肯尼亚基西郡在采用基于青蒿素的联合疗法时代新型疟原虫 falciparum k13 基因多态性。

Novel Plasmodium falciparum k13 gene polymorphisms from Kisii County, Kenya during an era of artemisinin-based combination therapy deployment.

机构信息

Department of Medical Microbiology and Immunology, Kampala International University Western Campus, Bushenyi, Uganda.

School of Health Sciences, Kisii University, Kisii, Kenya.

出版信息

Malar J. 2023 Mar 9;22(1):87. doi: 10.1186/s12936-023-04517-2.

DOI:10.1186/s12936-023-04517-2

PMID:36894982

原文链接:https://pmc.ncbi.nlm.nih.gov/articles/PMC9996564/

Abstract

BACKGROUND

Currently, chemotherapy stands out as the major malaria intervention strategy, however, anti-malarial resistance may hamper global elimination programs. Artemisinin-based combination therapy (ACT) stands as the drug of choice for the treatment of Plasmodium falciparum malaria. Plasmodium falciparum kelch13 gene mutations are associated with artemisinin resistance. Thus, this study was aimed at evaluating the circulation of P. falciparum k13 gene polymorphisms from Kisii County, Kenya during an era of ACT deployment.

METHODS

Participants suspected to have malaria were recruited. Plasmodium falciparum was confirmed using the microscopy method. Malaria-positive patients were treated with artemether-lumefantrine (AL). Blood from participants who tested positive for parasites after day 3 was kept on filter papers. DNA was extracted using chelex-suspension method. A nested polymerase chain reaction (PCR) was conducted and the second-round products were sequenced using the Sanger method. Sequenced products were analysed using DNAsp 5.10.01 software and then blasted on the NCBI for k13 propeller gene sequence identity using the Basic Local Alignment Search Tool (BLAST). To assess the selection pressure in P. falciparum parasite population, Tajima' D statistic and Fu & Li's D test in DnaSP software 5.10.01 was used.

RESULTS

Out of 275 enrolled participants, 231 completed the follow-up schedule. 13 (5.6%) had parasites on day 28 hence characterized for recrudescence. Out of the 13 samples suspected of recrudescence, 5 (38%) samples were positively amplified as P. falciparum, with polymorphisms in the k13-propeller gene detected. Polymorphisms detected in this study includes R539T, N458T, R561H, N431S and A671V, respectively. The sequences have been deposited in NCBI with bio-project number PRJNA885380 and accession numbers SAMN31087434, SAMN31087433, SAMN31087432, SAMN31087431 and SAMN31087430 respectively.

CONCLUSIONS

WHO validated polymorphisms in the k13-propeller gene previously reported to be associated with ACT resistance were not detected in the P. falciparum isolates from Kisii County, Kenya. However, some previously reported un-validated k13 resistant single nucleotide polymorphisms were reported in this study but with limited occurrences. The study has also reported new SNPs. More studies need to be carried out in the entire country to understand the association of reported mutations if any, with ACT resistance.

摘要

背景

目前，化疗是治疗疟疾的主要干预策略，但抗疟药物的耐药性可能会阻碍全球消除疟疾计划的实施。青蒿素类复方疗法（ACT）是治疗恶性疟原虫疟疾的首选药物。恶性疟原虫kelch13 基因突变与青蒿素类药物耐药性有关。因此，本研究旨在评估基西郡肯尼亚在 ACT 应用时代恶性疟原虫 k13 基因突变的流行情况。

方法

招募疑似患有疟疾的参与者。采用显微镜法确认疟原虫。对疟疾阳性患者进行青蒿琥酯-甲氟喹（AL）治疗。对 3 天后仍检测到寄生虫的参与者的血液保存在滤纸上。采用 chelex 悬浮法提取 DNA。进行巢式聚合酶链反应（PCR），然后使用 Sanger 法对第二轮产物进行测序。使用 DNAsp 5.10.01 软件对测序产物进行分析，然后使用基本局部比对搜索工具（BLAST）在 NCBI 上对 kelch13 螺旋桨基因序列进行同源性比对。为了评估恶性疟原虫种群中的选择压力，我们在 DnaSP 软件 5.10.01 中使用 Tajima' D 统计量和 Fu & Li 的 D 检验。

结果

在 275 名入组的参与者中，有 231 名完成了随访计划。28 天时有 13 名（5.6%）出现寄生虫，因此被定性为复发。在 13 份疑似复发的样本中，有 5 份（38%）被确认为恶性疟原虫阳性，并检测到 kelch13-螺旋桨基因的多态性。本研究中检测到的多态性包括 R539T、N458T、R561H、N431S 和 A671V。这些序列已在 NCBI 中注册，生物项目编号为 PRJNA885380，登录号分别为 SAMN31087434、SAMN31087433、SAMN31087432、SAMN31087431 和 SAMN31087430。

结论

世卫组织先前已验证了与 ACT 耐药性相关的 kelch13 螺旋桨基因的多态性，但在肯尼亚基西郡的恶性疟原虫分离株中未检测到。然而，本研究报道了一些先前报道的未经验证的 kelch13 耐药性单核苷酸多态性，但发生频率有限。该研究还报告了新的 SNP。需要在全国范围内开展更多的研究，以了解如果存在任何与 ACT 耐药性相关的报道突变。