Analytical and clinical validation of multiplex droplet digital PCR assay for detecting pathogenic fungal infection in lungs
Analytical and clinical validation of multiplex droplet digital PCR assay for detecting pathogenic fungal infection in lungs
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DOI：
10.1080/21501203.2023.2296941
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发表时间：
2023-12-30
期刊：
影响因子：
--
通讯作者：
Wu，Wenjuan
中科院分区：
文献类型：
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作者：
Guo，Jian;Tian，Wenjie;Wu，Wenjuan
免疫功能低下宿主的肺部侵袭性真菌感染很难诊断，目前用于诊断或监测抗真菌治疗反应的工具具有固有的局限性。微滴式数字 PCR (ddPCR) 已成为一种很有前景的高灵敏度肺部病原体检测工具。本研究提出了一种新型 ddPCR 组合，用于快速、灵敏地鉴定肺部真菌病原体。首先，建立并评估了检测肺孢子菌、曲霉属和隐球菌属三个真菌属的ddPCR方法。然后，使用170份标本将ddPCR与qPCR的临床验证性能进行比较，并对结果不一致的6份标本进行宏基因组二代测序进一步验证，得到与ddPCR结果一致的结果。最后，用ROC曲线下面积（AUC）来评价ddPCR的效率。 qPCR 检测出 16 例（9.41%）曲霉菌和 6 例（3.53%）肺孢子虫，而 ddPCR 检测出 20 例（11.76%）曲霉菌和 8 例（4.71%）肺孢子虫。曲霉属、隐球菌属和肺囊虫属的 AUC 分别为 0.974、0.998 和 0.975。这些发现表明，ddPCR 检测是一种高度灵敏的方法，可用于识别侵袭性真菌肺部感染的病原体，并且是一种有前途的早期诊断工具。
Pulmonary invasive fungal infection in immunocompromised hosts is difficult to diagnose, and current tools for diagnosis or monitoring of response to antifungal treatments have inherent limitations. Droplet digital PCR (ddPCR) has emerged as a promising tool for pulmonary pathogen detection with high sensitivity. This study presents a novel ddPCR panel for rapid and sensitive identification of pulmonary fungal pathogens. First, a ddPCR method for detecting three fungal genera, includingPneumocystis,Aspergillus, andCryptococcus, was established and evaluated. Then, the clinical validation performance of ddPCR was compared with that of qPCR using 170 specimens, and the 6 specimens with inconsistent results were further verified by metagenomics next-generation sequencing, which yielded results consistent with the ddPCR findings. Finally, the area under the ROC curve (AUC) was used to evaluate the efficiency of ddPCR. While the qPCR identified 16 (9.41%) cases ofAspergillusand 6 (3.53%) cases ofPneumocystis, ddPCR detected 20 (11.76%)Aspergilluscases and 8 (4.71%)Pneumocystiscases. The AUC forAspergillus,Cryptococcus, andPneumocystiswas 0.974, 0.998, and 0.975, respectively. These findings demonstrated that the ddPCR assay is a highly sensitive method for identifying pathogens responsible for invasive fungal pulmonary infections, and is a promising tool for early diagnosis.