Pathogen-dependent diagnostic performance of plasma metagenomic next-generation sequencing in HIV-associated opportunistic infections

Background Opportunistic infections (OIs) remain a major cause of morbidity and mortality among people living with HIV (PLWH), particularly in patients with advanced immunosuppression. Blood metagenomic next-generation sequencing (mNGS) has emerged as a rapid and minimally invasive approach for pathogen detection; however, its diagnostic performance may vary substantially across pathogens because of differences in microbial dissemination patterns and circulating microbial cell-free DNA (cfDNA) burden. The pathogen-dependent diagnostic characteristics of blood mNGS in HIV-associated opportunistic infections remain insufficiently defined. Methods We retrospectively analyzed HIV-infected patients who underwent plasma mNGS testing for suspected opportunistic infections at a tertiary referral center between January 2020 and July 2025. Patients were classified according to a composite clinical reference standard into Talaromycosis (TM), tuberculosis (TB), Pneumocystis jirovecii pneumonia (PJP), and other opportunistic infections. Pathogen-specific detection performance and false-negative and false-positive results were evaluated. Results A total of 87 HIV-infected patients with opportunistic infections were included. Blood mNGS demonstrated high overall pathogen detection sensitivity (91.46%) and positive predictive value (94.94%). Diagnostic performance differed markedly among pathogens. Blood mNGS showed excellent sensitivity for disseminated fungal infections, including TM (97.73%) and PJP (93.33%), whereas sensitivity was lower for TB (86.67%) and other localized or intracellular infections (62.50%). False-negative results predominantly occurred in infections characterized by tissue compartmentalization or low circulating microbial burden, including cryptococcal meningitis and Mycobacterium avium complex infection. In contrast, false-positive Pneumocystis detections likely reflected colonization or low-level microbial translocation rather than invasive disease. Conclusion The diagnostic performance of blood mNGS in HIV-associated opportunistic infections is strongly pathogen-dependent. Disseminated infections associated with high circulating microbial cfDNA burden demonstrate superior blood-based detection sensitivity, whereas localized or intracellular infections remain diagnostically challenging. These findings provide a biologically informed framework for interpreting blood mNGS results in immunocompromised patients and support the role of blood mNGS as a rapid adjunctive diagnostic tool in HIV-associated opportunistic infections.