Antifolate resistance markers and complexity of infection in children with sickle cell anaemia receiving malaria chemoprevention in Uganda: a molecular sub-study of the CHEMCHA randomised trial

Background Sickle cell anaemia (SCA) is a major public health problem in Uganda. Monthly sulfadoxine-pyrimethamine (SP) remains the standard malaria chemoprevention regimen despite widespread antifolate resistance in East Africa. We assessed Pfdhfr and Pfdhps resistance mutations and the complexity of infection (COI) among children with SCA receiving malaria chemoprevention. Methods This prospective study of antifolate resistance was nested within the CHEMCHA randomised controlled trial comparing monthly SP with weekly dihydroartemisinin-piperaquine (DP) for malaria chemoprevention. Participants were children with SCA aged 6 months to 15 years and attending Jinja and Kitgum hospitals in Uganda. Dried blood spots were collected at enrolment, study closeout, and during febrile episodes. Parasite DNA from microscopy-confirmed clinical malaria samples was genotyped by amplicon sequencing for Pfdhfr (N51I, C59R, S108N, and I164L) and Pfdhps (A437G, K540E, A581G, A613S, and A613T). COI was estimated using MOIRE. Results Complete Pfdhfr/Pfdhps haplotypes were resolved for 7-28 isolates per site and time point, and COI was estimated for 67 samples. The quintuple mutant (Pfdhfr IRN/Pfdhps GE) accounted for 85.7-100% of isolates across sites and time points; A613S and A613T were not detected. Pfdhfr I164L was detected in 2/9 isolates in Jinja at baseline and 1/8 at endline. The Pfdhps A581G mutant allele was absent at baseline (0/44 isolates) but was found in 1/8 isolates in Jinja and 3/34 in Kitgum at endline, including quintuple-plus-581G (sextuple) haplotypes in 1/8 and 2/27 isolates, respectively. Most infections were monoclonal (51/67, 76.1%). Conclusions The high prevalence of quintuple antifolate-resistant haplotypes and the detection of higher-level resistance markers, including emerging sextuple mutants carrying Pfdhfr I164L and Pfdhps A581G, call into question the continued use of SP for chemoprevention in children with SCA in Uganda and support evaluation of alternative regimens.