{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"indexed":{"date-parts":[[2026,9,30]],"date-time":"2026-09-30T13:38:16Z","timestamp":1790775496571,"version":"4.1.0"},"reference-count":28,"publisher":"Public Library of Science (PLoS)","issue":"6","license":[{"start":{"date-parts":[[2021,6,17]],"date-time":"2021-06-17T00:00:00Z","timestamp":1623888000000},"content-version":"vor","delay-in-days":0,"URL":"http:\/\/creativecommons.org\/licenses\/by\/4.0\/"}],"content-domain":{"domain":["www.plosone.org"],"crossmark-restriction":false},"short-container-title":["PLoS ONE"],"abstract":"<jats:p>\n                    Classification of clinical symptoms and diagnostic microbiology are essential to effectively employ antimicrobial therapy for lower respiratory tract infections (LRTIs) in a timely manner. Empirical antibiotic treatment without microbial identification hinders the selective use of narrow-spectrum antibiotics and effective patient treatment. Thus, the development of rapid and accurate diagnostic procedures that can be readily adopted by the clinic is necessary to minimize non-essential or excessive use of antibiotics and accelerate patient recovery from LRTI-induced damage. We developed and validated a multiplex real-time polymerase chain reaction (mRT-PCR) assay with good analytical performance and high specificity to simultaneously detect four bacterial pathogens causing pneumonia:\n                    <jats:italic>Klebsiella pneumoniae<\/jats:italic>\n                    ,\n                    <jats:italic>Pseudomonas aeruginosa<\/jats:italic>\n                    ,\n                    <jats:italic>Staphylococcus aureus<\/jats:italic>\n                    , and\n                    <jats:italic>Moraxella catarrhalis<\/jats:italic>\n                    . The analytical performance of mRT-PCR against target pathogens was evaluated by the limit of detection (LOD), specificity, and repeatability. Two hundred and ten clinical specimens from pneumonia patients were processed using an automatic nucleic acid extraction system for the \u201crespiratory bacteria four\u201d (RB4) mRT-PCR assay, and the results were directly compared to references from bacterial culture and\/or Sanger sequencing. The RB4 mRT-PCR assay detected all target pathogens from sputum specimens with a coefficient of variation ranging from 0.29 to 1.71 and conservative LOD of DNA corresponding to 5 \u00d7 10\n                    <jats:sup>2<\/jats:sup>\n                    copies\/reaction. The concordance of the assay with reference-positive specimens was 100%, and additional bacterial infections were detected from reference-negative specimens. Overall, the RB4 mRT-PCR assay showed a more rapid turnaround time and higher performance that those of reference assays. The RB4 mRT-PCR assay is a high-throughput and reliable tool that assists decision-making assessment and outperforms other standard methods. This tool supports patient management by considerably reducing the inappropriate use of antibiotics.\n                  <\/jats:p>","DOI":"10.1371\/journal.pone.0253402","type":"journal-article","created":{"date-parts":[[2021,6,17]],"date-time":"2021-06-17T13:38:11Z","timestamp":1623937091000},"page":"e0253402","update-policy":"https:\/\/doi.org\/10.1371\/journal.pone.corrections_policy","source":"Crossref","is-referenced-by-count":23,"title":["Development of a multiplex real-time PCR assay for the simultaneous detection of four bacterial pathogens causing pneumonia"],"prefix":"10.1371","volume":"16","author":[{"ORCID":"https:\/\/orcid.org\/0000-0003-0115-5170","authenticated-orcid":true,"given":"Ho Jae","family":"Lim","sequence":"first","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Eun-Rim","family":"Kang","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Min Young","family":"Park","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Bo