Simultaneous identification of DNA and RNA pathogens using metagenomic sequencing in cases of severe acute respiratory infection

Metagenomic next-generation sequencing (mNGS) is a valuable technique for identifying pathogens. However, conventional mNGS requires the separate processing of DNA and RNA genomes, which can be resource- and time-intensive. To mitigate these impediments, we propose a novel method called DNA/RNA cosequencing that aims to enhance the efficiency of pathogen detection. DNA/RNA cosequencing uses reverse transcription of total nucleic acids extracted from samples by using random primers, without removing DNA, and then employs mNGS. We applied this method to 85 cases of severe acute respiratory infections (SARI). Influenza virus was identified in 13 cases (H1N1: seven cases, H3N2: three cases, unclassified influenza type: three cases) and was not detected in the remaining 72 samples. Bacteria were present in all samples. Pseudomonas aeruginosa, Klebsiella pneumoniae, and Acinetobacter baumannii were detected in four influenza-positive samples, suggesting coinfections. The sensitivity and specificity for detecting influenza A virus were 73.33% and 95.92%, respectively. A κ value of 0.726 indicated a high level of concordance between the results of DNA/RNA cosequencing and SARI influenza virus monitoring. DNA/RNA cosequencing enhanced the efficiency of pathogen detection, providing a novel capability to strengthen surveillance and thereby prevent and control infectious disease outbreaks.