This is the second characterization of key anti-malarial drug resistance associated genetic polymorphisms in P. falciparum field isolates in Ujjain, Madhya Pradesh, Central India. Samples were collected 3 and 4 years after the implementation of ASP as first line antimalarial in Ujjain (2012) and 5 years after the first base-line study was conducted. From these data, the frequency of in vivo chloroquine and SP resistance in the study area and an indication of the speed at which resistant genotypes accumulate can be inferred.
The most encouraging finding was that the frequency of the previously identified key SP resistance associated alleles had not increased significantly despite the presumed increased SP drug pressure. In the previous study, 90% (70/78) and 96% (75/78) of samples had pfdhfr 59R and 108N mutations, respectively, compared to 96% in this study. Similarly, 100% (76/76) and 9% (7/77) of samples had the pfdhps 436F and 437G mutations, respectively in the previous study compared to 100% and 10% in the current study. Furthermore, the triple mutant pfdhfr 59R108N plus pfdhps 437G haplotype was also stable at a frequency of 10% in the current study compared to 8% (6/76) prior to introduction of ASP. As in the previous study no alleles at pfdhfr codons 51 or 164 nor at pfdhps codons 540, 581 or 613 that are associated with high levels of SP resistance were found. The results suggests that mutations associated with SP resistance have not accumulated rapidly since the introduction of ASP 3 to 4 years previously.
However, the majority of samples 68/83 had a double mutation (pfdhfr 59R + 108N), triple mutations were found in 12% of samples and unlike the previous study a quadruple mutation (pfdhfr 50R59R108N and pfdhps 437G) was found. Moreover, the triple pfdhfr 50R59R108N, that was not seen prior to the introduction of ASP was seen in 5% of samples (4/87) possibly indicating that this haplotype is beginning to accumulate. In a more western part of Madhya Pradesh state, the pfdhfr 108N, 59R and 51I frequencies were 80%, 57% and 32%, respectively and the frequency of triple pfdhfr mutations were 0%, 2%, and 3% in 2012, 2013 and 2014. The frequency of pfdhps double mutations were 0, 3 and 8.5% the same respective years . The numbers are small but point in the same direction as our data possibly indicating that more resistant haplotypes are evolving in Madhya Pradesh. However, the risk of importing highly SP resistant P. falciparum is perhaps greater than the risk of local evolution as the frequency of pfdhfr + pfdhps with 6 or 7 mutations correlating to highly SP resistant parasites were 38% (85/226) in a study conducted in 2014–2016 only slightly further west, in West Bengal .
In the same West Bengal study various pfdhfr and pfdhps genotypes were tested for in vitro susceptibility . Approximately half the parasites with pfdhfr 59R108N had IC50 values suggestive of pyrimethamine resistance. Samples with pfdhps 437G or 436A had sulfadoxine IC50 values ranging up to resistance level. Half the samples with 436A437G had IC50 values suggesting resistance. The effect of the pfdhps 436F mutation only on IC50 values was not assessed but has previously been shown to modulate sulfadoxine susceptibility . The 82% frequency of pfdhfr 59R108N plus pfdhps 436F and the 11% frequency pfdhfr 59R108N plus pfdhps 436F437G thus suggests that the parasites are at least SP tolerant verging on resistant at our site. In line with these data, triple mutation pfdhfr 59R108N + pfdhps 437G have been associated with SP treatment failure in India .
Similar to findings in western Madhya Pradesh and Southwestern India but unlike findings in Norteastern India and West Bengal there were no mutations in the Kelch-13 propeller domain suggesting that parasites remained artemisinin susceptible at our site [2, 29, 32, 33]. ASP efficacy was 99.6% in the Madya Pradesh study and 84% in the West Bengal study [2, 29]. The lack of mutations linked to artemisinin resistance or high degree of SP resistance suggests that the ASP efficacy at our site should be closer to the 99% found in Madhya Pradesh. The results thus supports the continued use of ASP at the study site assuming continued direct or indirect monitoring of ASP efficacy.
In this study and our previous study we found that pfdhps 436F was fixed . As discussed previously its existence prior to the introduction of ASP suggests that it is a wild type allele in our study area. Interestingly it was not noted further east in Madhya Pradesh but even further east in Orissa, the pfdhps F436A437K540A581A613 haplotype was found in 52% of samples . Furthermore, the F436A437K540A581A613 and F436G437K540A581A613 haplotypes were found in 44% and 21% of samples at the same time as 33% carried the S436A437K540A581A613 haplotypes in study from kolkata . The 436F allele is thus not uncommon in India and its frequency varies.
The evolution of SP resistance in Asia has been shown to be ordered . It appears to start with two initial mutations in pfdhfr (108N and then 59R), followed by two in pfdhps 437G and then either 540E or 581G. A third mutation then accumulates in each of pfdhfr and pfdhps . In Ujjain, pfdhfr 50R appears to be developing in P. falciparum that already have pfdhfr 108N59R and pfdhps 436F concurrently with development of the pfdhfr (108N and then 59R) pfdhps 436F437G haplotype at our site. The numbers are small and no conclusions should be made. However, the pfdhps 436F allele has been shown to modulate sulfadoxine susceptibility  and was not detected in the study assessing the ordered evolution of SP resistance causing SNPs . Its potential effect in the ordered accumulation of SNPs may thus be significant thought this has not been shown.
The 100% frequency of pfcrt 76T is significantly higher compared to the 96% (80/84) frequency prior to introduction of ASP (fishers exact p = 0.04). Similarly, though not significantly the pfmdr1 86Y frequency increased from 16% (13/83) prior to ASP introduction to 28% in the current study. This clearly indicates that chloroquine is not a viable treatment option. The pfcrt 76T and pfmdr1 86Y alleles are linked to lower lumefantrine IC 50 values suggesting that artemether-lumefantrine is a good second line treatment in the case of ASP failure.
Finally, the poor PCR outcome in DNA extracted from RDTs is in line with other studies indicating the difficulty of extracting sufficient DNA . Optimally blood for this type of survey should therefore be collected by other means so as not to introduce potential bias such as not being able to analyse samples with low parasite density.