Genomic RNA and DNA next generation sequencing (mNGS) for the diagnosis of neonatal sepsis---based on host and pathogen plasma genomics detection

Abstract

Background Neonatal sepsis is difficult to confirm because clinical signs are nonspecific and blood culture is slow and insensitive. Metagenomic next-generation sequencing (mNGS) of plasma can detect pathogen nucleic acids without culture, while plasma RNA may capture the host response. Whether combined DNA and RNA plasma mNGS improves diagnosis of neonatal sepsis is unclear. Methods We prospectively enrolled 168 neonates with suspected sepsis who underwent standard evaluation and paired plasma DNA and RNA mNGS. Cases were adjudicated, blinded to sequencing, as confirmed (culture-positive) sepsis (n = 42), clinical (culture-negative) sepsis (n = 51), suspected sepsis/rule-out (n = 45), or noninfectious controls (n = 30). Microbial reads were classified after host subtraction and background correction. Plasma RNA was used for differential expression and a host diagnostic classifier. An integrated model combined the host score with microbial abundance. Results Blood culture was positive in 25.0% of the cohort. Combined DNA+RNA mNGS was positive in 51.2% overall and in 90.5% of confirmed sepsis, and identified a plausible pathogen in 60.8% of clinical sepsis, including fastidious bacteria, fungi, and RNA viruses. DNA mNGS recovered the cultured species in 85.7% of culture-positive cases; RNA mNGS added several exclusive viral diagnoses. Plasma RNA reproduced a sepsis host program (neutrophil activation up, MHC class II/lymphoid programs down). The host classifier achieved a validation AUC of 0.79; the integrated host–microbe model reached 0.93, called 97.6% of culture-confirmed cases positive, and predicted sepsis in 74.5% of culture-negative clinical cases, with 86.7% specificity in controls. Conclusions Dual DNA/RNA plasma mNGS increases pathogen yield over culture and, from the same small-volume sample, provides a biologically coherent host signature. Integrating both layers improves classification of confirmed and culture-negative neonatal sepsis and should be interpreted with conventional microbiology and clinical context.