Chloroquine Clinical Failures in P. falciparum Malaria Are Associated with Mutant Pfmdr-1, Not Pfcrt in Madagascar

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.

Association between Prevalence of Chloroquine Resistance and Unusual Mutation in pfmdr-I and pfcrt Genes in India

“…The former accepted view revealed that the major role of CQ resistance was determined by polymorphisms in the pfcrt gene that encoded a transporter, which promoted drug efflux from the parasite digestive vacuole in its mutated forms, 8 whereas pfmdr-I mutation modulates the level of in vitro CQ resistance. 20 In our study, we combined in vitro and in vivo tests as well as molecular genotyping at pfcrt and pfmdr-I loci.…”

“…The correlation between pfmdr-I genotypes and quinoline resistance has often generated conflicting results. It has been suggested that pfmdr-I 86Y can be correlated with increased CQ resistance in parasites that originated from different areas of the world, 7,8 but in India, field studies had not corroborated these findings, and the results of the P. falciparum genetic cross indicated that CQ resistance did not depend on the pfmdr-I gene. 12,21 Different groups of workers showed that, in India, CQ-resistant P. falciparum and the CQ treatment failure mainly occurred because of the mutation in the pfcrt and not the pfmdr-I gene.…”

“…The elevated frequency of pfmdr-I mutant alleles (YYD, NFD, YFD, and YFY) associated with CQ resistance and the low frequency of pfcrt mutant genotypes might account for the high rate of late clinical failure of CQ treatment. 8,23 pfcrt mutations were known to have an effect on CQ resistance, which was proven by genetic cross-breeding between sensitive and resistant parasites. 13 However, the unbalanced numbers of genotypes here did not allow us to draw any conclusion on the impact of pfcrt mutations.…”

“…5,6 In addition, the pfmdr-I gene located on chromosome 5 has mutations that can play a significant role in P. falciparum resistance to various antimalarials, such as mefloquine, quinine, and artemisinin derivatives. 7 Specific combinations of pfcrt and pfmdr-I alleles, resulting in varying responses to CQ, seems geographically restricted, 8 which may explain why some field studies reported an association between pfmdr-I polymorphisms and CQ resistance, 9,10 whereas other studies did not. 11 Studies from different geographical areas of the world suggest that the point mutation of asparagine to tyrosine in codon 86 (N86 to 86Y) of the pfmdr-I gene is associated with CQ resistance.…”

Étude de la résistance dePlasmodium falciparumaux antipaludiques au sein du réseau international des Instituts Pasteur (RIIP-Palu)

“…trouve que nos données indiquent que certains marqueurs pertinents pour prédire la résistance aux antipaludiques dans certaines régions (comme l'Afrique par exemple) doivent être réévalués pour des contextes particuliers comme Madagascar [22] ou la Guyane française [23]. Ces données indiquent que l'évolution des populations parasitaires diffère dans différents contextes épidémiologiques et géographiques.…”