评估多重实时 PCR 在埃塞俄比亚消除目标低传播环境下疟疾诊断中的性能。
Evaluating performance of multiplex real time PCR for the diagnosis of malaria at elimination targeted low transmission settings of Ethiopia.
机构信息
Department of Medical Laboratory Sciences, College of Health Sciences, Addis Ababa University, Addis Ababa, Ethiopia.
Malaria and Neglected Tropical Diseases Research Team, Ethiopian Public Health Institute, P.O. Box: 1242, Addis Ababa, Ethiopia.
出版信息
Malar J. 2022 Jan 6;21(1):9. doi: 10.1186/s12936-021-04029-x.
BACKGROUND
Malaria incidence has declined in Ethiopia in the past 10 years. Current malaria diagnostic tests, including light microscopy and rapid antigen-detecting diagnostic tests (RDTs) cannot reliably detect low-density infections. Studies have shown that nucleic acid amplification tests are highly sensitive and specific in detecting malaria infection. This study took place with the aim of evaluating the performance of multiplex real time PCR for the diagnosis of malaria using patient samples collected from health facilities located at malaria elimination targeted low transmission settings in Ethiopia.
METHODS
A health facility-based, cross-sectional survey was conducted in selected malaria sentinel sites. Malaria-suspected febrile outpatients referred to laboratory for malaria testing between December 2019 and March 2020 was enrolled into this study. Sociodemographic information and capillary blood samples were collected from the study participants and tested at spot with RDTs. Additionally, five circles of dry blood spot (DBS) samples on Whatman filter paper and thick and thin smear were prepared for molecular testing and microscopic examination, respectively. Multiplex real time PCR assay was performed at Ethiopian Public Health Institute (EPHI) malaria laboratory. The performance of multiplex real time PCR assay, microscopy and RDT for the diagnosis of malaria was compared and evaluated against each other.
RESULTS
Out of 271 blood samples, multiplex real time PCR identified 69 malaria cases as Plasmodium falciparum infection, 16 as Plasmodium vivax and 3 as mixed infections. Of the total samples, light microscopy detected 33 as P. falciparum, 18 as P. vivax, and RDT detected 43 as P. falciparum, 17 as P. vivax, and one mixed infection. Using light microscopy as reference test, the sensitivity and specificity of multiplex real time PCR were 100% (95% CI (93-100)) and 83.2% (95% CI (77.6-87.9)), respectively. Using multiplex real time PCR as a reference, light microscopy and RDT had sensitivity of 58% (95% CI 46.9-68.4) and 67% (95% CI 56.2-76.7); and 100% (95% CI 98-100) and 98.9% (95% CI 96-99.9), respectively. Substantial level of agreement was reported between microscopy and multiplex real time PCR results with kappa value of 0.65.
CONCLUSIONS
Multiplex real-time PCR had an advanced performance in parasite detection and species identification on febrile patients' samples than did microscopy and RDT in low malaria transmission settings. It is highly sensitive malaria diagnostic method that can be used in malaria elimination programme, particularly for community based epidemiological samples. Although microscopy and RDT had reduced performance when compared to multiplex real time PCR, still had an acceptable performance in diagnosis of malaria cases on patient samples at clinical facilities.
背景
在过去的 10 年中，埃塞俄比亚的疟疾发病率有所下降。目前的疟疾诊断检测方法，包括显微镜检查和快速抗原检测诊断试验（RDT），无法可靠地检测低密度感染。研究表明，核酸扩增检测在检测疟疾感染方面具有高度的敏感性和特异性。本研究旨在评估多重实时 PCR 用于诊断疟疾的性能，使用从位于疟疾消除目标低传播地区的卫生机构采集的患者样本进行研究。
方法
在选定的疟疾哨点进行了一项基于卫生机构的横断面调查。2019 年 12 月至 2020 年 3 月期间，因疑似疟疾发热而被转诊至实验室进行疟疾检测的门诊患者被纳入本研究。从研究参与者中收集社会人口统计学信息和毛细血管血样，并在现场使用 RDT 进行检测。此外，还在 Whatman 滤纸上制备了 5 圈干血斑（DBS）样本，用于分子检测和显微镜检查，分别。在埃塞俄比亚公共卫生研究所（EPHI）疟疾实验室进行多重实时 PCR 检测。比较和评估了多重实时 PCR 检测、显微镜检查和 RDT 对疟疾的诊断性能。
结果
在 271 份血样中，多重实时 PCR 检测出 69 例疟原虫感染为恶性疟原虫感染，16 例为间日疟原虫感染，3 例为混合感染。在所有样本中，显微镜检查出 33 例为恶性疟原虫，18 例为间日疟原虫，RDT 检测出 43 例为恶性疟原虫，17 例为间日疟原虫，1 例为混合感染。以显微镜检查为参考检测方法，多重实时 PCR 的敏感性和特异性分别为 100%（95%CI（93-100））和 83.2%（95%CI（77.6-87.9））。以多重实时 PCR 为参考，显微镜检查和 RDT 的敏感性分别为 58%（95%CI 46.9-68.4）和 67%（95%CI 56.2-76.7）；100%（95%CI 98-100）和 98.9%（95%CI 96-99.9）。显微镜检查和多重实时 PCR 结果之间的一致性水平较高，kappa 值为 0.65。
结论
在低疟疾传播地区，与显微镜检查和 RDT 相比，多重实时 PCR 在检测发热患者样本中的寄生虫检测和物种鉴定方面具有更高的性能。它是一种高度敏感的疟疾诊断方法，可用于疟疾消除计划，特别是用于社区为基础的流行病学样本。尽管与多重实时 PCR 相比，显微镜检查和 RDT 的性能有所降低，但在临床设施对患者样本进行疟疾诊断时仍具有可接受的性能。
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