{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"indexed":{"date-parts":[[2026,11,7]],"date-time":"2026-11-07T04:45:30Z","timestamp":1794026730066,"version":"4.8.0"},"reference-count":122,"publisher":"MDPI AG","issue":"16","license":[{"start":{"date-parts":[[2025,8,8]],"date-time":"2025-08-08T00:00:00Z","timestamp":1754611200000},"content-version":"vor","delay-in-days":0,"URL":"https:\/\/creativecommons.org\/licenses\/by\/4.0\/"}],"content-domain":{"domain":[],"crossmark-restriction":false},"short-container-title":["Diagnostics"],"abstract":"<jats:p>Metagenomic next-generation sequencing (mNGS) is transforming infectious disease diagnostics by enabling simultaneous, hypothesis-free detection of a broad array of pathogens\u2014including bacteria, viruses, fungi, and parasites\u2014directly from clinical specimens such as cerebrospinal fluid, blood, and bronchoalveolar lavage fluid. Unlike traditional culture and targeted molecular assays, mNGS serves as a powerful complementary approach, capable of identifying novel, fastidious, and polymicrobial infections while also characterizing antimicrobial resistance (AMR) genes. These advantages are particularly relevant in diagnostically challenging scenarios, such as infections in immunocompromised patients, sepsis, and culture-negative cases. Despite its potential, mNGS remains underutilized in clinical microbiology due to persistent gaps between its technical capabilities and routine diagnostic adoption. This review addresses key translational challenges that limit the broader implementation of mNGS, especially in resource-constrained and critical care settings. We provide a comprehensive overview of the entire workflow\u2014from sample processing and host DNA depletion to sequencing platforms and downstream bioinformatics\u2014and highlight sources of variability, including contamination, human DNA interference, and inconsistencies in resistance gene annotation. Additionally, we explore the ethical, legal, and privacy implications of host genomic data, as well as economic and regulatory obstacles hindering mNGS integration into standard clinical practice. To illustrate clinical relevance, we examine real-world evidence from large-scale trials such as MATESHIP, GRAIDS, DISQVER, and NGS-CAP. Finally, we outline future directions involving artificial intelligence, multi-omics integration, cloud-based analytics, and portable sequencing technologies for point-of-care diagnostics. By addressing both current limitations and emerging innovations, this review offers a translational framework for integrating mNGS into precision diagnostics and infection management across diverse healthcare environments.<\/jats:p>","DOI":"10.3390\/diagnostics15161991","type":"journal-article","created":{"date-parts":[[2025,8,8]],"date-time":"2025-08-08T15:30:52Z","timestamp":1754667052000},"page":"1991","update-policy":"https:\/\/doi.org\/10.3390\/mdpi_crossmark_policy","source":"Crossref","is-referenced-by-count":80,"title":["Metagenomic Next-Generation Sequencing in Infectious Diseases: Clinical Applications, Translational Challenges, and Future Directions"],"prefix":"10.3390","volume":"15","author":[{"ORCID":"https:\/\/orcid.org\/0000-0001-7726-9532","authenticated-orcid":false,"given":"Ayman","family":"Elbehiry","sequence":"first","affiliation":[{"name":"Department of Public Health, College of Applied Medical Sciences, Qassim University, P.O. Box 6666, Buraydah 51452, Saudi Arabia"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-2294-9837","authenticated-orcid":false,"given":"Adil","family":"Abalkhail","sequence":"additional","affiliation":[{"name":"Department of Public Health, College of Applied Medical Sciences, Qassim University, P.O. Box 6666, Buraydah 51452, Saudi Arabia"}],"role":[{"vocabulary":"crossref","role":"author"}]}],"member":"1968","published-online":{"date-parts":[[2025,8,8]]},"reference":[{"key":"ref_1","doi-asserted-by":"crossref","first-page":"629","DOI":"10.1016\/S0140-6736(21)02724-0","article-title":"Global burden of bacterial antimicrobial resistance in 2019: A systematic analysis","volume":"399","author":"Murray","year":"2022","journal-title":"Lancet"},{"key":"ref_2","doi-asserted-by":"crossref","first-page":"100249","DOI":"10.1016\/j.modpat.2023.100249","article-title":"Emerging antimicrobial resistance","volume":"36","author":"Flynn","year":"2023","journal-title":"Mod. Pathol."},{"key":"ref_3","doi-asserted-by":"crossref","first-page":"26","DOI":"10.2174\/1871526523666230725103902","article-title":"An overview of the global alarming increase of multiple drug resistant: A major challenge in clinical diagnosis","volume":"24","author":"Alara","year":"2024","journal-title":"Infect. 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