Blood mNGS: an effective non-invasive diagnostic tool for Pneumocystis jirovecii pneumonia

Pneumocystis jirovecii pneumonia (PJP) is a life-threatening opportunistic infection. Colonization is prevalent but cannot be reliably distinguished from active infection by conventional methods. Metagenomic next-generation sequencing (mNGS) is a promising diagnostic tool, but the value of blood mNGS for diagnosis, microbial community comparison, and outcome-related associations in PJP remains unclear.

We analyzed 73 suspected PJP patients with paired BALF and blood mNGS. Using strict diagnostic criteria, patients were classified as: PJP (n = 50) and P. jirovecii colonization (PJC, n = 23). Bioinformatic analyses compared compartment-specific microbiota. BALF-blood concordance and associations between P. jirovecii load and outcomes were evaluated.

BALF showed higher α-diversity than blood (both Shannon and Simpson, P < 0.001), whereas β-diversity showed no compartmental segregation. BALF identified 216 species versus 43 in blood; however, the top-10 species were concordantly ranked (90% concordance). Blood mNGS distinguished PJP from PJC with an AUC of 0.80 (specificity 95.7%, sensitivity 62.0% at RPM > 4.8), outperforming BALF mNGS (AUC 0.76), blood PCR (AUC 0.64) and BALF PCR (AUC 0.73). Gram-negative bacteria accounted for a large proportion of blood taxa (75% of top 20 taxa), while BALF showed additional fungal taxa including Aspergillus fumigatus. LEfSe identified matrix-specific taxa: oral commensals in PJC-BALF. Blood P. jirovecii load correlated positively with LDH (r = 0.34, P = 0.0035), CRP (r = 0.34, P = 0.0031), and BDG (r = 0.26, P = 0.025), and was higher in non-survivors (P < 0.05).

Blood mNGS may serve as a non-invasive, highly specific complementary tool for PJP diagnosis and broader microbiological assessment.