Clinical and molecular monitoring ofPlasmodium falciparumresistance to antimalarial drug (artesunate+sulphadoxine-pyrimethamine) in two highly malarious district of Madhya Pradesh, Central India from 2012–2014

Abstract

The spread of P. falciparum resistant strain has led to a significant resurgence of malaria morbidity and mortality. The current cornerstone in malaria treatment in India is Artemisinin based Combination (Artesunate + Sulphadoxine-Pyrimethamine) Therapy (ACT) for treatment of uncomplicated P. falciparum malaria since 2010. In the present study we assessed the therapeutic efficacy of ACT and molecular monitoring of antimalarial resistance. Therapeutic efficacy was determined by in vivo method using 28 days follow-up. Molecular genotyping of dihydrofolate reductase (dhfr), dihydropteroate synthase (dhps) and kelch13 genes were analyzed. msp-1 and msp-2 genotyping were used to differentiate recrudescence. Therapeutic efficacy of ACT was determined in 237 patients over the three year period. Most of the patients showed adequate clinical and parasitological response (99.6%). Molecular study revealed that 72% parasites were of mutant genotype (27.2% single mutants, 43.5% double mutants and 1.3% triple mutants) for pfdhfr while pfdhps showed 78.2% wild type alleles and 21.8% mutants (18.1% single mutants and 3.7% double mutants). Analysis of total 135 samples revealed mutation in k13 gene along with non-synonymous single mutation at codon M579T (1.5%) and double mutations at codon M579T & N657H in 37%. ACT remains effective for the treatment of uncomplicated P. falciparum malaria in Madhya Pradesh, Central India. However, increasing mutation in pfdhfr (particularly triple mutations) and pfdhps may reduce susceptibility to partner drug SP and mutation in k13 propeller gene, highlighting the need for continuous monitoring of the efficacy of ACT.

Stable high frequencies of sulfadoxine-pyrimethamine resistance associated mutations and absence of K13 mutations in Plasmodium falciparum 3 and 4 years after the introduction of artesunate plus sulfadoxine-pyrimethamine in Ujjain, Madhya Pradesh, India

“…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 [29]. The numbers are small but point in the same direction as our data possibly indicating that more resistant haplotypes are evolving in Madhya…”

“…In line with these data, triple mutation pfdhfr 59R108N + pfdhps 437G have been associated with SP treatment failure in India[31].Similar to findings in western Madhya Pradesh and Southwestern India but unlike findings inNorteastern 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 delayed parasite clearance when treated with artemisinin 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 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[31].Similar to findings in western Madhya Pradesh and Southwestern India but unlike findings inNorteastern 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].…”

Stable high frequencies of sulfadoxine-pyrimethamine resistance associated mutations and absence of K13 mutations in Plasmodium falciparum 3 and 4 years after the introduction of artesunate plus sulfadoxine-pyrimethamine in Ujjain, Madhya Pradesh, India

“…Similar to ndings in western Madhya Pradesh and Southwestern India but unlike ndings in Norteastern India and West Bengal there were no mutations in the kelch-13 propeller domain suggesting that parasites remained artemisinin susceptible at the study site [2,29,32,33]. ASP e cacy was 99.6% in the Madya Pradesh study and 84% in the West Bengal study [2,29].…”

“…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 [29]. The numbers are small but point in the same direction as our data possibly indicating that more resistant haplotypes are evolving in Madhya Pradesh.…”