Occurrence of septuple and elevated Pfdhfr-Pfdhps quintuple mutations in a general population threatens the use of sulfadoxine-pyrimethamine for malaria prevention during pregnancy in eastern-coast of Tanzania

Abstract

Background: Plasmodium falciparum dihydrofolate reductase (Pfdhfr) and dihydropteroate synthetase (Pfdhps) mutations compromise the effectiveness of sulfadoxine-pyrimethamine (SP) for treatment of uncomplicated malaria, and are likely to impair the efficiency of intermittent preventive treatment during pregnancy (IPTp). This study was conducted to determine the level of Pfdhfr-Pfdhps mutations, a decade since SP was limited for IPTp use in pregnant women in Tanzania. Methods: P. falciparum genomic DNA was extracted from dried blood spots prepared from a finger prick. Extracted DNA were sequenced using a single MiSeq lane by combining all PCR products. Genotyping of Pfdhfr and Pfdhps mutations were done using bcftools whereas custom scripts were used to filter and translate genotypes into SP resistance haplotypes. Results: The Pfdhfr was analyzed from 445 samples, the wild type (WT) Pfdhfr haplotype NCSI was detected in 6 (1.3%) samples. Triple Pfdhfr IRNI (mutations are bolded and underlined) haplotype was dominant, contributing to 84% (number [n] = 374) of haplotypes while 446 samples were studied for Pfdhps, WT for Pfdhps (SAKAA) was found in 6.7% (n = 30) in samples. Double Pfdhps haplotype (SGEAA) accounted for 83% of all mutations at Pfdhps gene. Of 447 Pfdhfr-Pfdhps combined genotypes, only 0.9% (n = 4) samples contained WT gene (SAKAA-NCSI). Quintuple (five) mutations, SGEAA-IRNI accounted for 71.4% (n = 319) whereas 0.2% (n = 1) had septuple (seven) mutations (AGKGS-IRNI). The overall prevalence of Pfdhfr K540E was 90.4% (n = 396) while Pfdhps A581G was 1.1% (n = 5). Conclusions: This study found high prevalence of Pfdhfr-Pfdhps quintuple and presence of septuple mutations. Mutations at Pfdhfr K540E and Pfdhps A581G, major predictors for IPTp-SP failure were within the recommended WHO range. Abandonment of IPTp-SP is recommended in settings where the Pfdhfr K540E prevalence is > 95% and Pfdhps A581G is > 10% as SP is likely to be not effective. Nonetheless, saturation in Pfdhfr and Pfdhps haplotypes is alarming, a search for alternative antimalarial drug for IPTp in the study area is recommended.