Kyung","family":"Kim","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Min Jin","family":"Kim","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-1450-8150","authenticated-orcid":true,"given":"Sunkyung","family":"Jung","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Kyoung Ho","family":"Roh","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Nackmoon","family":"Sung","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Jae-Hyun","family":"Yang","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Min-Woo","family":"Lee","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Sun-Hwa","family":"Lee","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-6153-9646","authenticated-orcid":true,"given":"Yong-Jin","family":"Yang","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]}],"member":"340","published-online":{"date-parts":[[2021,6,17]]},"reference":[{"issue":"6","key":"pone.0253402.ref001","doi-asserted-by":"crossref","first-page":"920","DOI":"10.1016\/j.jmii.2019.10.009","article-title":"Performance of a multiplex PCR pneumonia panel for the identification of respiratory pathogens and the main determinants of resistance from the lower respiratory tract specimens of adult patients in intensive care units","volume":"52","author":"SH Lee","year":"2019","journal-title":"J Microbiol Immunol Infect"},{"key":"pone.0253402.ref002","doi-asserted-by":"crossref","first-page":"4834396","DOI":"10.1155\/2019\/4834396","article-title":"Bacterial Aetiologies of Lower Respiratory Tract Infections among Adults in Yaounde, Cameroon","volume":"2019","author":"S Tchatchouang","year":"2019","journal-title":"Biomed Res Int"},{"issue":"1","key":"pone.0253402.ref003","doi-asserted-by":"crossref","first-page":"75","DOI":"10.1136\/thx.2004.027441","article-title":"Aetiological role of viral and bacterial infections in acute adult lower respiratory tract infection (LRTI) in primary care","volume":"61","author":"DD Creer","year":"2006","journal-title":"Thorax"},{"issue":"2","key":"pone.0253402.ref004","doi-asserted-by":"crossref","first-page":"108","DOI":"10.5578\/tt.68421","article-title":"Comparative analysis of the patients with community-acquired pneumonia (CAP) and health care-associated pneumonia (HCAP) requiring hospitalization","volume":"67","author":"S Kara","year":"2019","journal-title":"Tuberk Toraks"},{"issue":"6","key":"pone.0253402.ref005","doi-asserted-by":"crossref","first-page":"839","DOI":"10.1055\/s-0036-1592075","article-title":"Community-Acquired Pneumonia in the Asia-Pacific Region","volume":"37","author":"JH Song","year":"2016","journal-title":"Semin Respir Crit Care Med."},{"issue":"1","key":"pone.0253402.ref006","doi-asserted-by":"crossref","first-page":"179","DOI":"10.1186\/s12890-019-0944-1","article-title":"Severe community-acquired pneumonia in general medical wards: outcomes and impact of initial antibiotic selection","volume":"19","author":"P Wongsurakiat","year":"2019","journal-title":"BMC Pulm Med"},{"issue":"2","key":"pone.0253402.ref007","doi-asserted-by":"crossref","first-page":"134","DOI":"10.1016\/j.jmii.2011.09.004","article-title":"Antimicrobial resistance of Moraxella catarrhalis isolates in Taiwan","volume":"45","author":"SF Hsu","year":"2012","journal-title":"J Microbiol Immunol Infect"},{"key":"pone.0253402.ref008","doi-asserted-by":"crossref","first-page":"326","DOI":"10.1016\/j.ijid.2020.03.024","article-title":"Evaluation of the BioFire FilmArray Pneumonia Panel for rapid detection of respiratory bacterial pathogens and antibiotic resistance genes in sputum and endotracheal aspirate specimens","volume":"95","author":"IY Yoo","year":"2020","journal-title":"Int J Infect Dis"},{"issue":"8","key":"pone.0253402.ref009","doi-asserted-by":"crossref","first-page":"788 e1","DOI":"10.1016\/j.cmi.2015.05.004","article-title":"Development of two real-time multiplex PCR assays