Saturation of Pfdhfr and Pfdhps haplotypes in general population increasingly threatens the benefits of using sulfadoxine‑pyrimethamine for intermittent preventive treatment in pregnant women in Tanzania

Abstract Background : High levels of Plasmodium falciparum resistance prompted withdrawal of sulfadoxine-pyrimethamine (SP) as the first-line treatment for uncomplicated malaria in Tanzania. However, SP was limited for intermittent preventive treatment during pregnancy (IPTp) especially where there is moderate to high malaria transmission. This study reports the patterns of P. falciparum dihydrofolate reductase ( Pfdhfr ) and dihydropteroate synthetase ( Pfdhps ) mutations. Methods: Parasite genomic DNA was extracted from dried blood spots prepared by finger prick. Batched samples (384) were sequenced in a single MiSeq lane combining all PCR products. Samples were de-plexed using the multiplexing adapters and individual CRAM files were aligned to a modified amplicon reference genome. Genotyping of Pfdhfr and Pfdhps mutations were done using bcftools as well as custom scripts to filter and translate genotypes into drug resistance haplotypes. Results: The Pfdhfr was analyzed from 445 samples, the wild type (WT) Pfdhfr haplotype NCSI was detected in only six (1.3%) samples. Triple Pfdhfr IRN I haplotype was dominant, contributing to 84% (n=374) of haplotypes. The total of 446 samples were studied for Pfdhps . WT for Pfdhps was found in 6.7% (n=30) of all samples detected. Double Pfdhps haplotype (S GE AA) accounted for 83% of mutations of the Pfdhps gene. The overall prevalence of K540E was 90.4% (n=396) while A581G was 1.1% (n=5). Additionally, 91.4% (n=447) genotypes where detected from 489 sequenced samples. Of 447 genotypes detected only 0.9% (n=4) of samples were WT (SAKAA-NCSI). Quintuple mutation, S GE AA- IRN I accounted 71.4% of concomitant Pfdhfr/Pfdhps mutations where 0.2% (n=1) had septuple mutation, AG K GS - IRN I. Conclusions : Despite the high prevalence of mutations in Pfdhfr and Pfdhps gene but the current mutations at Pfdhfr K540E and Pfdhps A581G are within the recommended WHO range, stopping IPTp-SP is recommended in areas where the Pfdhfr K540E prevalence is >95 % and Pfdhps A581G is >10 % as SP is likely to be ineffective). Nevertheless, saturation in Pfdhfr and Pfdhps haplotypes is alarming, therefore screening for alternative antimalarial drug for IPTp-SP is recommended.