The prevalence of molecular markers of resistance to sulfadoxine-pyrimethamine among pregnant women at first antenatal clinic attendance and delivery in the forest-savannah area of Ghana

Intermittent preventive treatment during pregnancy with sulfadoxine-pyrimethamine (IPTp-SP) is used to prevent malaria and associated unfavorable maternal and foetal outcomes in pregnancy in moderate to high malaria transmission areas. Effectiveness of IPTp-SP is, however, threatened by mutations in the Plasmodium falciparum dihydrofolate reductase (Pfdhfr) and dihydropteroate synthase (Pfdhps) genes which confer resistance to pyrimethamine and sulfadoxine, respectively. This study determined the prevalence of molecular markers of SP resistance among pregnant women in a high malaria transmission area in the forest-savannah area of Ghana. Genomic DNA was extracted from 286 P. falciparum-positive dried blood spots obtained from pregnant women aged ≥18 years (255 at first Antenatal Care (ANC) clinic visit and 31 at delivery from 2017 to 2019) using Chelex 100. Mutations in Pfdhfr and Pfdhps genes were detected using molecular inversion probes and next generation sequencing. In the Pfdhfr gene, single nucleotide polymorphisms (SNPs) were detected in 83.1% (157/189), 92.0% (173/188) and 91.0% (171/188) at codons 51, 59, and 108 respectively in samples collected at first ANC visit, while SNPs were detected in 96.6 (28/29), 96.6% (28/29) and 96.8% (30/31) in isolates collected at delivery. The Pfdhfr triple mutant N51I, C59R and S108N (IRN) was carried by 80.5% (128/159) and 96.5% (28/29) of the typed isolates collected at ANC visit and at delivery respectively. In the Pfdhps gene, SNPs were detected in 0.6% (1/174), 76.2% (138/181), 33.2% (60/181), 1.2% (2/174), 0% (0/183), and 16.6% (27/173) at codons 431, 436, 437, 540, 581 and 613 respectively in samples collected at ANC, and 0% (0/25), 72% (18/25), 40% (10/25), 3.6% (1/25), 0% (0/29) and 7.4% (2/27) in samples collected at delivery. Quadruple mutant Pfdhfr N51I, C59R, and S108N + Pfdhps A437G (IRN-GK) was present in 25.8% (33/128) and 34.8% (8/23) of isolates at ANC and at delivery respectively. Quintuple mutant alleles Pfdhfr N51I, C59R, and S108N + Pfdhps A437G and K540E (IRN-GE) were detected in 0.8% (1/128) and 4.4% (1/23) of samples collected at ANC and at delivery respectively. No mutations were identified at Pfdhfr codons 16 or 164 or Pfdhps 581. There is a high prevalence of Pfdhfr triple mutant P. falciparum infections among pregnant women in the study area. However, prevalence of the combined Pfdhfr/Pfdhps quadruple and quintuple mutants IRN-GK and IRN-GE respectively prior to commencement of IPTp-SP were low, and no Pfdhps A581G mutant was detected, indicating that SP is still likely to be efficacious for IPTp-SP in the forest-savannah area in the middle belt of Ghana.