AS + SP remained efficacious for the treatment of P. falciparum across diverse sites in central and eastern India. Molecular genotyping of dhfr and dhps genes indicate the dominance of isolates with dhfr double mutation and dhps wild type suggesting the ongoing development of antifolate resistance.
Despite the presence of mutations in dhfr and dhps gene, AS + SP treatment remained successful. This may be due to several reasons: first, the action of artemisinin in combination therapy, which kills parasites rapidly and reduces SP drug pressure on the parasite, preserving the latter’s efficacy; second, the rarity of dhfr-dhps quintuple mutations, which are virtually synonymous with SP treatment failure, in the population studied. However, it is likely that in the course of time such haplotypes may develop and spread in the face of sustained selection pressure; third, host immunity contributes to treatment efficacy and the study population was dominated by persons of tribal ethnicity living in highly endemic areas, who would have high immunity. The findings of this study were similar to those of Mishra et al. who reported AS + SP combination as a safe and effective treatment for uncomplicated falciparum malaria in India during 2009–2010 across 22 sites . This study contributes ‘baseline’ data on antifolate resistance as the bulk of samples were collected in 2007 prior to the widespread scale-up of AS + SP.
SP is extensively used, widely available, inexpensive, and slowly eliminated from the body. These factors have promoted the emergence and spread of SP-resistant parasites. Mutations in dhfr gene are related to pyrimethamine resistance and develop stepwise, starting with the mutation at codon S108N (Ser-Asn), followed by subsequent mutations at codon 59 (Cys-Arg), 51 (Asn-Ile) and 164 (Ile-Leu). In India, the most common dhfr mutation is S108N, followed by C59R, and N51I [17, 19, 20]. The results of this study showed a similar pattern with the exception of an absence of mutation in codon N51I. It was observed the I164L mutation associated with high levels of pyrimethamine resistance . Previous studies in Assam, in north-east India and Odisha in eastern India, documented the dhfr triple mutant Asn108 + Ile51 + Arg59 associated with pyrimethamine resistance . These combinations of mutations were not detected in the present study sites which are in nearby states. The dhfr double mutations (S108N + C59R) were the most prevalent here similar to other reported results . This study did not detect any quadruple mutations in dhfr such as those reported from the Andaman and Nicobar Islands by Ahmed  and Das et al.. The success rate of dhps gene amplification was low compared to amplification of the dhfr gene; however, isolates where it was successfully amplified were wild type. Other authors have also reported the dominance of wild type dhps genotype in India [14,17,22,]. The prevalence of dhps single mutation (K540E or A581G) and double mutations (540 + 580) were low and triple mutant dhps alleles were not detected though reports exist, again, from the Andaman and Nicobar Islands .
Regional variations of dhfr-dhps haplotypes were extensive, with some haplotypes common among Jharkhand, Odisha and West Garo Hills, while others were site specific. The highest number of haplotypes was present in Odisha, which contributes the highest proportion of the reported malaria burden in the country. Thus, high transmission generates higher diversity. In low-transmission areas in contrast, self-recombination leads to greater fixation of haplotypes. Ahmed et al. described similar regional differences where more diverse genotypes were seen in the higher transmission states of Assam and Odisha. In addition to transmission, drug pressure varies across states. In low-transmission settings a greater proportion of infections are likely to be symptomatic, and since these areas also tend to be better developed, more cases will have access to care leading to high selection pressure. Anderson et al. suggest resistance to SP appears to arise de novo much less frequently but spreads rapidly since gametocyte production is stimulated by this drug. High-level pyrimethamine-resistant alleles have emerged in independent foci from which they have rapidly spread to the nearby areas. In contrast, Vinayak et al. found single origin for dhfr-resistant alleles in the Thai-Cambodia region, while dhps-resistant alleles had multiple origins. Lumb et al. reported several unique haplotypes among mutant dhps alleles and concluded that the dhps alleles in India have evolved from multiple genetic backgrounds.
Delayed parasite clearance time is the most important signal for artemisinin resistance . The parasite clearance was rapid at all sites in this study. This rapid clearance of parasitaemia is suggestive of continued artemisinin effectiveness in India though better inference could be made with precise data on parasite clearance rates. No statistically significant association between parasite clearance time and increased mutations in dhfr and dhps was observed, which was not surprising if the artemisinin component drives clearance. Since the treatment efficacy was high, it was not possible to correlate molecular results with subsequent treatment failure.
The present study had several limitations. Foremost was a follow-up of only 28 days. As the partner drug (SP) has a long half-life, extended follow-up for 42 days could have detected potential late treatment failures emerging with the elimination of residual drug levels. Second, only 25 patents were enrolled in West Garo Hills because enough eligible patients were not found at the recruitment site during the transmission season. Third, the lack of treatment failures restricted the ability to determine the relationship between molecular markers and AS + SP failure. Fourth, the parasite clearance time (PCT) was determined based on a daily blood smear. Finally, while three sites provided some geographic coverage, the results may not be generalizable across India given the size of the country and diversity of malaria ecotypes. The diversity of findings in dhfr and dhps mutations alone suggests the need for routine monitoring especially in locations with a high prevalence of markers associated with anti-malarial drug resistance. One potential solution, which would minimize the need for expensive in vivo trials, is the targeting of such studies using molecular surveillance .