Faculty of Medicine, Department of Human Genetics, University of Debrecen, Nagyerdei krt. 98, Debrecen, H-4032, Hungary.
Faculty of Science and Technology, Department of Biotechnology and Microbiology, University of Debrecen, Debrecen, Hungary.
BMC Infect Dis. 2018 Aug 13;18(1):393. doi: 10.1186/s12879-018-3283-6.
Fungal bloodstream infections (BSI) may be serious and are associated with drastic rise in mortality and health care costs. Candida spp. are the predominant etiological agent of fungal sepsis. The prompt and species-level identification of Candida may influence patient outcome and survival. The aim of this study was to develop and evaluate the CanTub-simplex PCR assay coupled with T calling and subsequent high resolution melting (HRM) analysis to barcode seven clinically relevant Candida species.
Efficiency, coefficient of correlation and the limit of reliable detection were estimated on purified Candida EDTA-whole blood (WB) reference panels seeded with Candida albicans, Candida glabrata, Candida parapsilosis, Candida tropicalis, Candida krusei, Candida guilliermondii, Candida dubliniensis cells in a 6-log range. Discriminatory power was measured on EDTA-WB clinical panels on three different PCR platforms; LightCycler®96, LightCycler® Nano, LightCycler® 2.0. Inter- and intra assay consistencies were also calculated.
The limit of reliable detection proved to be 0.2-2 genomic equivalent and the method was reliable on broad concentration ranges (10-10 CFU) providing distinctive melting peaks and characteristic HRM curves. The diagnostic accuracy of the discrimination proved to be the best on Roche LightCycler®2.0 platform. Repeatability was tested and proved to be % C.V.: 0.14 ± 0.06 on reference- and % C.V.: 0.14 ± 0.02 on clinical-plates accounting for a very high accuracy. Reproducibility was % C.V.: 0.11 between reference- and % C.V.: 0.12between clinical-panels which is highly acceptable.
Our assay demonstrates recent advances on T calling and HRM analysis for the molecular identification of relevant Candida species. This unique, simplex PCR assay may be capable to outperform conventional phenotypic methods by reducing time and providing accurate and reliable results directly from blood (2 h) or from whole blood culture bottles (12-24 h).
真菌性血流感染（BSI）可能很严重，并与死亡率和医疗保健成本的大幅上升有关。念珠菌属是真菌性败血症的主要病原体。念珠菌的快速和种水平鉴定可能会影响患者的预后和生存。本研究旨在开发和评估 CanTub-simplex PCR 检测方法，结合 T 呼叫和随后的高分辨率熔解（HRM）分析，对七种临床相关念珠菌进行条码化。
采用纯化的念珠菌 EDTA-全血（WB）参考面板，在 6 个对数范围内用念珠菌 albicans、Candida glabrata、Candida parapsilosis、Candida tropicalis、Candida krusei、Candida guilliermondii 和 Candida dubliniensis 细胞接种，估计效率、相关系数和可靠检测的极限。在三个不同的 PCR 平台（LightCycler®96、LightCycler®Nano、LightCycler®2.0）上，对 EDTA-WB 临床板进行了区分能力的测量。还计算了板内和板间的一致性。
可靠检测的极限被证明为 0.2-2 个基因组当量，该方法在广泛的浓度范围内（10-10 CFU）可靠，提供独特的熔解峰和特征 HRM 曲线。区分的诊断准确性在罗氏 LightCycler®2.0 平台上被证明是最好的。重复性测试并证明重复性变异系数（CV）：参考板为 0.14 ± 0.06，临床板为 0.14 ± 0.02，这代表了非常高的准确性。参考板之间的再现性变异系数（CV）为 0.11%，临床板之间的再现性变异系数（CV）为 0.12%，这是高度可接受的。
我们的检测方法证明了 T 呼叫和 HRM 分析在鉴定相关念珠菌种方面的最新进展。这种独特的 simplex PCR 检测方法可能能够通过减少时间并直接从血液（2 小时）或全血培养瓶（12-24 小时）提供准确可靠的结果，从而优于传统的表型方法。
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