Plasmodium falciparum isolates in India exhibit a progressive increase in mutations associated with sulfadoxine-pyrimethamine resistance

Abstract

The combination of sulfadoxine-pyrimethamine (SP) is used as a second line of therapy for the treatment of uncomplicated chloroquine-resistant Plasmodium falciparum malaria. Resistance to SP arises due to certain point mutations in the genes for the dihydrofolate reductase (DHFR) and dihydropteroate synthetase (DHPS) enzymes of the parasite. We have analyzed these mutations in 312 field isolates of P. falciparum collected from different parts of India to assess the effects of drug pressure. The rate of mutation in the gene for DHFR was found to be higher than that in the gene for DHPS, although the latter had mutations in more alleles. There was a temporal rise in the number of isolates with double dhfr mutations and single dhps mutations, resulting in an increased total number of mutations in the loci for DHFR and DHPS combined over a 5-year period. During these 5 years, the number of isolates with drug-sensitive genotypes decreased and the number of isolates with drug-resistant genotypes (double DHFR mutations and a single DHPS mutation) increased significantly. The number of isolates with the triple mutations in each of the genes for the two enzymes (for a total of six mutations), however, remained very low, coinciding with the very low rate of SP treatment failure in the country. There was a regional bias in the mutation rate, as isolates from the northeastern region (the state of Assam) showed higher rates of mutation and more complex genotypes than isolates from the other regions. It was concluded that even though SP is prescribed as a second line of treatment in India, the mutations associated with SP resistance continue to be progressively increasing.

FIG. 1.

Mutations in the loci for DHFR (A) and DHPS (B) of P. falciparum isolates from India. n, number of isolates analyzed. The amino acid sequence is shown at the top of each lane, where mutated amino acids are shown in boldface. The mutated codons are shaded.

FIG. 2.

Mutation rates in the P. falciparum DHFR enzyme sequence among two groups of isolates (groups A and B). A comparison of the rates of mutation in individual codons between the two groups is shown in the inset. The number of samples in each group is shown in parentheses. The chi-square test with Yates' correction was used to compare values between two groups (*, P < 0.05; **, not significant; ***, not applicable).

FIG. 3.

Mutation rates in the P. falciparum DHPS enzyme sequence among two groups of isolates. A comparison of the rates of mutation in individual codons between the two groups is shown in the inset. The asterisks are explained in the legend to Fig. 2.

FIG. 4.

Regional distributions of P. falciparum DHFR genotypes among Indian isolates. Mutated amino acids are shown in boldface. n, number of isolates analyzed.

FIG. 5.

Group and regional (inset) distributions of the total numbers of mutations in the DHFR and DHPS loci. n, number of isolates.

FIG. 6.

Expected SP sensitivities among isolates from two groups of patients and their regional distributions (inset). The expected clinical sensitivity was derived from the DHFR and DHPS genotypes combined, as described in the text.