This study traces the origins of chloroquine-resistant and chloroquine-susceptible parasites in Malawi. We demonstrated that chloroquine-resistant parasites in Malawi had haplotypes that were nearly identical to that of a chloroquine-resistant isolate from Southeast Asia at microsatellite loci flanking pfcrt.
The Asian origin of chloroquine resistance in Africa has been demonstrated elsewhere in other African countries [21
], and our results extend the observation to include Malawi. The selective valley around chloroquine-resistant parasites in Malawi is ~80 kb upstream and 30 kb downstream of pfcrt,
except for an area of decreased heterozygosity at the microsatellite locus 29 kb upstream of pfcrt
in both chloroquine-resistant and chloroquine-susceptible isolates, which is likely due to the lack of variation at this marker. The selection valley was narrower than that observed by Wootton et al [8
] in chloroquine-resistant parasites from Asia. This is likely due to more extensive parasite diversity in Africa than in Asia and more frequent recombination of nonidentical parasite lineages in Africa.
These results support the hypothesis that the return of chloroquine-susceptible malaria in Malawi following the removal of chloroquine drug pressure represents a reexpansion of a heterogeneous population of susceptible parasites that persisted in Malawi during the period when chloroquine was used. The predominant chloroquine-susceptible pfcrt
genotype observed in Malawi contains the expected amino acid sequence at all polymorphic codons within the gene, suggesting that chloroquine susceptibility did not arise from a back mutation at codon 76. Diversity in the regions flanking pfcrt
did not differ between chloroquine-susceptible parasites circulating in 2005 and chloroquine-susceptible parasites circulating at the time of chloroquine drug pressure, which indicates that chloroquine susceptibility did not result from propagation of a single chloroquine-susceptible parasite with increased fitness. An isolate from a single infection had a chloroquine-resistant allele at position 220 but otherwise had the expected chloroquine-susceptible haplotype, as has been reported for a small percentage of parasites in India [22
]. The infection was successfully treated with chloroquine.
These findings have important implications for our understanding of the effect of the removal of drug pressure on the evolution and ecology of drug resistance. Drug-susceptible organisms may regain predominance as long as there is a population that survives despite prolonged drug pressure in the region that favors resistant parasites. This observation does not imply that parasites survive exposure to the drug; rather, it suggests that some parasites are not exposed to lethal drug concentrations, which supports the existence of a reservoir that is removed from drug pressure. In the case of countries where malaria is endemic, transmission is high, and acquired immunity is extensive, asymptomatic adults, who rarely become ill and who generally do not receive therapy, may provide such a reservoir for susceptible parasites to persist in the population. If nascent malaria elimination efforts in Africa result in lower transmission and less acquired immunity, then this reservoir may shrink and eventually disappear, allowing resistant alleles to become fully fixed in the population, as has been observed in both South America [23
] and Southeast Asia [7
]. It will be important to monitor the effects of malaria control and elimination interventions on parasite population diversity in order to track and predict the patterns of the rise and fall of drug resistance.
The renaissance of chloroquine-susceptible parasites was not expected. Earlier evidence had suggested that chloroquine-resistant parasites may have a survival advantage. In vitro, ex vivo, and ecological studies indicated that chloroquine-resistant parasites formed more gametocytes than did chloroquine-susceptible parasites, suggesting that resistance may increase infectiousness [24
]. Buckling et al [25
] found that the isolates of P. falciparum
in vitro and Plasmodium chabaudi
in mice that survived the nonlethal drug pressure with chloroquine were more likely to form gametocytes and more likely to be infectious to mosquitoes than untreated parasites. Our study did not examine the sexual stage of the parasite life cycle, but the rapid decline in the prevalence of chloroquine resistance in Malawi implies that any increase in infectiousness among resistant organisms must have been outweighed by other survival advantages of the susceptible genotypes.
