Mbituyumuremyi, A. | bioRxiv Science

bioRxiv2026

Prevalence of chloroquine and antifolate drug resistance markers in Plasmodium falciparum parasites in Rwanda, 2018

BackgroundAntimalarial drug efficacy is threatened by the development of drug resistance. In Rwanda, high levels of resistance have been reported in the past, including mutations in the Pfk13 gene associated with partial resistance to artemisinin, leading to delayed parasite clearance of Artemether-Lumefantrine (AL). Therefore, continuous molecular surveillance of drug resistance markers is required. Here, we report polymorphisms in the Pfcrt, Pfdhps, and Pfdhfr genes in samples collected in Rwanda in 2018. MethodsSamples from three sites (Masaka, Rukara, and Bugarama) were collected during a therapeutic efficacy study (TES) conducted in 2018. Targeted amplification of resistance genes of interest was performed by PCR, and products were sequenced by Sanger sequencing. ResultsThe K76T Pfcrt mutation was found in 10% of the samples, only in Bugarama. For Pfdhps, the A437G, K540E and A581G mutations were detected in 94%, 94% and 55% of the samples, respectively. For Pfdhfr, the N51I, S198N, C59R and I164L mutations were found in 99%, 99%, 88% and 16% of samples, respectively. The latter was only detected in Masaka and Rukara. ConclusionsWe confirm the slow and partial recovery of chloroquine susceptibility in the country. All previously reported Pfk13 mutants were Pfcrt wild type, thus possibly selected by AL. The prevalence of Pfdhps and Pfdhfr mutants, including those with the highly resistant I164L mutation, is high and increasing, despite the absence of drug pressure. It is imperative to closely monitor resistance and efficacy of AL and other treatment options as part of the malaria surveillance program and resistance mitigation strategy in the country.

genomics↗

bioRxiv2025

Assessment of different techniques and markers to distinguish recrudescence from new infection in an antimalarial therapeutic efficacy study conducted in Rwanda

IntroductionAccurate estimation of antimalarial drug efficacy against P. falciparum requires PCR correction to distinguish recrudescence from new infection in recurrent infections. Genotyping length polymorphic or SNP-rich markers, and various decision algorithms based on match counting or probabilistic approaches may be used for this purpose. In this study, we compared several markers and decision algorithms using samples collected in a therapeutic efficacy study conducted in Rwanda to identify the most suitable and robust approaches for PCR correction. MethodsWe optimized nested PCR assays to genotype four microsatellites and assessed their sensitivity in detecting minority clones with laboratory parasite strain mixtures. We analyzed patient samples by capillary electrophoresis and amplicon deep sequencing and assessed the diversity and allelic frequency of msp1, msp2, glurp, microsatellites (Poly-, PfPK2, TA40, TA81), and SNP-rich markers (ama1-D2, ama1-D3, cpmp, cpp, csp). We then classified the samples into recrudescence or new infection based on different marker combinations (msp1/msp2/glurp, msp1/msp2/microsatellite, and SNP-rich markers) and three decision algorithms (WHO algorithm, 2/3 algorithm, and a Bayesian approach), and compared our results with previously published findings. ResultsAmong microsatellites, TA40 and PfPK2 had the highest sensitivity in detecting minority clones; however, their intra-assay reproducibility was found to be limited. The 3D7 allelic family of the msp2 gene, glurp and SNP-rich markers had the highest genetic diversity. In distinguishing recrudescence from new infection, the WHO algorithm identified the fewest recrudescences across all marker combinations. Conversely, the 2/3 algorithm identified the highest number of recrudescences, and the algorithm based on Bayesian statistics yielded intermediate results. We observed the least variability in classification results among the different algorithms for the SNP-rich markers ama1-D2/ama1-D3/cpmp combination. In our study, replacing glurp with one of the four analyzed microsatellites or using SNP-rich markers did not significantly alter the final number of recrudescent infections. InterpretationOur findings emphasize the importance of using assays with high sensitivity in detecting minority clones, and markers with high diversity and low allelic frequency for accurate PCR correction. The SNP-rich markers method yielded the most consistent results in detecting recrudescences, regardless of the decision algorithm used. Therefore, it holds great potential for performing reliable PCR correction. Further evaluation of decision algorithms based on probabilistic approaches, compared to match-counting methods, is essential to ensure the accuracy and consistency of PCR-corrected drug efficacy estimations.

molecular biology↗