Origins of the Recent Emergence of Plasmodium falciparum Pyrimethamine Resistance Alleles in Madagascar

The combination of sulfadoxine-pyrimethamine is recommended for use as intermittent preventive treatment of malaria during pregnancy and is deployed in Africa. The emergence and the spread of resistant parasites are major threats to such an intervention. We have characterized the Plasmodium falciparum dhfr (pfdhfr) haplotypes and flanking microsatellites in 322 P. falciparum isolates collected from the Comoros Islands and Madagascar. One hundred fifty-six (48.4%) carried the wild-type pfdhfr allele, 19 (5.9%) carried the S108N single-mutation allele, 30 (9.3%) carried the I164L single-mutation allele, 114 (35.4%) carried the N51I/C59R/S108N triple-mutation allele, and 3 (1.0%) carried the N51I/C59R/S108N/I164L quadruple-mutation allele. Microsatellite analysis showed the introduction from the Comoros Islands of the ancestral pfdhfr triple mutant allele of Asian origin and its spread in Madagascar. Evidence for the emergence on multiple occasions of the I164L single-mutation pfdhfr allele in Madagascar was also obtained. Thus, the conditions required to generate mutants with quadruple mutations are met in Madagascar, representing a serious threat to current drug policy.Despite the increasing financial support for the control of malaria (16, 31), malaria remains a major cause of morbidity and mortality in many developing countries in the tropical world (10). In the Indian Ocean region, where the burden of malaria is restricted to the Comoros Archipelago and Madagascar (33), various intervention strategies are currently being implemented (35). The use of effective and well-tolerated antimalarial drugs is the mainstay of the armory for the control and elimination of Plasmodium falciparum malaria. Artemisinin combination therapies (ACTs) are used for the first-line treatment of P. falciparum infections, and the antifolate sulfadoxine-pyrimethamine (SP) combination is recommended for the intermittent preventive treatment of malaria during pregnancy (IPTp) (3). Indeed, SP, effective in reducing placental malaria and low birth weight, acts as a competitive inhibitor of two enzymes in the parasite''s folate synthesis pathway: dihydrofolate reductase (DHFR) and dihydropteroate synthetase (DHPS), respectively (9). Nevertheless, the emergence and the spread of SP-resistant parasites remain major threats that could render this intervention ineffective (19). Amino acid changes at positions 51, 59, 108, and 164 in the P. falciparum dhfr (pfdhfr) gene are strongly associated with pyrimethamine treatment failures (6). Field surveys and experimental studies suggest a stepwise process of accumulation of mutations. The single S108N mutation confers increased in vitro resistance to pyrimethamine (∼20-fold), and subsequent mutations at position 51 (N51I) or 59 (C59R) further increase it (11). Parasites with a triple-mutation allele (51I/59R/108N) have markedly reduced in vitro susceptibility to pyrimethamine, and the presence of the triple-mutation allele increases the risk of SP therapeutic failure. Finally the quadruple-mutation allele, which carries an additional mutation at position 164 (I164L), is highly resistant to pyrimethamine, abrogating the clinical efficacy of SP, as observed in Southeast Asia and South America (26).Recent progress in molecular population genetic studies has greatly facilitated our understanding of the emergence and geographical spread of drug-resistant lineages. In particular, it has been demonstrated that the emergence and dissemination of pyrimethamine-resistant parasites in Africa in the 1990s resulted from the migration of a few resistant mutants from Southeast Asia (29). Indeed, analysis of the microsatellite regions flanking the P. falciparum pfdhfr gene has clearly revealed that in Africa, the pfdhfr triple-mutation allele (I51/R59/N108) associated with pyrimethamine resistance harbored microsatellite haplotypes identical to those found in Southeast Asia (12, 13, 24, 29).In the context of the Indian Ocean, SP resistance has been widely reported in the Comoros Islands (23, 25, 28, 32), whereas SP is still effective in Madagascar (18). However, recent studies performed in Madagascar have shown that the situation is deteriorating and have demonstrated the introduction of P. falciparum multidrug-resistant parasites into Madagascar from the Comoros Islands (17), the rapid rise in the frequency of P. falciparum parasites with both pfdhfr and dhps mutations, and the alarming emergence of the single pfdhfr 164L allele from isolates collected during the last 3 years (2).In order to better understand the origin of the SP-resistant genotypes circulating in the region and determine the importance of gene flow in parasite populations with regard to SP resistance between Africa, the Comoros Islands, and Madagascar, we have characterized the pfdhfr genotype and flanking microsatellite haplotypes of a collection of P. falciparum samples from these areas. Our results confirm that pyrimethamine resistance in Madagascar is essentially related to the introduction from the Comoros Islands of the ancestral pfdhfr triple-mutation allele of Asian origin. Interestingly, however, the I164L single-mutation pfdhfr allele was observed in multiple lineages in areas restricted to the Southeast Madagascar, suggesting local pressure to generate this allele. The coexistence in the same transmission area of mutants with triple mutations and the single I164L mutation indicates that the local emergence of a mutant with quadruple mutations is a likely event that deserves reinforced surveillance.