ONT sequencing identifies a high prevalence of sensitive, triple mutant and single mutant parasites within an ANC population in Nigeria.

Background: Malaria in pregnancy is a major public health issue, particularly among vulnerable populations in malaria-endemic sub-Saharan African countries. To mitigate its risks, WHO recommends sulphadoxine-pyrimethamine (SP) for chemoprevention and artemisinin-based combination therapy (ACT) to treat uncomplicated malaria. These interventions have helped to alleviate the risk associated with malaria in pregnancy; however, in the context of the emergence of SP- and ACT-resistant , maintained efficacy is under threat. Molecular surveillance is a reliable tool to monitor the emergence of resistance where molecular markers are known. Thus, the objective of the study was to use a multiplexed amplicon Oxford Nanopore sequencing approach to assess the molecular markers for antimalarial resistance among pregnant women in Nigeria.

Methods: Dried blood spots (DBS) were collected from pregnant women who received IPTp-SP at the enrollment and follow-up visits. genomic DNA was extracted by the Chelex method and 18S qPCR was used to detect parasite DNA in each sample. With nested PCR assays, fragments of , , , , and genes were amplified and multiplexed amplicon-based sequencing was conducted on the minION Oxford Nanopore Technology.

Result: In total, 251 pregnant women were enrolled in the study and 457 DBS samples were collected. genomic DNA was detected in 12% (56/457) of the samples, 31 at baseline and the remaining during the follow-up visits. , , , , were successfully sequenced in a single run. Notably, k13 artemisinin resistance mutations were absent, the frequencies of and SP resistance haplotypes, for pyrimethamine resistance and IKA/IAKA associated with sulphadoxine resistance were 82% (36/44) and 64% (27/42), respectively, and the CV resistant haplotype was at approximately 22% (7/32).

Conclusion And Recommendations: Here a multiplexed amplicon-based ONT assay established that triple mutant -IRN, double mutant -SG haplotypes and the chloroquine sensitive strain were prevalent among pregnant women in Nigeria.