CLINICAL METAGENOMICS IN MICROBIOLOGY DIAGNOSTICS: ANALYTICAL CHALLENGES, QUALITY ASSURANCE, AND EMERGING FUTURE-READY APPROACHES

Rawat Deepanshu

Deepanshu Rawat Assistant Professor, Faculty of Paramedical and Allied Health Sciences, Motherhood University Roorkee, Haridwar, Uttarakhand 247661 (India)
drawat95@gmail.com

Abstract

Metagenomic sequencing has quickly developed into a revolutionary method in clinical microbiology by making it possible to identify and characterise pathogens directly from clinical specimens without the need for culture. In contrast to focused PCR tests, which need previous information of suspected species, metagenomics facilitates the wide identification of bacteria, viruses, fungi, and parasites and can offer insights into strain-level epidemiology and antibiotic resistance determinants. However, obstacles such low microbial biomass, host DNA dominance, contamination, variable extraction efficiency, bioinformatics complexity, interpretative ambiguity, and expense continue to limit routine application in diagnostic laboratories.The modern metagenomic workflows—sample preparation, nucleic acid extraction, library building, sequencing techniques, and computational pipelines—are summarised in this study, which also emphasises the crucial quality assurance and control procedures needed for accurate reporting. Meningitis/encephalitis, respiratory infections, sepsis, bloodstream infections, and outbreak investigations are among the clinical applications we cover. We also assess translation obstacles including standardisation, regulatory frameworks, reference databases, and result interpretation.

Keywords: Clinical metagenomics, Next-generation sequencing, Pathogen detection, Bioinformatics, Quality control,antimicrobial resistance, Diagnostic microbiology

Author Biography

Deepanshu Rawat, Assistant Professor, Faculty of Paramedical and Allied Health Sciences, Motherhood University Roorkee, Haridwar, Uttarakhand 247661 (India)

Assistant Professor, Faculty of Paramedical and Allied Health Sciences, Motherhood University Roorkee, Haridwar, Uttarakhand 247661 (India)

References

1. Chiu, C. Y., & Miller, S. A. (2019). Clinical metagenomics. Nature Reviews Genetics, 20(6), 341–355.
2. Xu, C., & others. (2025). Effect of interpretation of positive metagenomic next-generation sequencing results on clinical decision-making. Open Forum Infectious Diseases, 12(2), ofaf076.
3. Yin, C., & others. (2024). Pathogenic detection by metagenomic next-generation sequencing in spinal infections. Orthopedic Surgery.
4. Miller, S., & others. (2020). Retrospective review of clinical utility of shotgun metagenomic sequencing in cerebrospinal fluid. Journal of Clinical Microbiology, 59(2), e02593-20.
5. Li, Z., & others. (2024). Diagnostic performance of metagenomic sequencing in infectious diseases. Journal of Infection.
6. Cao, X., & others. (2025). Diagnostic performance and clinical utility of metagenomic next-generation sequencing. Infection and Drug Resistance.
7. Hong, D. K., Blauwkamp, T., Kertesz, M., Bercovich, A., & Chow, R. J. (2021). Multicenter assessment of shotgun metagenomics for pathogen detection. BMC Biology, 19(1), 267.
8. Gast, D. C., & others. (2023). Clinical metagenomics for infectious diseases. Journal of Clinical Microbiology, 61(3), e0177622.
9. Simmon, K. E., & others. (2020). Advances and challenges in clinical metagenomics. Journal of Infectious Diseases, 221(Supplement_3), S318–S323.
10. others. (2025). Metagenomic next-generation sequencing in infectious disease diagnostics. Frontiers in Microbiology.
11. Chen, S., & others. (2025). Application of metagenomic next-generation sequencing in pulmonary infections. BMC Pulmonary Medicine.
12. Rajendhran, J., & others. (2024). Clinical metagenomics-based diagnostics for infectious diseases. Frontiers in Cellular and Infection Microbiology, 14, 1459621.
13. others. (2025). Shotgun metagenomic sequencing analysis as a diagnostic tool. Diagnostics, 15(12), 1456.
14. Govender, K. N., & others. (2021). Metagenomic sequencing as a pathogen-agnostic clinical diagnostic tool. PLoS Neglected Tropical Diseases, 15(8), e0009666.
15. Blauwkamp, T. A., & others. (2019). Analytical and clinical validation of a microbial cell-free DNA sequencing test for infectious disease. Nature Medicine, 25(7), 1188–1195.
16. Wilson, M. R., & others. (2019). Clinical metagenomic sequencing for diagnosis of meningitis and encephalitis. New England Journal of Medicine, 380(18), 1737–1746.
17. Gu, W., & others. (2021). Optimized viral metagenomics for clinical diagnosis. Genome Biology, 22(1), 41
18. Charalampous, T., & others. (2019). Nanopore metagenomics enables rapid clinical diagnosis of bacterial lower respiratory tract infection. Nature Medicine, 25(11), 1854–1860.
19. Street, T. L., & others. (2020). How to approach the diagnosis and management of lower respiratory tract infection. Journal of Clinical Microbiology, 58(7), e00421-20.
20. Leo, Y. S., & others. (2022). Clinical validation of metagenomic next-generation sequencing in routine diagnostic practice. Clinical Infectious Diseases, 75(1), 107–115
21. Handelsman, J. (2004). Metagenomics: Application of genomics to uncultured microorganisms. Microbiology and Molecular Biology Reviews, 68(4), 669–685.
22. Chen, E. C., & others. (2017). Cross-talk between Akkermansiamuciniphila and intestinal epithelium controls diet-induced obesity. PNAS. (Early metagenomics influence)
23. Forbes, J. D., Knox, N. C., & Tang, P. (2019). The diagnostic accuracy of metagenomic next generation sequencing in routine clinical diagnosis. Clinical Chemistry.
24. Simner, P. J., & others. (2021). Development and clinical validation of a host gene expression test for distinguishing sepsis from non-infectious systemic inflammation. Journal of Clinical Microbiology.
25. Kalantar, K., & others. (2020). IDseq—an open source cloud-based pipeline and analysis service for metagenomic data. bioRxiv.
26. Farnaud, S., & others. (2023). Clinical metagenomics implementation challenges. Journal of Molecular Diagnostics.
27. Vissers, J. H., & others. (2024). Metagenomic next-generation sequencing outperforms cultures in identifying pathogens in pediatric osteoarticular infections. Clinical Infectious Diseases.
28. Zhang, H. C., & others. (2022). Metagenomic next-generation sequencing for the diagnosis of central nervous system infections: A real-world prospective observational study. Clinical Microbiology and Infection.
29. Wang, S., & others. (2021). Comparison of metagenomic next-generation sequencing and targeted detection for etiological diagnosis. Journal of Infection.
30. Parize, P., & others. (2017). Untargeted next-generation sequencing-based first-line diagnosis of infection in immunocompromised adults: A multicentre, blinded, prospective study. Clinical Microbiology and Infection, 23(8), 574.e1–574.e6.