AUTHOR=Adegbola Adebanjo Jonathan , Ndwiga Leonard , Wamae Kevin , Osoti Victor , Bolaji Oluseye Oladotun , Bejon Philip , Ochola-Oyier Lynette Isabella 

TITLE=ONT sequencing identifies a high prevalence of crt sensitive, triple mutant dhfr and single mutant dhps parasites within an ANC population in Nigeria

JOURNAL=Frontiers in Genetics

VOLUME=15

YEAR=2024

URL=https://www.frontiersin.org/journals/genetics/articles/10.3389/fgene.2024.1470156

DOI=10.3389/fgene.2024.1470156

ISSN=1664-8021

ABSTRACT=<sec><title>Background</title><p>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 <italic>Plasmodium falciparum</italic> 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 <italic>P. falciparum</italic>, 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.</p></sec><sec><title>Methods</title><p>Dried blood spots (DBS) were collected from pregnant women who received IPTp-SP at the enrollment and follow-up visits. <italic>P. falciparum</italic> genomic DNA was extracted by the Chelex<sup>®</sup> method and <italic>Pf</italic>18S qPCR was used to detect parasite DNA in each sample. With nested PCR assays, fragments of <italic>Pfdhps</italic>, <italic>Pfdhfr</italic>, <italic>Pfmdr1</italic>, <italic>Pfcrt</italic>, <italic>Pfk13</italic> and <italic>Pfama1</italic> genes were amplified and multiplexed amplicon-based sequencing was conducted on the minION Oxford Nanopore Technology.</p></sec><sec><title>Result</title><p>In total, 251 pregnant women were enrolled in the study and 457 DBS samples were collected. <italic>P. falciparum</italic> genomic DNA was detected in 12% (56/457) of the samples, 31 at baseline and the remaining during the follow-up visits. <italic>Pfama1</italic>, <italic>pfk13</italic>, <italic>Pfdhps</italic>, <italic>Pfdhfr</italic>, <italic>Pfmdr1 and Pfcrt</italic> were successfully sequenced in a single run. Notably, k13 artemisinin resistance mutations were absent, the frequencies of <italic>Pfdhfr</italic> and <italic>Pfdhps</italic> SP resistance haplotypes, <bold>IRN</bold> for pyrimethamine resistance and I<bold>SG</bold>KA/IA<bold>G</bold>KA associated with sulphadoxine resistance were 82% (36/44) and 64% (27/42), respectively, and the <italic>Pfcrt</italic> CV<bold>IET</bold> resistant haplotype was at approximately 22% (7/32).</p></sec><sec><title>Conclusion and recommendations</title><p>Here a multiplexed amplicon-based ONT assay established that triple mutant <italic>Pfdfhr</italic>-IRN, double mutant <italic>Pfdhps</italic>-SG haplotypes and the chloroquine sensitive strain were prevalent among pregnant women in Nigeria.</p></sec>