Adegbola Adebanjo Jonathan, Ndwiga Leonard, Wamae Kevin, Osoti Victor, Bolaji Oluseye Oladotun, Bejon Philip, Ochola-Oyier Lynette Isabella
Faculty of Pharmacy, Obafemi Awolowo University, Ile-Ife, Nigeria.
Kenya Medical Research Institute (KEMRI)-Wellcome Trust Research Programme (KWTRP), Kilifi, Kenya.
Front Genet. 2024 Oct 15;15:1470156. doi: 10.3389/fgene.2024.1470156. eCollection 2024.
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.
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.
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).
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.
妊娠疟疾是一个重大的公共卫生问题，在撒哈拉以南非洲疟疾流行国家的弱势群体中尤为突出。为降低其风险，世界卫生组织推荐使用周效磺胺-乙胺嘧啶（SP）进行化学预防，并使用青蒿素联合疗法（ACT）治疗非复杂性疟疾。这些干预措施有助于减轻与妊娠疟疾相关的风险；然而，在出现对SP和ACT耐药的情况下，维持疗效受到威胁。分子监测是在已知分子标记的情况下监测耐药性出现的可靠工具。因此，本研究的目的是使用多重扩增子牛津纳米孔测序方法评估尼日利亚孕妇中抗疟药物耐药性的分子标记。
在入组和随访时从接受间歇性预防治疗-周效磺胺-乙胺嘧啶（IPTp-SP）的孕妇中收集干血斑（DBS）。采用Chelex法提取基因组DNA，并使用18S qPCR检测每个样本中的寄生虫DNA。通过巢式PCR检测，扩增出 、 、 、 、 和 基因的片段，并在MinION牛津纳米孔技术上进行基于多重扩增子的测序。
本研究共纳入251名孕妇，收集了457份DBS样本。12%（56/457）的样本检测到基因组DNA，31份在基线时检测到，其余在随访期间检测到。 、 、 、 、 在一次运行中成功测序。值得注意的是，未发现k13青蒿素耐药突变，SP耐药单倍型 和 的频率分别为82%（36/44）和64%（27/42），与周效磺胺耐药相关的乙胺嘧啶耐药单倍型IKA/IAKA的频率约为22%（7/32）。
本研究通过基于多重扩增子的ONT检测方法确定，三重突变-IRN、双重突变-SG单倍型和氯喹敏感株在尼日利亚孕妇中普遍存在。
