Abstract

The successful application of metagenomic next-generation sequencing (mNGS; or clinical metagenomics) for the diagnosis of infectious diseases directly from a clinical specimen generated significant interest from a high-profile report nearly a decade ago [ [1] Wilson M.R. Naccache S.N. Samayoa E. Biagtan M. Bashir H. Yu G. Salamat S.M. Somasekar S. Federman S. Miller S. Sokolic R. Garabedian E. Candotti F. Buckley R.H. Reed K.D. Meyer T.L. Seroogy C.M. Galloway R. Henderson S.L. Gern J.E. DeRisi J.L. Chiu C.Y. Actionable diagnosis of neuroleptospirosis by next-generation sequencing. N Engl J Med. 2014; 370: 2408-2417 Crossref PubMed Scopus (578) Google Scholar ]. Several case series have since emerged, generating further interest, and building momentum for clinical use. Metagenomic NGS holds promise to improve and streamline the diagnosis of infectious diseases due to its untargeted nature, facilitating organism detection without a priori knowledge of the differential diagnosis. However, mNGS requires more advanced molecular and bioinformatics expertise and is more expensive than conventional microbiologic testing. Currently, mNGS test ordering is driven by local institutional practice, often ad hoc as a test of last resort, and with inconsistent oversight by laboratory medicine and infectious diseases specialists [ [2] Simner P.J. Miller S. Carroll K.C. Understanding the Promises and Hurdles of Metagenomic Next-Generation Sequencing as a Diagnostic Tool for Infectious Diseases. Clin Infect Dis. 2018; 66: 778-788 Crossref PubMed Scopus (321) Google Scholar ]. The evidence base supporting clinical mNGS has been largely retrospective and limited by the lack of standardized definitions, variable mNGS timing in the care pathway, and need for stratification by specific patient population or clinical syndrome (Table 1). Reviewing this evidence, we note that several studies report that the positive clinical impact of plasma mNGS is strongest in immunocompromised hosts. However, positive impact varied widely across studies, and negative impact has also been reported. Furthermore, most studies highlight the unmet need for prospective studies and clinical correlation of plasma mNGS results, both of which we seek to address with the current study design. Table 1Review of plasma mNGS studies assessing test performance and/or clinical impact StudyReference a Number, BCx: blood culture; FN: febrile neutropenia; FUO: fever of unknown origin; GI: gastrointestinal; GM: galactomannan; IC: immunocompromised; ID: infectious diseases; IE: infective endocarditis; IFI: invasive fungal infection; Micro: microbiology; mNGS: metagenomic next-generation sequencing; MPM: molecules per microlitre; NA: not available; Neg: negative; NPA: negative percent agreement; Poly: polymicrobial; Pos: positivity; PPA: positive percent agreement; Resp: respiratory; Sen: sensitivity; SOTR: solid organ transplant recipient; Spe: specificity. Sites (location) a Number, BCx: blood culture; FN: febrile neutropenia; FUO: fever of unknown origin; GI: gastrointestinal; GM: galactomannan; IC: immunocompromised; ID: infectious diseases; IE: infective endocarditis; IFI: invasive fungal infection; Micro: microbiology; mNGS: metagenomic next-generation sequencing; MPM: molecules per microlitre; NA: not available; Neg: negative; NPA: negative percent agreement; Poly: polymicrobial; Pos: positivity; PPA: positive percent agreement; Resp: respiratory; Sen: