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Background  In Zambia the first-line treatment for uncomplicated malaria is artemisinin combination therapy (ACT), with artemether-lumefantrine currently being used. However, the antifolate regimen, sulphadoxine-pyrimethamine (SP), remains the treatment of choice in children weighing less than 5 kg and also in expectant mothers. SP is also the choice drug for intermittent preventive therapy in pregnancy and serves as stand-by treatment during ACT stock outs. The current study assessed the status of  Plasmodium falciparum  point mutations associated with antifolate drug resistance in the area around Macha.    Methods  A representative sample of 2,780 residents from the vicinity of Macha was screened for malaria by microscopy. At the same time, blood was collected onto filter paper and dried for subsequent  P. falciparum  DNA analysis. From 188 (6.8%) individuals that were thick film-positive, a simple random sub-set of 95  P. falciparum  infections were genotyped for DHFR and DHPS antifolate resistance mutations, using nested PCR and allele-specific restriction enzyme digestion.    Results   Plasmodium falciparum  field samples exhibited a high prevalence of antifolate resistance mutations, including the DHFR triple (Asn-108 + Arg-59 + Ile-51) mutant (41.3%) and DHPS double (Gly-437 + Glu-540) mutant (16%). The quintuple (DHFR triple + DHPS double) mutant was found in 4 (6.5%) of the samples. Levels of mutated parasites showed a dramatic escalation, relative to previous surveys since 1988. However, neither of the Val-16 and Thr-108 mutations, which jointly confer resistance to cycloguanil, was detectable among the human infections. The Leu-164 mutation, associated with high grade resistance to both pyrimethamine and cycloguanil, as a multiple mutant with Asn-108, Arg-59 and (or) Ile-51, was also absent.    Conclusion  This study points to escalating levels of  P. falciparum  antifolate resistance in the vicinity of Macha. Continued monitoring is recommended to ensure timely policy revisions before widespread resistance exacts a serious public health toll.

Escalating Plasmodium falciparum antifolate drug resistance mutations in Macha, rural Zambia