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

Abstract

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.

Keywords: COVID-19; SARS-CoV-2; Unexplained pneumonia; diagnosis; molecular.

Figure 1.

Molecular diagnostic approaches in this patient and comparisons of updated diagnostic methods. (A) Real-time amplification curve for detecting SARS-CoV-2. The left curve pointed to this sample and the right one meant the positive control. (B) Phylogenetic tree based on N gene of coronavirus including SARS-CoV-2 genomes (blue labels) and this sample (red label). (C) Genome coverage and depth compared with SARS-CoV-2 Wuhan-Hu-1 (GenBank MN908947.2) genome. (D) Genome similarity analysis with other 13 β family coronaviruses. The numbers showed the percentage of nt identity. (E) Comparisons of characteristics of updated diagnostic methods including rapid diagnostic tests (RDT), NAATs, multiplex respiratory virus infection assays and mNGS.