sensitivity; SOTR: solid organ transplant recipient; Spe: specificity. participants a Number, BCx: blood culture; FN: febrile neutropenia; FUO: fever of unknown origin; GI: gastrointestinal; GM: galactomannan; IC: immunocompromised; ID: infectious diseases; IE: infective endocarditis; IFI: invasive fungal infection; Micro: microbiology; mNGS: metagenomic next-generation sequencing; MPM: molecules per microlitre; NA: not available; Neg: negative; NPA: negative percent agreement; Poly: polymicrobial; Pos: positivity; PPA: positive percent agreement; Resp: respiratory; Sen: sensitivity; SOTR: solid organ transplant recipient; Spe: specificity. tests % peds % IC Study design Indication for testing (%) ID/micro approval required Primary Outcome Secondary Outcomes Pos rate (% poly) Main findings Benamu et al. 2022 [ [13] Benamu E. Gajurel K. Anderson J.N. Lieb T. Gomez C.A. Seng H. Aquino R. Hollemon D. Hong D.K. Blauwkamp T.A. Kertesz M. Blair L. Bollyky P.L. Medeiros B.C. Coutre S. Zompi S. Montoya J.G. Deresinski S. Plasma Microbial Cell-free DNA Next-generation Sequencing in the Diagnosis and Management of Febrile Neutropenia. Clin Infect Dis. 2022; 74: 1659-1668 Crossref PubMed Scopus (28) Google Scholar ] 1 (Stanford, USA) 55 151 0 100% Prospective observational FN (100%) NA, tested if fit study criteria Diagnostic performance compared to composite reference (conventional micro, radiology, and clinical adjudication by ID) Diagnostic performance on subsequent testing 85% (61%) Sen 92%/Spe 70% Time to diagnosis Earlier time to diagnosis in 87% Anticipated change in antimicrobial management Potential early optimization of antimicrobials in 47% Hill et al. 2020 [ [14] Hill J.A. Dalai S.C. Hong D.K. Ahmed A.A. Ho C. Hollemon D. Blair L. Maalouf J. Keane-Candib J. Stevens-Ayers T. Boeckh M. Blauwkamp T.A. Fisher C.E. Liquid Biopsy for Invasive Mold Infections in Hematopoietic Cell Transplant Recipients With Pneumonia Through Next-Generation Sequencing of Microbial Cell-Free DNA in Plasma. Clin Infect Dis. 2021; 73: e3876-e3883 Crossref PubMed Scopus (21) Google Scholar ] 1 (Seattle, USA) 114 114 0 100% Retrospective observational Suspected pulmonary IFI (100%) NA, tested if fit study criteria Diagnostic performance compared to conventional micro, clinical adjudication for discrepant cases - 51% (5/75) p/p pulmonary IFI: Sen 51%/Spe 100% p/p non-Aspergillus IFI: Sen 79% p/p Aspergillus IFI: Sen 31% mNGS complementary to GM Rossoff et al. 2019 [ [15] Rossoff J. Chaudhury S. Soneji M. Patel S.J. Kwon S. Armstrong A. Muller W.J. Noninvasive Diagnosis of Infection Using Plasma Next-Generation Sequencing: A Single-Center Experience. Open Forum Infect Dis. 2019; 6 Crossref PubMed Scopus (61) Google Scholar ] 1 (Chicago, USA) 79 100 100% 76% Retrospective chart review Suspected IFI (55%) No, but 94% ordered by ID Diagnostic performance compared to conventional micro, clinical adjudication for discrepant cases - 70.0% (33%) Sen 92%/Spe 64% (overall) Fever/sepsis (22%) Sen 93%/Spe 59% (IC) Prolonged/recurrent fever (18%) 56 (80%) clinically relevant Lymphadenopathy (4%) 14 identified by mNGS only Niles et al, 2020 [ [16] Niles D.T. Wijetunge D.S.S. Palazzi D.L. Singh I.R. Revell P.A. Plasma Metagenomic Next-Generation Sequencing Assay for Identifying Pathogens: a Retrospective Review of Test Utilization in a Large Children's Hospital. J Clin Microbiol. 