FG16, Unit for Mycotic and Parasitic Agents and Mycobacteria, Robert Koch Institute, Berlin, Germany.
Westerdijk Fungal Biodiversity Institute (WI-KNAW), Utrecht, The Netherlands.
Mycoses. 2023 Sep;66(9):774-786. doi: 10.1111/myc.13603. Epub 2023 May 11.
In-house real-time PCR (qPCR) is increasingly used to diagnose the so-called endemic mycoses as commercial assays are not widely available.
To compare the performance of different molecular diagnostic assays for detecting Histoplasma capsulatum and Coccidioides spp. in five European reference laboratories.
Two blinded external quality assessment (EQA) panels were sent to each laboratory that performed the analysis with their in-house assays. Both panels included a range of concentrations of H. capsulatum (n = 7) and Coccidioides spp. (n = 6), negative control and DNA from other fungi. Four laboratories used specific qPCRs, and one laboratory a broad-range fungal conventional PCR (cPCR) and a specific cPCR for H. capsulatum with subsequent sequencing.
qPCR assays were the most sensitive for the detection of H. capsulatum DNA. The lowest amount of H. capsulatum DNA detected was 1-4 fg, 0.1 pg and 10 pg for qPCRs, specific cPCR and broad-range cPCR, respectively. False positive results occurred with high concentrations of Blastomyces dermatitidis DNA in two laboratories and with Emergomyces spp. in one laboratory. For the Coccidioides panel, the lowest amount of DNA detected was 1-16 fg by qPCRs and 10 pg with the broad-range cPCR. One laboratory reported a false positive result by qPCR with high load of Uncinocarpus DNA.
All five laboratories were able to correctly detect H. capsulatum and Coccidioides spp. DNA and qPCRs had a better performance than specific cPCR and broad-range cPCR. EQAs may help standardise in-house molecular tests for the so-called endemic mycoses improving patient management.
由于商业检测方法并不广泛可用，内部实时 PCR（qPCR）越来越多地用于诊断所谓的地方性真菌病。
比较五个欧洲参考实验室中不同分子诊断检测方法对荚膜组织胞浆菌和粗球孢子菌的检测性能。
每个实验室都收到了两个盲法外部质量评估（EQA）试剂盒，这些试剂盒包含了一系列荚膜组织胞浆菌（n=7）和粗球孢子菌（n=6）的浓度，阴性对照和其他真菌的 DNA。四个实验室使用了特定的 qPCR，一个实验室使用了广谱真菌常规 PCR（cPCR）和针对荚膜组织胞浆菌的特异性 cPCR，随后进行测序。
qPCR 检测方法对荚膜组织胞浆菌 DNA 的检测最敏感。qPCR、特异性 cPCR 和广谱 cPCR 检测到的荚膜组织胞浆菌 DNA 的最低量分别为 1-4 fg、0.1 pg 和 10 pg。两个实验室的高浓度皮炎芽生菌 DNA 和一个实验室的Emergomyces spp. 导致假阳性结果。对于粗球孢子菌检测板，qPCR 检测到的最低 DNA 量为 1-16 fg，广谱 cPCR 检测到的最低 DNA 量为 10 pg。一个实验室报告了一个 qPCR 假阳性结果，该结果与高负荷的 Uncinocarpus DNA 有关。
五个实验室均能够正确检测荚膜组织胞浆菌和粗球孢子菌 DNA，qPCR 的性能优于特异性 cPCR 和广谱 cPCR。EQA 可能有助于标准化所谓的地方性真菌病的内部分子检测，从而改善患者管理。
