Comparative analysis of metagenomic next-generation sequencing for pathogenic identification in clinical body fluid samples

Objectives This study aims to evaluate and compare the effectiveness of metagenomic next-generation sequencing (mNGS) in identifying pathogens from clinical body fluid samples, with a specific focus on the application of microbial cell-free DNA (cfDNA) mNGS. Methods A total of 125 clinical body fluid samples were collected. All samples underwent mNGS targeting whole-cell DNA (wcDNA), with 30 samples also analyzed for cfDNA mNGS and 41 subjected to 16S rRNA NGS for comparative analysis. Patient clinical data, including culture results, were obtained from electronic medical records. Results In comparison to cfDNA mNGS, the mean proportion of host DNA in wcDNA mNGS was 84%, significantly lower than the 95% observed in cfDNA mNGS (p < 0.05). Using culture results as a reference, concordance rates were 63.33% (19/30) for wcDNA mNGS and 46.67% (14/30) for cfDNA mNGS. Additionally, wcDNA mNGS showed greater consistency in bacterial detection with culture results, achieving a rate of 70.7% (29/41) compared to 58.54% (24/41) for 16S rRNA NGS. The sensitivity and specificity of wcDNA mNGS for pathogen detection in body fluid samples were 74.07% and 56.34%, respectively, when compared to culture results. Conclusion Whole-cell DNA mNGS demonstrates significantly higher sensitivity for pathogen detection and identification compared to both cfDNA mNGS and 16S rRNA NGS in clinical body fluid samples, particularly those associated with abdominal infections. However, the compromised specificity of wcDNA mNGS highlights the necessity for careful interpretation in clinical practice.