Post Graduate Institute of Medical Education and Research, Chandigarh, 160012, India.
Aarupadai Veedu Medical College and Hospital (AVMC&H), VMRF-DU, Puducherry, 607402, India.
Sci Rep. 2024 Oct 27;14(1):25608. doi: 10.1038/s41598-024-76479-7.
Chloroquine is still used as a first-line treatment for uncomplicated Plasmodium vivax malaria in India and resistance to this therapy can act as a major hurdle for malaria elimination. It is difficult to monitor drug-efficacy and drug resistance through in vivo and in vitro studies in case of Plasmodium vivax so analysis of molecular markers serves as an important tool to track resistance. Molecular methods that are currently in use for detecting single nucleotide polymorphisms in resistant genes including Polymerase chain reaction (PCR), Realtime-Polymerase chain reaction require highly sophisticated labs and are time consuming. So, with this background the study has been designed to optimize Loop Mediated Isothermal Amplification Assay to detect single nucleotide polymorphisms in chloroquine resistance gene of Plasmodium vivax in field settings. Eighty-eight Plasmodium vivax positive samples were collected. Pvmdr1 gene was amplified for all the samples and sequenced. Obtained sequences were analyzed for the presence of single nucleotide polymorphisms in the target gene. Further Loop Mediated Isothermal Amplification Assay primer sets were designed for the target mutants and the assay was optimized. Clinical as well as analytical sensitivity and specificity for the assay was calculated. Double mutants with variations at T958M and F1076L were detected in 100% of the Plasmodium vivax clinical isolates with haplotype M958 Y976 Y1028 L1076. Designed primers for Loop Mediated Isothermal Amplification Assay successfully detected both the mutants (T958M and F1076L) in 100% of the isolates and do not show cross-reactivity with other strains. So, the assay was 100% sensitive and specific for detecting single nucleotide polymorphisms in the target Pvmdr1 gene. Limit of detection was found to be 0.9 copies/µl and lowest DNA template concentration detected by designed assay was 1.5 ng/µL. Observed prevalence of single nucleotide polymorphisms in Pvmdr 1 gene is indicating a beginning of trend towards chloroquine resistance in Plasmodium vivax. The present study optimized LAMP for detecting single nucleotide polymorphisms in Plasmodium vivax cases in field settings, thus would help in finding significant hubs of emerging chloroquine drug resistance and ultimately helping in the management of suitable antimalarial drug policy.
氯喹在印度仍被用作治疗无并发症间日疟原虫的一线药物，而对这种治疗方法的耐药性可能是消除疟疾的主要障碍。由于难以通过体内和体外研究来监测间日疟原虫的药物疗效和耐药性，因此分析分子标记物是跟踪耐药性的重要工具。目前用于检测耐药基因中的单核苷酸多态性的分子方法，包括聚合酶链反应（PCR）、实时聚合酶链反应，都需要高度复杂的实验室和耗时的操作。因此，基于这一背景，本研究旨在优化环介导等温扩增分析，以检测现场间日疟原虫氯喹耐药基因中的单核苷酸多态性。共收集了 88 份间日疟原虫阳性样本。对所有样本进行 Pvmdr1 基因扩增和测序。对目标基因中的单核苷酸多态性进行分析。进一步设计了针对目标突变的环介导等温扩增分析引物对，并对该分析进行了优化。计算了该分析的临床和分析灵敏度和特异性。在 100%的间日疟原虫临床分离株中检测到 T958M 和 F1076L 双突变，这些分离株的单倍型为 M958 Y976 Y1028 L1076。用于环介导等温扩增分析的设计引物成功地在 100%的分离株中检测到这两种突变（T958M 和 F1076L），并且与其他菌株没有交叉反应。因此，该分析对检测目标 Pvmdr1 基因中的单核苷酸多态性具有 100%的灵敏度和特异性。检测限为 0.9 拷贝/µl，设计的分析检测到的最低 DNA 模板浓度为 1.5 ng/µL。观察到 Pvmdr 1 基因中的单核苷酸多态性的流行率表明间日疟原虫对氯喹的耐药性开始出现趋势。本研究优化了环介导等温扩增分析，用于检测现场间日疟原虫病例中的单核苷酸多态性，因此有助于发现新兴氯喹耐药性的重要中心，并最终有助于管理合适的抗疟药物政策。