2020; 58 Crossref PubMed Scopus (21) Google Scholar ] 1 (Houston, USA) 60 60 100% 62% Retrospective chart review Lung lesion (27%) No Diagnostic performance compared to conventional micro Time to diagnosis 63% (42%) PPA 61%/NPA 58% Unclear (20%) Conventional time to diagnosis 3.5 days earlier FN (10%) Change in therapy When mNGS identified new organism, no change in therapy in 74% Sepsis (10%) Lee et al. 2020[ [17] Lee R.A. Al Dhaheri F. Pollock N.R. Sharma T.S. Assessment of the Clinical Utility of Plasma Metagenomic Next-Generation Sequencing in a Pediatric Hospital Population. J Clin Microbiol. 2020; 58 Crossref Scopus (32) Google Scholar ] 1 (Boston, USA) 54 59 100% 56% Retrospective chart review Resp (31%) Yes Clinical impact of mNGS compared to conventional micro Diagnostic performance compared to conventional micro 49% (35%) Clinical impact in 14% FUO (19%) Organism clinical relevance IC status associated with significant clinical impact Multisite (15%) Effect of treatment on mNGS result PPA 53%/NPA 79% Cardiac (14%) Interpretation of MPM of plasma Hogan et al. 2021[ [18] Hogan C.A. Yang S. Garner O.B. Green D.A. Gomez C.A. Dien Bard J. Pinsky B.A. Banaei N. Clinical Impact of Metagenomic Next-Generation Sequencing of Plasma Cell-Free DNA for the Diagnosis of Infectious Diseases: A Multicenter Retrospective Cohort Study. Clin Infect Dis. 2021; 72: 239-245 Crossref PubMed Scopus (89) Google Scholar ] 5 (Stanford, Los Angeles (2), New York, Salt Lake City, USA) 82 98 52.40% 65% Retrospective chart review FUO (23%) Initially no then yes through study period Clinical impact of mNGS compared to conventional micro Clinical impact of mNGS compared to conventional micro for repeat testing 61.0% (50%) No impact 86.6% Resp (13%) Pos impact 7.3% Sepsis (10%) Neg impact 3.7% IE (9%) FN (7%) Wilke et al. 2021[ [19] Wilke J. Ramchandar N. Cannavino C. Pong A. Tremoulet A. Padua L.T. Harvey H. Foley J. Farnaes L. Coufal N.G. Clinical application of cell-free next-generation sequencing for infectious diseases at a tertiary children's hospital. BMC Infect Dis. 2021; 21: 552 Crossref PubMed Scopus (7) Google Scholar ] 1 (San Diego, USA) 110 142 100% 33% Retrospective chart review Clinical symptoms suggestive of infection (48%) Yes Diagnostic performance compared to conventional micro Clinical impact of mNGS compared to conventional micro for repeat testing 74% (56%) PPA 90%/NPA 52% Focal imaging finding (27%) Clinical impact in 32% Shishido et al. 2022[ [20] Shishido A.A. Noe M. Saharia K. Luethy P. Clinical impact of a metagenomic microbial plasma cell-free DNA next-generation sequencing assay on treatment decisions: a single-center retrospective study. BMC Infect Dis. 2022; 22: 372 Crossref PubMed Scopus (6) Google Scholar ] 1 (Baltimore, USA) 80 80 0% 56% Retrospective chart review Resp (31%) Yes Clinical impact of mNGS compared to conventional micro - 61% (51%) No impact 55% Sepsis (15%) Pos impact 43% IE (13%) Neg impact 3% FUO (10%) Highest impact in SOTR, sepsis, and individuals on antimicrobial therapy for <7 days Niles et al. 2022[ [21] Niles D.T. Revell P.A. Ruderfer D. Marquez L. McNeil J.C. Palazzi D.L. Clinical Impact of Plasma Metagenomic Next-generation Sequencing in a Large Pediatric Cohort. Pediatr Infect Dis J. 2022; 41: 166-171 Crossref PubMed Scopus (4) Google Scholar ] 1 (Houston, USA) 169 169 100% 76% Retrospective chart review Deep-seated infection (49%) No Clinical impact of mNGS compared to conventional micro Clinical impact of mNGS compared to conventional micro