{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"indexed":{"date-parts":[[2026,5,7]],"date-time":"2026-05-07T18:13:06Z","timestamp":1778177586860,"version":"3.51.4"},"reference-count":20,"publisher":"Public Library of Science (PLoS)","issue":"5","license":[{"start":{"date-parts":[[2026,5,7]],"date-time":"2026-05-07T00:00:00Z","timestamp":1778112000000},"content-version":"vor","delay-in-days":0,"URL":"http:\/\/creativecommons.org\/licenses\/by\/4.0\/"}],"funder":[{"name":"Johns Hopkins Center of Excellence for Influenza Research and Response","award":["HHS 75N93021C00045"],"award-info":[{"award-number":["HHS 75N93021C00045"]}]},{"name":"Johns Hopkins Center of Excellence for Influenza Research and Response","award":["HHS N272201400007C"],"award-info":[{"award-number":["HHS N272201400007C"]}]},{"DOI":"10.13039\/100000002","name":"National Institutes of Health","doi-asserted-by":"publisher","award":["NIH R35-GM130151"],"award-info":[{"award-number":["NIH R35-GM130151"]}],"id":[{"id":"10.13039\/100000002","id-type":"DOI","asserted-by":"publisher"}]}],"content-domain":{"domain":["www.plosone.org"],"crossmark-restriction":false},"short-container-title":["PLoS One"],"abstract":"<jats:sec id=\"sec001\">\n                    <jats:title>Objectives<\/jats:title>\n                    <jats:p>Accurate diagnosis of existing and emerging respiratory pathogens is important. We evaluated the capability of unbiased metagenomic next generation sequencing (mNGS) to identify pathogenic RNA viruses from two cohorts of nasopharyngeal (NP) swabs previously tested by commercial multiplex respiratory diagnostics.<\/jats:p>\n                  <\/jats:sec>\n                  <jats:sec id=\"sec002\">\n                    <jats:title>Methods<\/jats:title>\n                    <jats:p>NP swabs (N\u2009=\u2009100) in viral transport media (VTM) were assessed using mNGS for this study. Cohort 1 (N\u2009=\u200952) consisted of symptomatic individuals who tested negative for SARS-CoV-2, influenza A\/B, and RSV by the Xpert Xpress CoV-2\/Flu\/RSV Plus multiplex respiratory virus panel and were tested by mNGS for undetected pathogens. Cohort 2 (N\u2009=\u200948) included symptomatic individuals who were positive (N\u2009=\u200926) or negative (N\u2009=\u200922) by the ePlex RP2 multiplex respiratory pathogen panel. Samples were positive for influenza A (N\u2009=\u20098), rhinovirus\/enterovirus (N\u2009=\u20095), RSV (N\u2009=\u20094), adenovirus (N\u2009=\u20093), parainfluenza (N\u2009=\u20092), seasonal coronaviruses (N\u2009=\u20092), and human metapneumovirus (N\u2009=\u20091), as well as a rhinovirus\/enterovirus\/human metapneumovirus co-infected sample (N\u2009=\u20091). mNGS results were compared with ePlex RP2 findings, and symptomatic negative samples were evaluated for additional pathogen detection.<\/jats:p>\n                  <\/jats:sec>\n                  <jats:sec id=\"sec003\">\n                    <jats:title>Results<\/jats:title>\n                    <jats:p>\n                      Cohort 1 contained 8% (4\/52) viral and 19% (10\/52) bacterial reads. In cohort 2, positive concordance between ePlex RP2 and mNGS was 31% (8\/26). mNGS did not identify any viral reads in ePlex RP2-negative samples. However, it detected other microbial reads, such as\n                      <jats:italic>Acanthamoeba castellanii,<\/jats:italic>\n                      in 21% (10\/48) of samples.\n                    <\/jats:p>\n                  <\/jats:sec>\n                  <jats:sec id=\"sec004\">\n                    <jats:title>Conclusion<\/jats:title>\n                    <jats:p>In this study, targeted multiplex amplification methods demonstrated better overall sensitivity in NPs of symptomatic respiratory individuals than mNGS. Other mNGS approaches may produce different results. This study suggests that mNGS may offer adjunctive information, including the detection of rare pathogens, which may be helpful in some clinical contexts.<\/jats:p>\n                  <\/jats:sec>","DOI":"10.1371\/journal.pone.0347750","type":"journal-article","created":{"date-parts":[[2026,5,7]],"date-time":"2026-05-07T17:35:03Z","timestamp":1778175303000},"page":"e0347750","update-policy":"https:\/\/doi.org\/10.1371\/journal.pone.corrections_policy","source":"Crossref","is-referenced-by-count":0,"title":["Comparison of unbiased metagenomic next generation sequencing to targeted multiplex diagnostic assays for the detection of respiratory viruses"],"prefix":"10.1371","volume":"21","author":[{"given":"Justin","family":"Hardick","sequence":"first","affiliation":[],"role":[{"role":"author","vocabulary":"crossref"}]},{"ORCID":"https:\/\/orcid.org\/0000-0001-8089-2819","authenticated-orcid":true,"given":"Raghavendran","family":"Anantharam","sequence":"additional","affiliation":[],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Jennifer","family":"Lu","sequence":"additional","affiliation":[],"role":[{"role":"author","vocabulary":"crossref"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-8859-7432","authenticated-orcid":true,"given":"Steven L.","family":"Salzberg","sequence":"additional","affiliation":[],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Richard