Geographic patterns of Plasmodium falciparum drug resistance distinguished by differential responses to amodiaquine and chloroquine

Abstract

Chloroquine (CQ) resistance (CQR) in Plasmodium falciparum originated from at least six foci in South America, Asia, and Oceania. Malaria parasites from these locations exhibit contrasting resistance phenotypes that are distinguished by point mutations and microsatellite polymorphisms in and near the CQR transporter gene, pfcrt, and the multidrug resistance transporter gene, pfmdr1. Amodiaquine (AQ), a 4-aminoquinoline related to CQ, is recommended and often used successfully against CQ-resistant P. falciparum in Africa, but it is largely ineffective across large regions of South America. The relationship of different pfcrt and pfmdr1 combinations to these drug-resistant phenotypes has been unclear. In two P. falciparum genetic crosses, particular pfcrt and pfmdr1 alleles from South America interact to yield greater levels of resistance to monodesethylamodiaquine (MDAQ; the active metabolite of AQ) than to CQ, whereas a pfcrt allele from Southeast Asia and Africa is linked to greater CQ than MDAQ resistance with all partner pfmdr1 alleles. These results, together with (i) available haplotype data from other parasites; (ii) evidence for an emerging focus of AQ resistance in Tanzania; and (iii) the persistence of 4-aminoquinoline-resistant parasites in South America, where CQ and AQ use is largely discontinued, suggest that different histories of drug use on the two continents have driven the selection of distinct suites of pfcrt and pfmdr1 mutations. Increasing use of AQ in Africa poses the threat of a selective sweep of highly AQ-resistant, CQ-resistant parasites with pfcrt and pfmdr1 mutations that are as advantaged and persistent as in South America.

Fig. 1.

In vitro CQ and MDAQ responses of individual P. falciparum clones. Mean CQ IC₅₀ values of parents (arrows) and progeny from the 7G8×GB4 (A) and HB3×Dd2 (B) crosses and mean MDAQ IC₅₀ values of parents (arrows) and progeny from the 7G8×GB4 (C) and HB3×Dd2 (D) crosses are shown. The mean values and standard errors are from at least six independent drug assays in the absence (red, for 7G8×GB4 and blue, for HB3×Dd2) or presence (black) of 0.8 μM VP. Inherited pfcrt and pfmdr1 alleles are indicated above each group.

Fig. 2.

QTL analysis of in vitro drug responses from the 7G8×GB4 and HB3×Dd2 genetic crosses. Results of QTL scans using the IC₅₀ values of CQ (A) and MDAQ (B) are shown. Each graph represents the logarithm of odds scores of the individual markers ordered along the 14 P. falciparum chromosomes. Dashed lines correspond to the 95% C.I. threshold (significant) and 63% C.I. threshold (suggestive) calculated from 1,000 permutations. Small differences on the exact placement of peaks reflect differences in the genetic distances of linkage markers observed in the 7G8×GB4 and HB3×Dd2 crosses.

Fig. 3.

Average in vitro CQ and MDAQ responses of P. falciparum clones grouped according to their pfcrt and pfmdr1 haplotypes. Average IC₅₀ responses to CQ (A) and MDAQ (B) are shown. IC₅₀ values and 95% C.I.s are indicated along with fold increases over the group of parasites carrying the pfcrt ^HB3and pfmdr1^HB3 alleles. Note the log scale of IC₅₀ values along the vertical axes.

Fig. 4.

AQ introduction and PfCRT types in malaria-endemic regions. Parasites with the CQ-sensitive PfCRT type (wild-type CVMNK parasites) are prevalent in most malaria endemic areas except in large areas of South America. P. falciparum parasites with the CVIET (i.e., Dd2 or GB4) PfCRT type are found frequently in Africa, areas of Southeast Asia, and Northern areas of South America, where resistance to CQ is high. Parasites with the SVMNT (i.e., 7G8) PfCRT types are found frequently in South America and other regions where AQ was frequently used. One of the earliest recorded uses of AQ was in 1946 in the South Pacific against P. vivax malaria [personal communication from L. T. Coggeshall cited by WHO (50) and Payne et al. (62)]. Between 1946 and 1951 AQ was introduced in India (63), Brazil (50, 64, 65), the Philippines (50, 66), Ecuador (67), Panama (68), Bolivia (69), and Honduras (70). Other regions of more recent use of AQ and prevalence of PfCRT- type SVMNT include PNG (71) and Tanzania (51). Data points have been included only if confirmed by more than a single observation; details and references for the individual points are provided in Table S6.