The resurgence of susceptible parasites in the absence of drug pressure is best explained by a fitness cost associated with drug resistance that allows susceptible organisms to outcompete resistant organisms in the absence of drug pressure. Resistant organisms often evolve compensatory mutations that restore a variable degree of fitness to susceptible organisms [28
]. In the case of chloroquine-resistant malaria, although the global spread of these parasites took decades, the return of susceptibility in Malawi was rapid—detectable within a year after drug pressure was removed [3
]. P. falciparum
with resistant forms of pfcrt
are now undetectable by standard PCR methods at our study site and at many sites throughout the country (Dzinjalamala et al, unpublished data, 2007), although chloroquine-resistant parasites have been detected at a very low prevalence on the border with Tanzania [29
] and highly sensitive techniques have suggested the persistence of minority variant resistant parasites at a similar study site [30
]. The rapid disappearance of chloroquine resistance points to a high fitness cost of the resistance-conferring K76T mutation for which neither the constellation of other pfcrt
mutations that do not cause resistance in the absence of K76T nor other mutations elsewhere in the genome adequately compensate.
The dramatic return of chloroquine-susceptible falciparum following the removal of chloroquine drug pressure in Malawi has not occurred in Southeast Asia or South America, where treatment policies were changed well before policies were changed in Africa. A modest increase in chloroquine-susceptible malaria had been documented in China following reduction of chloroquine use [31
]. The reasons for these different patterns may lie in parasite, host, and/or environmental factors. Chloroquine resistance may have persisted in Asia and South America because of lower transmission (which is accompanied by smaller effective population sizes and less frequent polyclonal infections, and thus fewer opportunities for recombination, leading to fixation of the resistant genotype in the population), higher proportions of the parasite population being subjected to drug pressure in relatively nonimmune human populations, and/or persistent drug pressure with chloroquine or amodiaquine [32
]. It is also possible that the genetic backgrounds of parasites from Asia and South America have unique characteristics that favor the emergence or support the spread of resistance [33
]. There is some evidence of impaired DNA repair mechanisms in Asian parasites, compared with African parasites [35
]. New genomic epidemiological approaches and the exploration of rapid mutator or impaired DNA repair phenotypes may lead to the identification of a genetic or evolutionary basis for the consistent finding of emerging drug resistance in Asia that spreads across continents.
The results of this study have important implications for the future of antimalarial drug use in Africa. Almost all countries where malaria is endemic are now recommending the use of artemisinin-based combination therapy in place of the older drugs typically used as monotherapy—chloroquine and sulfadoxine-pyrimethamine. In a recent multinational survey, sub-Saharan African countries were found to have maintained parasite diversity with respect to pfcrt
in that susceptible parasites are still present in the population, although they are in the minority [37
]. We anticipate that as artemisinin-based combination therapies are successfully deployed throughout Africa, chloroquine-susceptible malaria will return to the region, a trend that has been documented in Kenya [38
If chloroquine susceptibility does become widespread in Africa, the possibility of using chloroquine in the future promises to be a welcome addition to the limited armamentarium in the battle against malaria. However, any reintroduction of chloroquine for the treatment or prevention of malaria should be planned with attention to the evolution of drug resistance. Chloroquine could become an attractive option for use as a tool for malaria prevention in specific targeted populations, as it is safe and well tolerated, has a long duration of action to maximize the period of protection, and has a resistance profile that differs from that of most other drugs currently being used. The extent to which the use of chloroquine (alone or in combination) for intermittent preventive treatment in select vulnerable groups, such as pregnant women or infants, would contribute to the reemergence of chloroquine resistance is not known.
If chloroquine is used again for the treatment of malaria, it should be used in combination with a partner drug to protect against resistance. The combination of chloroquine plus an artemisinin derivative represents a gross mismatch of drug pharmacokinetic and pharmacodynamic profiles, with the artemisinins clearing from the blood in just a few hours and chloroquine being detectable for at least 4–6 weeks. In the presence of high rates ofmalaria transmission, chloroquine will effectively be present as monotherapy once the 3-d course of artemisinins is completed [39
]. We are currently evaluating the efficacy of chloroquine combined with drugs with different pharmacokinetic and pharmacodynamic profiles for the treatment of falciparum malaria in Malawi, with the goal of identifying combinations that could deter the reemergence of resistance.