Era of molecular diagnosis for pathogen identification of unexplained pneumonia, lessons to be learned

Unexplained pneumonia (UP) caused by a novel coronavirus SARS-CoV-2 (severe acute respiratory syndrome coronavirus 2) emerged in China in late December 2019 and has infected more than 9000 cases by 31 January 2020. Shanghai reported the first imported case of COVID-19 (Coronavirus Disease 2019) in 20 January 2020. A combinative approach of real-time RT–PCR, CRISPR-based assay and metagenomic next-generation sequencing (mNGS) were used to diagnose this unexplained pneumonia patient. Real-time RT–PCR and CRISPR-based assay both reported positive. This sample belonged to Betacoronavirus and shared a more than 99% nucleotide (nt) identity with the Wuhan SARS-CoV-2 isolates. We further compared pros and cons of common molecular diagnostics in UP. In this study, we illustrated the importance of combining molecular diagnostics to rule out common pathogens and performed mNGS to obtain unbiased potential pathogen result for the diagnosis of UP.

2019年12月末，中国出现由严重急性呼吸综合征冠状病毒2（severe acute respiratory syndrome coronavirus 2，SARS-CoV-2）引发的不明原因肺炎（Unexplained pneumonia, UP）；截至2020年1月31日，该病毒累计感染病例已超9000例。2020年1月20日，上海市报告首例新型冠状病毒感染（Coronavirus Disease 2019, COVID-19）输入性病例。研究团队采用实时逆转录聚合酶链反应（real-time RT–PCR）、基于CRISPR的检测方法（CRISPR-based assay）与宏基因组下一代测序（metagenomic next-generation sequencing, mNGS）联合检测策略，对该不明原因肺炎患者进行诊断，其中实时逆转录聚合酶链反应与基于CRISPR的检测结果均呈阳性。该样本归类为乙型冠状病毒（Betacoronavirus），与武汉分离的SARS-CoV-2毒株的核苷酸（nucleotide, nt）序列同源性超过99%。本研究对比了常见分子诊断技术在不明原因肺炎检测中的优缺点，阐明了联合使用分子诊断技术以排除常见病原体的重要性，并通过宏基因组下一代测序获取无偏倚的潜在病原体检测结果，用于不明原因肺炎的诊断。