The aim of this work was to define the cause of sulfadoxine/pyrimethamine (SP) treatment failure in Plasmodium falciparum infections in a malaria-endemic zone of India. Samples were collected from 176 patients in Kolkata from November 2008 to July 2009. In vitro susceptibility testing was performed on all isolates. Parasite DNA was extracted, and PCR and restriction fragment length polymorphism (RFLP) analysis of different codons of the dhfr gene (16, 51, 59, 108 and 164) and dhps gene (436, 437, 540, 581 and 613) were performed. Finally, sequencing of the products was performed to confirm the mutations. The in vivo treatment response to SP among the 176 patients was determined. A novel mutation of isoleucine was observed at codon 108 of the dhfr gene, which was highly correlated with in vitro SP resistance as well as early treatment failure. A double dhfr mutation (108I+51I) was observed in 77.3% of isolates, and triple mutation of the dhps gene was observed in 18.2% of isolates. In this endemic zone, SP treatment failure is due to a novel dhfr mutation (108I+51I) and any one of the dhps mutations (S436A, A437G, A581G or A613T/S). An increase in these mutations was highly correlated with SP resistance (P < 0.0001).
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