for repeat testing 64% (47%) No impact 82% Resp (30%) Pos impact 12% GI (11%) Neg impact 5% Lymphadenopathy (7%) Highest yield in IC (56 vs 30%) Vijayvargiya et al. 2022[ [22] Vijayvargiya P. Feri A. Mairey M. Rouillon C. Jeraldo P.R. Esquer Garrigos Z. Thoendel M.J. Greenwood-Quaintance K.E. Sohail M.R. Sampathkumar P. Spychalla M.T. Stewart A.K. Patnaik M.M. Tande A.J. Cruveiller S. Hannet I. Beurdeley P. Patel R. Metagenomic shotgun sequencing of blood to identify bacteria and viruses in leukemic febrile neutropenia. PLoS One. 2022; 17e0269405 Crossref PubMed Scopus (1) Google Scholar ] 1 (Rochester, USA) 20 60 0% 100% Prospective observational FN (100%) NA, tested if fit study criteria Clinical impact of metagenome shotgun sequencing compared to conventional micro - 15% ‘clinically relevant’ (0%) No concordance with BCx for bacterial targets supports non-infectious etiology of FN Vissichelli et al. 2023 [ [23] Vissichelli N.C. Morales M.K. Kolipakkam B. Bryson A. Sabo R.T. Toor A.A. Cell-free next-generation sequencing impacts diagnosis and antimicrobial therapy in immunocompromised hosts: A retrospective study. Transpl Infect Dis. 2023; 25e13954 Crossref PubMed Scopus (1) Google Scholar ] 1 (Richmond, USA) 36 36 0% 92% Retrospective chart review Pneumonia (72%) NA Clinical impact of mNGS compared to conventional micro - 58% (NA) Higher impact in immunocompromised hosts; driven by antimicrobial initiation and de-escalation Fever (67%) Hepatosplenic lesions (5%) Invasive sinusitis (3%) Schulz et al. 2022[ [24] Schulz E. Grumaz S. Hatzl S. Gornicec M. Valentin T. Huber-Krassnitzer B. Kriegl L. Uhl B. Deutsch A. Greinix H. Krause R. Neumeister P. Pathogen Detection by Metagenomic Next-Generation Sequencing During Neutropenic Fever in Patients With Hematological Malignancies. Open Forum Infect Dis. 2022; 9: ofac393 Crossref PubMed Scopus (2) Google Scholar ] 1 (Graz, Austria) 60 97 0% 100% Prospective observational FN (100%) NA, tested if fit study criteria Diagnostic performance compared to blood cultures - 43% (36%) PPA 85%/NPA 63% Sen bacteria 40% Sen fungi 19% Grumaz et al. 2019[ [25] Grumaz S. Grumaz C. Vainshtein Y. Stevens P. Glanz K. Decker S.O. Hofer S. Weigand M.A. Brenner T. Sohn K. Enhanced Performance of Next-Generation Sequencing Diagnostics Compared With Standard of Care Microbiological Diagnostics in Patients Suffering From Septic Shock. Crit Care Med. 2019; 47: e394-e402 Crossref PubMed Scopus (60) Google Scholar ] 1 (Heidelberg, Germany) 48 239 0% NA Retrospective chart review Sepsis (100%);Abdominal source (90%), lung source (8%), genitourinary (2%) NA Diagnostic performance compared to conventional micro Change in therapy would have been warranted 72% at sepsis onset; 71% overall (56%) 95% of the mNGS findings were plausible; 53% would have led to change in clinical intervention a Number, BCx: blood culture; FN: febrile neutropenia; FUO: fever of unknown origin; GI: gastrointestinal; GM: galactomannan; IC: immunocompromised; ID: infectious diseases; IE: infective endocarditis; IFI: invasive fungal infection; Micro: microbiology; mNGS: metagenomic next-generation sequencing; MPM: molecules per microlitre; NA: not available; Neg: negative; NPA: negative percent agreement; Poly: polymicrobial; Pos: positivity; PPA: positive percent agreement; Resp: respiratory; Sen: sensitivity; SOTR: solid organ transplant recipient; Spe: specificity. 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