Stable high frequencies of sulfadoxine-pyrimethamine resistance associated mutations and absence of K13 mutations inPlasmodium falciparum3 and 4 years after the introduction of artesunate plus sulfadoxine-pyrimethamine in Ujjain, Madhya Pradesh, India

Background Artesunate plus sulfadoxine-pyrimethamine (ASP) is first-line treatment for uncomplicatedPlasmodium falciparummalaria in most of India, except for six North-eastern provinces where treatment failure rates were high. In Ujjain, central India, the frequency of mutations associated with increased drug tolerance, but not overt resistance to sulfadoxine and pyrimethamine were 9% and > 80%, respectively, in 2009 and 2010, just prior to the introduction of ASP. The frequency of drug resistance associated mutations in Ujjain in 2015-2016 after 3-4 years of ASP use, are reported. Methods Blood samples from patients withP. falciparummono-infection verified by microscopy were collected on filter-paper at all nine major pathology laboratories in Ujjain city. Codonspfdhfr16-185,pfdhps436-632 and K13 407-689 were identified by sequencing.PfcrtK76T andpfmdr1N86Y were identified by restriction fragment length polymorphism. Results Sulfadoxine-pyrimethamine resistance-associatedpfdhfr108 N and 59R alleles were found in 100/104 (96%) and 87/91 (96%) samples, respectively.Pfdhps437G was found in 10/105 (10%) samples. Double mutantpfdhfr59R + 108 N were found in 75/81 (93%) samples. Triple mutantpfdhfr59R + 108 N andpfdhps437G were found in 6/78 (8%) samples. Chloroquine-resistance-associatedpfcrt76T was found in 102/102 (100%).Pfmdr1N86 and 86Y were identified in 83/115 (72%) and 32/115 (28%) samples, respectively. Conclusion The frequency ofP. falciparumwith reduced susceptibility to sulfadoxine-pyrimethamine remained high, but did not appear to have increased significantly since the introduction of ASP. No polymorphisms in K13 associated with decreased artemisinin susceptibility were found. ASP probably remained effective, supporting continued ASP use.