I-369F was due to impurities consisting of the active erythro alkaloids. 50 ratios. Earlier, we demonstrated that among CQ-resistant clones derived from 106/1 K76 , a positive correlation exists between the CQ IC 50 and hydrophobicity of the PfCRT position 76 residue (16).…”

“…The log (Ϫ)/(ϩ)-isomer ratios clearly decreased as the PfCRT position 76 residue becomes more hydrophobic (P Ͻ 0.0001) for all CA pairs. We also conducted a t test between the mean log IC 50 ratios of the N and T residue, as the I value could leverage the regression due to its more extreme position on the plot. All N versus T mean log IC 50 ratios were significantly different (t test, P Ͻ 0.05), except for the CD/CN pair, which differ from the other CA in their lack of the quinoline methoxy group.…”

Plasmodium falciparum Na+/H+ Exchanger (pfnhe-1) Genetic Polymorphism in Indian Ocean Malaria-Endemic Areas

“…Among the 595 studied sequences, we have observed 29 different alleles, including 8 new alleles (27%). By compiling our data with previous published sequences available in GenBank, [14][15][16][17][18][19][20][21][22][23][24] we estimate that 101 different ms4760 alleles have been described to date. However, in most publications, the numbering of the ms4760 alleles did not always taking into account the previously described alleles, making data comparison difficult.…”

“…The mechanism underlying QN resistance is not well-understood, and it is probably complex and multigenic. Since the seminal work by Ferdig and others 13 in 2004, 11 studies have been conducted in different countries to evaluate implication of ms4760 polymorphisms in QN resistance, [14][15][16][17][18][19][20][21][22][23][24] and conflicting data have been reported, likely because of the different geographical origin of parasites (implying different genetic backgrounds), the type of parasites used (fresh isolates, culture-adapted strains, and reference lines), and the method used to assess in vitro QN susceptibility. 21,25 In this context and to extend our previous work regarding ms4760 polymorphisms in Plasmodium falciparum parasites circulating in malaria-endemic areas in the Indian Ocean, 14 we have analyzed ms4760 sequences from 595 isolates (Madagascar, N = 345; Comoros, N = 250).…”