For the effective care, rehabilitation, and protection of patients, recognizing and characterizing microorganisms that cause infection are essential. In the diagnostic laboratory, however, not all bacterial species can be cultured successfully. Genomics and whole-genome sequencing (WGS) can greatly enhance human knowledge and understanding of infectious diseases. The ability to assess the microbial community without the need to culture the species has created the ever-growing field of metagenomics and microbiome analysis. Currently, the principal possible applications of WGS in the diagnostic microbiology laboratory for characterizing bacterial pathogen are identification, typing, detection of resistance, and virulence gene detection. In addition, next-generation sequencing (NGS) has helped understand the genome of SARS-CoV-2 early and provided insight into epidemiology, expansion of COVID-19, early and efficient production of the vaccine. The metagenomic sequencing (mNGS) microbial cell-free DNA testing for infection diagnosis is gaining traction. More specifically, for clinicians and specialists in the clinical microbiology community, paradigm shifts in understanding molecular diagnostics are required. A comprehensive clinical review should complement NGS's diagnostic study, which (a) demonstrates clinical effectiveness, (b) guides the use, and (c) exposes possible fields of misunderstood use. Both conventional culture-based technologies and molecular diagnostics have several strengths and limitations.
Bleeker-Rovers CP, Vos FJ, de Kleijn EMHA, et al. A prospective multicentre study on fever of unknown origin: the yield of a structured diagnostic protocol. Medicine 2007;86(1):26–38. DOI: 10.1097/MD.0b013e31802fe858.
Ewig S, Torres A, Angeles Marcos M, et al. Factors associated with unknown aetiology in patients with community-acquired pneumonia. Eur Respir J 2002;20(5):1254–1262. DOI: 10.1183/09031936.02.01942001.
Gu W, Miller S, Chiu CY. Clinical metagenomic next-generation sequencing for pathogen detection. Annu Rev Pathol 2019;14(1):319–338. DOI: 10.1146/annurev-pathmechdis-012418-012751.
Ruud HD, Erik B, Monika AC. Application of next generation sequencing in clinical microbiology and infection prevention. J Biotechnol 2017;243:16–24. DOI: 10.1016/j.jbiotec.2016.12.022.
Pallen MJ. Diagnostic metagenomics: potential applications to bacterial, viral and parasitic infections. Parasitology 2014;141(14):1856–1862. DOI: 10.1017/S0031182014000134.
Han D, Li R, Shi J, et al. Liquid biopsy for infectious diseases: a focus on microbial cell-free DNA sequencing. Theranostics 2020;10(12):5501–5513. DOI: 10.7150/thno.45554.
Bergholz TM, Moreno Switt AI, Wiedmann M. Omics approaches in food safety: fulfilling the promise? Trends Microbiol 2014;22(5):275–281. DOI: 10.1016/j.tim.2014.01.006.
Andrea I, Moreno S, Viviana T. Infectious diseases in the genomic era. Rev chil infectol 2015;32(5):571–576. DOI: 10.4067/S0716-10182015000600013.
Simner PJ, Miller S, Carroll KC. Understanding the promises and hurdles of metagenomic next-generation sequencing as a diagnostic tool for infectious diseases. Clin Infect Dis 2018;66(5):778–788. DOI: 10.1093/cid/cix881.
Greninger AL, Messacar K, Dunnebacke T, et al. Clinical metagenomic identification of Balamuthia Mandrillaris encephalitis and assembly of the draft genome: the continuing case for reference genome sequencing. Genome Med 2016;8(1):1. DOI: 10.1186/s13073-015-0257-9.
Wilson MR, Naccache SN, Samayoa E, et al. Actionable diagnosis of neuroleptospirosis by next-generation sequencing. New Engl J Med 2014;370(25):2408–2417. DOI: 10.1056/NEJMoa1401268.
Miao Q, Ma Y, Wang Q, et al. Microbiological diagnostic performance of metagenomic next-generation sequencing when applied to clinical practice. Clin Infect Dis 2018;67(Suppl_2):S231–S240. DOI: 10.1093/cid/ciy693.
Bruton OC, Apt L, Gitlin D, et al. Absence of serum gamma globulins. AMA Am. J Dis Child 1952;84:632–636.
Pannaraj PS, Pediatric Infectious Disease Society. Available at: https://www.pids.org/news/411-how-next-generation-sequencing-is-changing-pediatric-infectious-diseases.html.
