Amodiaquine Resistance in Plasmodium falciparum Malaria in Afghanistan Is Associated with the pfcrt SVMNT Allele at Codons 72 to 76

Abstract

Mutations in the Plasmodium falciparum genes pfcrt and pfmdr1 are selected by amodiaquine treatment in Africa. To examine the importance of these mutations in amodiaquine-treated Asian parasites, we determined pre-and posttreatment genotypes for amodiaquine treatment failures from a clinical trial in Afghanistan. The pfcrt codon 72 to 76 haplotype SVMNT was present in all samples tested, both before and after treatment. Amodiaquine did not clearly select for any pfmdr1 genotype, but a novel mutation, pfmdr1 N86F, was detected in four samples. We provide in vivo data to support the in vitro correlation between pfcrt SVMNT and increased resistance to the metabolite of amodiaquine.Amodiaquine (AQ), a 4-aminoquinoline related to chloroquine (CQ), has been used commonly as a monotherapy and now as a partner drug in artemisinin-based combination therapies (ACTs) for the treatment of uncomplicated Plasmodium falciparum malaria. In many countries, predominantly in Africa, the in vivo efficacy of AQ was found to be good even in the face of increasing CQ resistance (12). However, reports of AQ resistance have come from South America, Asia, and East Africa (7,8,10).Mutations in the P. falciparum chloroquine resistance transporter gene (pfcrt) and multidrug resistance gene 1 (pfmdr1) have been associated with clinical resistance to both CQ and AQ (13). The presence of the pfcrt codon 72 to 76 haplotype SVMNT (Ser-Val-Met-Asn-Thr) correlates with high-level resistance to the AQ metabolite desethylamodiaquine (DEAQ) in in vitro tests (14) and has been detected with a high prevalence in parasite populations from Brazil, Papua New Guinea, Laos, Iran, and India (9,18). Clinical trials in East Africa have also demonstrated high levels of in vivo resistance to AQ; in those studies, the parasites carried pfcrt codon 72 to 76 haplotype CVIET, and pfmdr1 polymorphisms 86Y, 184Y, and 1246Y were found to be selected after AQ treatment failure (5, 6, 11).A clinical trial performed in Nangahar Province, East Afghanistan, in 2002 and 2003 to explore possible replacement treatments for CQ showed very poor efficacies of both CQ and AQ monotherapy (adequate clinical and parasitological responses were seen in 11% and 9% of cases, respectively, by day 42) (4). Our aim in this study was to evaluate pre-and posttreatment samples from patients treated with AQ for pfcrt and pfmdr1 mutations and to determine which, if any, polymorphisms are associated with AQ treatment failure in Afghanistan. MATERIALS AND METHODSWe analyzed samples collected from AQ-treated participants during a clinical trial of AQ versus CQ versus sulfadoxine-pyrimethamine versus AQ plus artesunate in East Afghanistan between October 2002 and January 2003. The clinical and parasitological results of drug efficacy testing have been reported elsewhere (4). Amodiaquine (Basoquin) was supplied by Parke-Davis. Ethical approval for the in vivo study and collection of samples for genotyping was given by the LSHTM Ethics Committee and also locally, by the Ministry of Public Health, Afgh...

Rapid, Field-Deployable Method for Genotyping and Discovery of Single-Nucleotide Polymorphisms Associated with Drug Resistance in Plasmodium falciparum

“…The pfmdr1 locus is known to be present in multiple copies in some drug-resistant parasites, particularly those resistant to mefloquine (13,28). As part of this analysis, we observed different genotypes for the pfmdr1 locus in the Dd2 parasite and confirmed the presence of the newly reported pfmdr1 N86F mutation in Dd2 (4,9). This demonstrates the ability of these assays to detect mutations among loci subject to copy number variation, which has been implicated in some drug-resistant parasites (11,18,22).…”

Distribution Pattern of Plasmodium falciparum Chloroquine Transporter (pfcrt) Gene Haplotypes in Sri Lanka 1996–2006

“…3 Furthermore, several recent studies indicate that the SVMNT mutations may be sufficient to confer resistance to amodiaquine. 11,12 Although the current ACT regimen used in Sri Lanka is artemether-lumefantrine, the high prevalence of the SVMNT haplotype found in Sri Lanka cautions against the widespread adoption of amodiaquinebased ACTs in the country.…”

“…8,9 Furthermore, the haplotypes observed in codons 72-76 have been associated with different fitness costs, coresistance to amodiaquine, and geographic origins. [10][11][12] A previous study has investigated these markers in Sri Lanka, finding that, among 76 samples collected from patients in the Mannar district of Northern Province in 2002 and 2004, 76.3% had the T76 mutation. 7 However, no published studies from outside the Mannar district have yet measured the change in haplotype frequencies in Sri Lanka over time.…”

Pfcrt mutant haplotypes may not correspond with chloroquine resistance

“…Multiple studies in falciparum malaria-endemic areas have suggested that K76T mutant parasites are linked to chloroquine resistance in both in vitro and in vivo trials [3][4][5][6][7]. The K76T mutation has also been found to be associated with amodiaquine resistance and predictive treatment failure for both chloroquine and amodiaquine [8][9][10]. The association of the N86Y mutation in the pfmdr1 gene in chloroquine resistance has been debated; some studies suggest that its presence along with pfcrt mutations can cause higher levels of chloroquine resistance [11][12][13][14].…”