E.","family":"Rothman","sequence":"additional","affiliation":[],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Katherine Z. J.","family":"Fenstermacher","sequence":"additional","affiliation":[],"role":[{"role":"author","vocabulary":"crossref"}]},{"ORCID":"https:\/\/orcid.org\/0000-0003-3248-1761","authenticated-orcid":true,"given":"Andrew","family":"Pekosz","sequence":"additional","affiliation":[],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Annet","family":"Onzia","sequence":"additional","affiliation":[],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Lydia","family":"Nakiyingi","sequence":"additional","affiliation":[],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Yukari C.","family":"Manabe","sequence":"additional","affiliation":[],"role":[{"role":"author","vocabulary":"crossref"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-9038-134X","authenticated-orcid":true,"given":"Abraham J.","family":"Kandathil","sequence":"additional","affiliation":[],"role":[{"role":"author","vocabulary":"crossref"}]}],"member":"340","published-online":{"date-parts":[[2026,5,7]]},"reference":[{"issue":"4","key":"pone.0347750.ref001","doi-asserted-by":"crossref","first-page":"780","DOI":"10.1111\/irv.12967","article-title":"Viral co-infections are associated with increased rates of hospitalization in those with influenza","volume":"16","author":"KL Shannon","year":"2022","journal-title":"Influenza Other Respir Viruses"},{"issue":"2","key":"pone.0347750.ref002","doi-asserted-by":"crossref","first-page":"236","DOI":"10.1111\/irv.12907","article-title":"Impact of coinfection status and comorbidity on disease severity in adult emergency department patients with influenza B","volume":"16","author":"AJ Zapf","year":"2022","journal-title":"Influenza Other Respir Viruses"},{"key":"pone.0347750.ref003","doi-asserted-by":"crossref","first-page":"104737","DOI":"10.1016\/j.jcv.2021.104737","article-title":"Clinical performance of the GenMark Dx ePlex respiratory pathogen panels for upper and lower respiratory tract infections","volume":"135","author":"J Jarrett","year":"2021","journal-title":"J Clin Virol"},{"issue":"6","key":"pone.0347750.ref004","doi-asserted-by":"crossref","first-page":"77","DOI":"10.15585\/mmwr.mm7506a2","article-title":"Respiratory virus activity - United States, July 1, 2024-June 30, 2025","volume":"75","author":"BJ Silk","year":"2026","journal-title":"MMWR Morb Mortal Wkly Rep"},{"issue":"4","key":"pone.0347750.ref005","doi-asserted-by":"crossref","DOI":"10.1128\/spectrum.01248-22","article-title":"Comparative performance of the Luminex NxTAG respiratory pathogen panel, GenMark eSensor respiratory viral panel, and BioFire FilmArray respiratory panel","volume":"10","author":"EB Popowitch","year":"2022","journal-title":"Microbiol Spectr"},{"issue":"2","key":"pone.0347750.ref006","doi-asserted-by":"crossref","first-page":"115352","DOI":"10.1016\/j.diagmicrobio.2021.115352","article-title":"Identification of pathogens from the upper respiratory tract of adult emergency department patients at high risk for influenza complications in a pre-Sars-CoV-2 environment","volume":"100","author":"J Hardick","year":"2021","journal-title":"Diagn Microbiol Infect Dis"},{"issue":"10223","key":"pone.0347750.ref007","doi-asserted-by":"crossref","first-page":"514","DOI":"10.1016\/S0140-6736(20)30154-9","article-title":"A familial cluster of pneumonia associated with the 2019 novel coronavirus indicating person-to-person transmission: a study of a family cluster","volume":"395","author":"JF-W Chan","year":"2020","journal-title":"Lancet"},{"issue":"9","key":"pone.0347750.ref008","doi-asserted-by":"crossref","DOI":"10.1371\/journal.pntd.0012451","article-title":"Metagenomic next generation sequencing of plasma RNA for diagnosis of unexplained, acute febrile illness in Uganda","volume":"18","author":"AJ Kandathil","year":"2024","journal-title":"PLoS Negl Trop Dis"},{"issue":"1","key":"pone.0347750.ref009","doi-asserted-by":"crossref","first-page":"21032","DOI":"10.1038\/s41598-022-25314-y","article-title":"Diagnostic accuracy of metagenomic next-generation sequencing in diagnosing infectious diseases: a meta-analysis","volume":"12","author":"J Liu","year":"2022","journal-title":"Sci Rep"},{"issue":"5","key":"pone.0347750.ref010","doi-asserted-by":"crossref","DOI":"10.1002\/rmv.2375","article-title":"Detection of respiratory viruses directly from clinical samples using next-generation sequencing: a literature review of recent advances and potential for routine clinical use","volume":"32","author":"X Wang","year":"2022","journal-title":"Rev Med Virol"},{"issue":"1","key":"pone.0347750.ref011","doi-asserted-by":"crossref","first-page":"1","DOI":"10.7326\/M17-0085","article-title":"Presence of human hepegivirus-1 in a cohort of people who inject drugs","volume":"167","author":"AJ Kandathil","year":"2017","journal-title":"Ann Intern Med"},{"key":"pone.0347750.ref012","unstructured":"Xpert\u00ae Xpress CoV-2\/Flu\/RSV plus; 2023 [cited 2026 Mar 3]. Available from: https:\/\/www.fda.gov\/media\/152163\/download"},{"key":"pone.0347750.ref013","unstructured":"Cobas\u00ae eplex respiratory pathogen panel 2; 2024 [cited 2026 Mar 3]. 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