Kwong JC, Mccallum N, Sintchenko V, et al. Whole genome sequencing in clinical and public health microbiology. Pathology 2015;47(3):199–210. DOI: 10.1097/PAT.0000000000000235.
Goldberg B, Sichtig H, Geyer C, et al. Making the leap from research laboratory to clinic: challenges and opportunities for next generation sequencing in infectious disease diagnostics. mBio 2015;6(6):e01888-15. DOI: 10.1128/mBio.01888-15.
Lefterova MI, Suarez CJ, Banaei N, et al. Next-generation sequencing for infectious disease diagnosis and management: a report of the association for molecular pathology. J Mol Diagn 2015;17(6):623–634. DOI: 10.1016/j.jmoldx.2015.07.004.
Bentley DR, Balasubramanian S, Swerdlow HP, et al. Accurate whole human genome sequencing using reversible terminator chemistry. Nature 2008;456(7218):53–59. DOI: 10.1038/nature07517.
Rothberg JM, Hinz W, Rearick TM, et al. An integrated semiconductor device enabling non-optical genome sequencing. Nature 2011;475(7356):348–352. DOI: 10.1038/nature10242.
Greninger AL, Naccache SN, Federman S, et al. Rapid metagenomic identification of viral pathogens in clinical samples by real-time nanopore sequencing analysis. Genome Med 2015;7(1):99. DOI: 10.1186/s13073-015-0220-9.
Radford AD, Chapman D, Dixon L, et al. Application of next-generation sequencing technologies in virology. J Gen Virol 2012;93(Pt 9):1853–1868. DOI: 10.1099/vir.0.043182-0.
Weinstock GM. Genomic approaches to studying the human microbiota. Nature 2012;489(7415):250–256. DOI: 10.1038/nature11553.
Koren S, Schatz MC, Walenz BP, et al. Hybrid error correction and de novo assembly of single-molecule sequencing reads. Nat Biotechnol 2012;30(7):693–700. DOI: 10.1038/nbt.2280.
Gwinn M, MacCannell D, Armstrong GL. Next-generation sequencing of infectious pathogens. JAMA 2019;321(9):893–894. DOI: 10.1001/jama.2018.21669.
Naccache SN, Federman S, Veeraraghavan N, et al. A cloud-compatible bioinformatics pipeline for ultrarapid pathogen identification from next-generation sequencing of clinical samples. Genome Res 2014;24(7):1180–1192. DOI: 10.1101/gr.171934.113.
Wood DE, Salzberg SL. Kraken: ultrafast metagenomic sequence classification using exact alignments. Genome Biol 2014;15(3):R46. DOI: 10.1186/gb-2014-15-3-r46.
Flygare S, Simmon K, Miller C, et al. Taxonomer: an interactive metagenomics analysis portal for universal pathogen detection and host mRNA expression profiling. Genome Biol 2016;17(1):111. DOI: 10.1186/s13059-016-0969-1.
Pan W, Ngo TTM, Camunas-Soler J, et al. Simultaneously monitoring immune response and microbial infections during pregnancy through plasma cfRNA sequencing. Clin Chem 2017;63(11):1695–1704. DOI: 10.1373/clinchem.2017.273888.
Bolger AM, Lohse M, Usadel B. Trimmomatic: a flexible trimmer for Illumina sequence data. Bioinformatics 2014;30(15):2114–2120. DOI: 10.1093/bioinformatics/btu170.
Aanensen DM, Feil EJ, Holden MTG, et al. Whole-genome sequencing for routine pathogen surveillance in public health: a population snapshot of invasive staphylococcus aureus in Europe. mBio 2016;7(3):e00444-16. DOI: 10.1128/mBio.00444-16.
Gibson RM, Schmotzer CL, Quinones-Mateu ME. Next-generation sequencing to help monitor patients infected with HIV: ready for clinical use? Curr Infect Dis Rep 2014;16(4):401. DOI: 10.1007/s11908-014-0401-5.
Lataillade M, Chiarella J, Yang R, et al. Prevalence and clinical significance of HIV drug resistance mutations by ultra-deep sequencing in antiretroviral-naive subjects in the CASTLE study. PLoS One 2010;5(6):e10952. DOI: 10.1371/journal.pone.0010952.
Lurain NS, Chou S. Antiviral drug resistance of human cytomegalovirus. Clin Microbiol Rev 2010;23(4):689–712. DOI: 10.1128/CMR.00009-10.
