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BACKGROUND: While metagenomic next-generation sequencing (mNGS) has been acknowledged as a valuable diagnostic tool for infections, its clinical validity and impact on patient management when using fresh tissue samples remains uncertain. METHODS: We conducted a retrospective cross-sectional study involving patients who underwent tissue mNGS at a tertiary hospital in China from February 2021 to February 2024, aiming to assess its ability to detect plausible pathogens and its clinical validity and impact. FINDINGS: A total of 520 mNGS results from 508 patients were analysed, detecting plausible pathogens in 302 (58.1%) tests, including 260 single-pathogen and 42 (13.9%) multi-pathogen results. Rare pathogens, such as Balamuthia mandrillaris, Bartonella henselae, and Sporothrix globosa, were identified. Of the multi-pathogen results, 22 were with predominance of anaerobes. mNGS showed higher positivity in cases with high initial suspicion of infection than those used for ruling out infection (PR 1.961, 95% CI: 1.604-2.394) and in patients living with HIV (PR 1.312, 95% CI: 1.047-1.643) or solid organ transplant recipients (PR 1.346, 95% CI: 1.103-1.643) compared to immunocompetent individuals. Sensitivity and specificity for diagnosing confirmed/probable infections were 85.0% (95% CI: 76.7%-93.3%) and 93.7% (95% CI: 86.8%-100.0%), respectively. mNGS influenced clinical management in 258 (49.6%) cases by identifying new infections and in 112 (21.5%) by excluding infections. It prompted initiation (20.2%), modification (23.1%), or discontinuation (6.3%) of antimicrobial therapy. INTERPRETATION: mNGS demonstrates high diagnostic accuracy for tissue infections. Its impact on clinical management highlights the need to integrate it into current diagnostic practices. FUNDING: National Natural Science Foundation of China (No. 82472371), "Leading Geese" Research and Development Plan of Zhejiang Province (No. 2024C03218), and Pudong New Area Joint Project (PW2021D-09).
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