Molecular characterisation of drug-resistant Plasmodium falciparum from Thailand

Abstract

BackgroundThe increasing levels of Plasmodium falciparum resistance to chloroquine (CQ) in Thailand have led to the use of alternative antimalarials, which are at present also becoming ineffective. In this context, any strategies that help improve the surveillance of drug resistance, become crucial in overcoming the problem.MethodsIn the present study, we have established the in vitro sensitivity to CQ, mefloquine (MF), quinine (QUIN) and amodiaquine (AMQ) of 52 P. falciparum isolates collected in Thailand, and assessed the prevalence of four putative genetic polymorphisms of drug resistance, pfcrt K76T, pfmdr1 N86Y, pfmdr1 D1042N and pfmdr1 Y1246D, by PCR-RFLP.ResultsThe percentage of isolates resistant to CQ, MF, and AMQ was 96% (50/52), 62% (32/52), and 58% (18/31), respectively, while all parasites were found to be sensitive to QUIN. In addition, 41 (79%) of the isolates assayed were resistant simultaneously to more than one drug; 25 to CQ and MF, 9 to CQ and AMQ, and 7 to all three drugs, CQ, MF and AMQ. There were two significant associations between drug sensitivity and presence of particular molecular markers, i) CQ resistance / pfcrt 76T (P = 0.001), and ii) MF resistance / pfmdr1 86N (P < 0.001)Conclusionsi) In Thailand, the high levels of CQ pressure have led to strong selection of the pfcrt 76T polymorphism and ii) pfmdr1 86N appears to be a good predictor of in vitro MF resistance.

Carriage of Mutant Dihydrofolate Reductase and Dihydropteroate Synthase Genes among <b><i>Plasmodium falciparum</i></b> Isolates Recovered from Pregnant Women with Asymptomatic Infection in Lagos, Nigeria

“…Polymorphisms in the dhfr and dhps genes were genotyped by the PCR and restriction fragment length polymorphism method previously described by Lopes et al [ 21 ], Figueiredo et al [ 22 ] and Mendes et al [ 23 ]. For the dhfr gene, DNA fragments of 206, 320 and 504 bp were amplified by PCR for codons 51, 59 and 108, respectively, using codon-specific primers (table 1 ), while for the dhps gene, a common DNA fragment of 438 bp was amplified for codons 437 and 540, respectively.…”

Ex Vivo Activity of Endoperoxide Antimalarials, Including Artemisone and Arterolane, against Multidrug-Resistant Plasmodium falciparum Isolates from Cambodia

“…DNA samples were also genotyped at pfmdr1 codons 86, 184, 1034, and 1042 for single nucleotide polymorphism (SNP) analysis by real-time PCR on an ABI sequence detector 7000 (Applied Biosystems, Warrington, United Kingdom), as previously described (28). Samples were also probed for an additional pfmdr1 SNP (Y1246D) using a PCR-restriction fragment length polymorphism method (29). The clinical isolates were also screened for the K76T point mutation in the P. falciparum chloroquine resistance transporter gene 1 (pfcrt1) using real-time PCR (30).…”

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“…In fact, apart from changed expression, there is also evidence to suggest that single nucleotide polymorphisms in the pfmdr1gene may modulate parasite susceptibility to quinine, mefloquine and halofantrine. A mutation at codon 86 (N84Y) was associated with increased mefloquine sensitivity in P. falciparum from Thailand [52,53] and the Gambia [54]. Genetic crossing experiments have shown that polymorphisms at codon 184 (F to Y) and 1042 (D to N) in the Pgh-1 protein are associated with increased sensitivity to both mefloquine and halofantrine [55].…”