National Malaria Reference Centre, AP-HP, Hôpital Bichat - Claude Bernard, 46 Rue Henri Huchard, 75018, Paris, France.
University of Paris Cité, IRD, MERIT, 75006, Paris, France.
Malar J. 2022 Jun 27;21(1):204. doi: 10.1186/s12936-022-04219-1.
Malaria is an infectious disease considered as one of the biggest causes of mortality in endemic areas. This life-threatening disease needs to be quickly diagnosed and treated. The standard diagnostic tools recommended by the World Health Organization are thick blood smears microscopy and immuno-chromatographic rapid diagnostic tests. However, these methods lack sensitivity especially in cases of low parasitaemia and non-falciparum infections. Therefore, the need for more accurate and reliable diagnostic tools, such as real-time polymerase chain reaction based methods which have proven greater sensitivity particularly in the screening of malaria, is prominent. This study was conducted at the French National Malaria Reference Centre to assess sensitivity and specificity of two commercial malaria qPCR kits and two in-house developed qPCRs compared to LAMP.
183 blood samples received for expertise at the FNMRC were included in this study and were subjected to four different qPCR methods: the Biosynex Ampliquick Malaria test, the BioEvolution Plasmodium Typage test, the in-house HRM and the in-house TaqMan qPCRs. The specificity and sensitivity of each method and their confidence intervals were determined with the LAMP-based assay Alethia® Malaria as the reference for malaria diagnosis. The accuracy of species diagnosis of the Ampliquick Malaria test and the two in-house qPCRs was also evaluated using the BioEvolution Plasmodium Typage test as the reference method for species identification.
The main results showed that when compared to LAMP, a test with excellent diagnostic performances, the two in-house developed qPCRs were the most sensitive (sensitivity at 100% for the in-house TaqMan qPCR and 98.1% for the in-house HRM qPCR), followed by the two commercial kits: the Biosynex Ampliquick Malaria test (sensitivity at 97.2%) and the BioEvolution Plasmodium Typage (sensitivity at 95.4%). Additionally, with the in-house qPCRs we were able to confirm a Plasmodium falciparum infection in microscopically negative samples that were not detected by commercial qPCR kits. This demonstrates that the var genes of P. falciparum used in these in-house qPCRs are more reliable targets than the 18S sRNA commonly used in most of the developed qPCR methods for malaria diagnosis.
Overall, these results accentuate the role molecular methods could play in the screening of malaria. This may represent a helpful tool for other laboratories looking to implement molecular diagnosis methods in their routine analysis, which could be essential for the detection and treatment of malaria carriers and even for the eradication of this disease.
疟疾是一种传染病，被认为是流行地区最大的死亡原因之一。这种危及生命的疾病需要快速诊断和治疗。世界卫生组织推荐的标准诊断工具是厚血涂片显微镜检查和免疫层析快速诊断检测。然而，这些方法的灵敏度较差，特别是在低寄生虫血症和非恶性疟原虫感染的情况下。因此，需要更准确和可靠的诊断工具，例如基于实时聚合酶链反应的方法，这些方法已被证明在疟疾筛查方面具有更高的灵敏度。本研究在法国国家疟疾参考中心进行，以评估两种商业疟疾 qPCR 试剂盒和两种内部开发的 qPCR 与环介导等温扩增（LAMP）相比的灵敏度和特异性。
本研究纳入了 183 份在 FNMRC 进行专业检测的血液样本，并对四种不同的 qPCR 方法进行了检测：Biosynex Ampliquick 疟疾检测、BioEvolution Plasmodium Typage 检测、内部 HRM 和内部 TaqMan qPCRs。以基于 LAMP 的 Alethia® Malaria 检测作为疟疾诊断的参考，确定了每种方法的特异性和灵敏度及其置信区间。还使用 BioEvolution Plasmodium Typage 检测作为物种鉴定的参考方法，评估了 Ampliquick 疟疾检测和两种内部 qPCR 对物种诊断的准确性。
主要结果表明，与具有出色诊断性能的 LAMP 检测相比，两种内部开发的 qPCR 是最敏感的（内部 TaqMan qPCR 的灵敏度为 100%，内部 HRM qPCR 的灵敏度为 98.1%），其次是两种商业试剂盒：Biosynex Ampliquick 疟疾检测（灵敏度为 97.2%）和 BioEvolution Plasmodium Typage（灵敏度为 95.4%）。此外，使用内部 qPCR，我们能够在商业 qPCR 试剂盒未检测到的镜检阴性样本中确认恶性疟原虫感染。这表明，与用于疟疾诊断的大多数开发的 qPCR 方法中常用的 18S sRNA 相比，这些内部 qPCR 中使用的 Pf 疟原虫 var 基因是更可靠的靶标。
总的来说，这些结果强调了分子方法在疟疾筛查中的作用。这可能是其他实验室在常规分析中实施分子诊断方法的有用工具，对于检测和治疗疟疾携带者甚至消除这种疾病至关重要。