Zankari E. Comparison of the web tools ARG-ANNOT and ResFinder for detection of resistance genes in bacteria. Antimicrob Agents Chemother 2014;58(8):4986. DOI: 10.1128/AAC.02620-14.
Köser CU, Bryant JM, Becq J, et al. Whole-genome sequencing for rapid susceptibility testing of M. tuberculosis. N Engl J Med 2013;369(3):290–292. DOI: 10.1056/NEJMc1215305.
Salipante SJ, Hoogestraat DR, Abbott AN, et al. Coinfection of Fusobacterium nucleatum and Actinomyces israelii in mastoiditis diagnosed by next-generation DNA sequencing. J Clin Microbiol 2014;52(5):1789–1792. DOI: 10.1128/JCM.03133-13.
Athey TBT, Teatero S, Li A, et al. Deriving group A streptococcus typing information from short-read whole-genome sequencing data. J Clin Microbiol 2014;52(6):1871–1876. DOI: 10.1128/JCM.00029-14.
Zhou K, Lokate M, Deurenberg RH, et al. Characterization of a CTX-M-15 producing Klebsiella pneumoniae outbreak strain assigned to a novel sequence type (1427). Front Microbiol 2015;6:1250. DOI: 10.3389/fmicb.2015.01250.
Weterings V, Zhou K, Rossen JW, et al. An outbreak of colistin-resistant Klebsiella pneumoniae carbapenemase-producing klebsiella pneumoniae in the Netherlands with inter-institutional spread. Eur J Clin Microbiol Infect Dis 2013;34(8):1647–1655. DOI: 10.1007/s10096-015-2401-2.
Bathoorn E, Rossen JW, Lokate M, et al. Isolation of an NDM-5-producing ST16 Klebsiella pneumoniae from a Dutch patient without travel history abroad. Euro Surveil 2015;20(41):30040. DOI: 10.2807/1560-7917.ES.2015.20.41.30040.
Harrison EM, Paterson GK, Holden MTG, et al. Whole genome sequencing identifies zoonotic transmission of MRSA isolates with the novel mecA homologue mecC. EMBO Mol Med 2013;5(4):509–515. DOI: 10.1002/emmm.201202413.
Armstrong AE, Rossoff J, Hollemon D, et al. Cell-free DNA next-generation sequencing successfully detects infectious pathogens in pediatric oncology and hematopoietic stem cell transplant patients at risk for invasive fungal disease. Pediatr Blood Cancer 2019;66(7):e27734. DOI: 10.1002/pbc.27734.
Maschmeyer G. Invasive fungal disease: better survival through early diagnosis and therapeutic intervention. Expert Rev Anti Infect Ther 2011;9(3):279–281. DOI: 10.1586/eri.11.11.
Camargo JF, Ahmed AA, Lindner MS, et al. Next-generation sequencing of microbial cell-free DNA for rapid non-invasive diagnosis of infectious diseases in immunocompromised hosts. F1000 Res 2019;8:1194. DOI: 10.12688/f1000research. 19766.3.
Cheng AP, Burnham P, Lee JR, et al. A cell-free DNA metagenomic sequencing assay that integrates the host injury response to infection. PNAS 2019;116(37):18738–18744. DOI: 10.1073/pnas. 1906320116.
Xing X-W, Zhang J-T, Ma Y-B, et al. Metagenomic next-generation sequencing for diagnosis of infectious encephalitis and meningitis: a large, prospective case series of 213 patients. Front Cell Infect Microbiol 2020;10:88. DOI: 10.3389/fcimb.2020.00088.
Hu Z, Weng X, Xu C, et al. Metagenomic next-generation sequencing as a diagnostic tool for toxoplasmic encephalitis. Ann Clin Microbiol Antimicrob 2018;17(1):45. DOI: 10.1186/s12941-018-0298-1.
Wilson MR, O'Donovan BD, Gelfand JM, et al. Chronic meningitis investigated via metagenomic next-generation sequencing. JAMA Neurol 2018;75(8):947–955. DOI: 10.1001/jamaneurol.2018.0463.
Wilson MR, Sample HA, Zorn KC, et al. Clinical metagenomic sequencing for diagnosis of meningitis and encephalitis. N Engl J Med 2019;380(24):2327–2340. DOI: 10.1056/NEJMoa1803396.