Abstract
Molecular studies have demonstrated that mutations in the Plasmodium falciparum chloroquine resistance transporter gene (Pfcrt) play a major role in chloroquine resistance, while mutations in P. falciparum multidrug resistance gene (Pfmdr-1) act as modulator. In Madagascar, the high rate of chloroquine treatment failure (44%) appears disconnected from the overall level of in vitro CQ susceptibility (prevalence of CQ-resistant parasites <5%) or Pfcrt mutant isolates (<1%), strongly contrasting with sub-Saharan African countries. Previous studies showed a high frequency of Pfmdr-1 mutant parasites (>60% of isolates), but did not explore their association with P. falciparum chloroquine resistance. To document the association of Pfmdr-1 alleles with chloroquine resistance in Madagascar, 249 P. falciparum samples collected from patients enrolled in a chloroquine in vivo efficacy study were genotyped in Pfcrt/Pfmdr-1 genes as well as the estimation of the Pfmdr-1 copy number. Except 2 isolates, all samples displayed a wild-type Pfcrt allele without Pfmdr-1 amplification. Chloroquine treatment failures were significantly associated with Pfmdr-1 86Y mutant codon (OR = 4.6). The cumulative incidence of recurrence of patients carrying the Pfmdr-1 86Y mutation at day 0 (21 days) was shorter than patients carrying Pfmdr-1 86N wild type codon (28 days). In an independent set of 90 selected isolates, in vitro susceptibility to chloroquine was not associated with Pfmdr-1 polymorphisms. Analysis of two microsatellites flanking Pfmdr-1 allele showed that mutations occurred on multiple genetic backgrounds. In Madagascar, Pfmdr-1 polymorphism is associated with late chloroquine clinical failures and unrelated with in vitro susceptibility or Pfcrt genotype. These results highlight the limits of the current in vitro tests routinely used to monitor CQ drug resistance in this unique context. Gaining insight about the mechanisms that regulate polymorphism in Pfmdr1 remains important, particularly regarding the evolution and spread of Pfmdr-1 alleles in P. falciparum populations under changing drug pressure which may have important consequences in terms of antimalarial use management.
Highlights
Plasmodium falciparum resistance to chloroquine (CQ) has emerged at least from six independent foci (South East Asia, Venezuela, Colombia, Papua New Guinea, India and Philippines) in the late 1950s and in the 1960s [1,2,3]
In the isolates from Madagascar studied here which harboured a wild type Plasmodium falciparum chloroquine resistance transporter gene (Pfcrt), Pfmdr-1 polymorphism, in the absence of any noticeable gene amplification, seems to play a major role in late chloroquine clinical failures without affecting the overall level of in vitro CQ susceptibility. This situation contrasts with findings from multiple settings across the African continent, where CQ resistance depends primarily on mutations in Pfcrt gene and on additional mutations in Pfmdr-1 gene, which may increase the level of resistance afforded by Pfcrt
The association of Pfmdr-1 with CQ resistance was not found in some areas [10], possibly reflecting the fact that the impact of the Pfcrt/Pfmdr-1 combination mutation depends on the genetic background of the strain [23,24] as demonstrated by studies with genetically manipulated lines or recombinant progeny of experimental crosses [23,24] and the history of use of the antimalarial drugs
Summary
Plasmodium falciparum resistance to chloroquine (CQ) has emerged at least from six independent foci (South East Asia, Venezuela, Colombia, Papua New Guinea, India and Philippines) in the late 1950s and in the 1960s [1,2,3]. Molecular evolutionary studies have demonstrated that P. falciparum CQ-resistant parasites from South-East Asia have entered in East Africa (Kenya and Tanzania) in the late 1970s and spread across the African continent within two decades [2]. Based on molecular allele exchange studies and analysis of genetic crosses, it is today generally accepted that the major role in CQ resistance is determined by polymorphisms in Pfcrt, a gene encoding a transporter which promotes, in its mutated forms, drug efflux from the parasite digestive vacuole, while Pfmdr-1 modulates the level of in vitro CQ-resistance [3,8,9]. Mutations or amplifications of Pfmdr-1 gene can play a significant role in P. falciparum resistance to diverse antimalarials such as mefloquine, quinine or artemisinin derivatives [10]. Specific combinations of Pfcrt and Pfmdr-1 alleles, resulting in varying responses to CQ (and amodiaquine), appeared geographically restricted, which may explain why some field studies reported an association between Pfmdr-1 polymorphisms and CQ resistance and other studies did not [9]
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