Parasitology-Mycology Laboratory, Centre Hospitalier Universitaire Grenoble Alpes and University of Grenoble Alpes, Grenoble, France; "Molecular Biology" Pole of the National Reference Center for Toxoplasmosis, Montpellier, France.
"Molecular Biology" Pole of the National Reference Center for Toxoplasmosis, Montpellier, France; Parasitology-Medical Mycology Laboratory, Parasitology and Tropical Diseases Institute, University Hospitals and University of Strasbourg, Strasbourg, France.
J Mol Diagn. 2022 Jun;24(6):687-696. doi: 10.1016/j.jmoldx.2022.03.009. Epub 2022 Apr 20.
Real-time PCR plays a crucial role in the diagnosis of toxoplasmosis. In this multicenter study, the Toxoplasma RealCycler Universal assay was assessed for the diagnosis of toxoplasmosis by eight reference laboratories. DNAs from diverse clinical samples were included: 141 characterized samples from patients with different clinical forms of proven toxoplasmosis and 27 from patients without toxoplasmosis were tested in duplicate with the commercial assay. Final diagnosis was affirmed by each center by analysis of clinical settings and biological follow-up. Calibrated Toxoplasma gondii standards and 11 external quality control samples were also included. Discrepant results observed after the first run of commercial PCR were controlled by both reference and commercial PCR assays. Using the commercial assay, the detection threshold varied from 0.01 to 1 tachyzoites/mL, depending on the center. The relationship between crossing point and DNA concentration was linear over 4 log units (r > 0.99), and PCR efficiencies were satisfactory (89% to 104%). The results of the 11 external quality control samples were concordant after one retesting, but those for 3 clinical samples remained discrepant. Sensitivity and specificity were calculated at 97.8% (95% CI, 97.8%-100%) and 100% (95% CI, 87.2%-100%), respectively. Provided that PCRs are performed at least in duplicate to detect low parasitic loads, Toxoplasma RealCycler Universal PCR showed suitable performances to diagnose the different forms of toxoplasmosis.
实时 PCR 在弓形虫病的诊断中起着至关重要的作用。在这项多中心研究中，八种参考实验室评估了 Toxoplasma RealCycler Universal 检测法对弓形虫病的诊断价值。该检测法检测了来自不同临床样本的 DNA：141 个特征样本来自具有不同临床形式的确诊弓形虫病患者，27 个来自无弓形虫病的患者，每个临床中心均通过分析临床情况和生物学随访来确定最终诊断。此外，还纳入了校准的刚地弓形虫标准品和 11 个外部质量控制样本。在商业 PCR 第一轮运行中观察到的不一致结果，通过参考和商业 PCR 检测来控制。使用商业检测法，检测阈值因中心而异，从 0.01 到 1 个速殖子/ml。在 4 个对数单位范围内，检测点与 DNA 浓度之间的关系呈线性（r>0.99），PCR 效率令人满意（89%-104%）。经过一次重新检测，11 个外部质量控制样本的结果是一致的，但 3 个临床样本的结果仍不一致。敏感性和特异性分别计算为 97.8%（95% CI，97.8%-100%）和 100%（95% CI，87.2%-100%）。如果至少进行两次 PCR 以检测低寄生虫负荷，则 Toxoplasma RealCycler Universal PCR 显示出适合诊断不同形式弓形虫病的性能。