for the detection and quantification of eight key bacterial pathogens in lower respiratory tract infections","volume":"21","author":"NJ Gadsby","year":"2015","journal-title":"Clin Microbiol Infect"},{"issue":"2","key":"pone.0253402.ref010","doi-asserted-by":"crossref","first-page":"202","DOI":"10.1086\/648678","article-title":"Etiology of community-acquired pneumonia: increased microbiological yield with new diagnostic methods","volume":"50","author":"N Johansson","year":"2010","journal-title":"Clin Infect Dis"},{"issue":"2","key":"pone.0253402.ref011","doi-asserted-by":"crossref","first-page":"479","DOI":"10.1128\/JCM.02045-16","article-title":"Performance Evaluation of Allplex Respiratory Panels 1, 2, and 3 for Detection of Respiratory Viruses and Influenza A Virus Subtypes","volume":"55","author":"HJ Huh","year":"2017","journal-title":"J Clin Microbiol"},{"issue":"10","key":"pone.0253402.ref012","doi-asserted-by":"crossref","first-page":"1642","DOI":"10.3346\/jkms.2017.32.10.1642","article-title":"Clinical Characteristics of Macrolide-Resistant Mycoplasma pneumoniae from Children in Jeju","volume":"32","author":"YJ Kim","year":"2017","journal-title":"J Korean Med Sci."},{"issue":"6","key":"pone.0253402.ref013","doi-asserted-by":"crossref","first-page":"1911","DOI":"10.1128\/JCM.00389-14","article-title":"Multiplex real-time PCR assay for rapid detection of methicillin-resistant staphylococci directly from positive blood cultures","volume":"52","author":"HY Wang","year":"2014","journal-title":"J Clin Microbiol"},{"issue":"2","key":"pone.0253402.ref014","doi-asserted-by":"crossref","first-page":"111","DOI":"10.7774\/cevr.2018.7.2.111","article-title":"Evaluation of EZplex MTBC\/NTM Real-Time PCR kit: diagnostic accuracy and efficacy in vaccination","volume":"7","author":"S Lee","year":"2018","journal-title":"Clin Exp Vaccine Res"},{"issue":"6","key":"pone.0253402.ref015","doi-asserted-by":"crossref","first-page":"e40","DOI":"10.1093\/nar\/gkm051","article-title":"Dual priming oligonucleotide system for the multiplex detection of respiratory viruses and SNP genotyping of CYP2C19 gene","volume":"35","author":"JY Chun","year":"2007","journal-title":"Nucleic Acids Res"},{"key":"pone.0253402.ref016","doi-asserted-by":"crossref","first-page":"64","DOI":"10.1186\/s12866-015-0396-6","article-title":"Development of a multiplex-PCR probe system for the proper identification of Klebsiella variicola","volume":"15","author":"U Garza-Ramos","year":"2015","journal-title":"BMC Microbiol"},{"issue":"1","key":"pone.0253402.ref017","doi-asserted-by":"crossref","first-page":"121","DOI":"10.1016\/j.scitotenv.2007.02.039","article-title":"Application of real-time quantitative PCR for the detection of selected bacterial pathogens during municipal wastewater treatment","volume":"382","author":"KE Shannon","year":"2007","journal-title":"Sci Total Environ"},{"issue":"3","key":"pone.0253402.ref018","doi-asserted-by":"crossref","first-page":"590","DOI":"10.1016\/j.burns.2014.08.018","article-title":"Identification of methicillin-resistant Staphylococcus aureus (MRSA) strains isolated from burn patients by multiplex PCR","volume":"41","author":"EA Montazeri","year":"2015","journal-title":"Burns"},{"key":"pone.0253402.ref019","doi-asserted-by":"crossref","first-page":"2335","DOI":"10.2147\/IDR.S213296","article-title":"The prevalence of respiratory pathogens in adults with community-acquired pneumonia in an outpatient cohort.","volume":"12","author":"J Chen","year":"2019","journal-title":"Infect Drug Resist"},{"key":"pone.0253402.ref020","doi-asserted-by":"crossref","first-page":"382","DOI":"10.3389\/fcimb.2019.00382","article-title":"Design and Evaluation of a Novel Multiplex Real-Time PCR Melting Curve Assay for the Simultaneous Detection of Nine Sexually Transmitted Disease Pathogens in Genitourinary Secretions","volume":"9","author":"XM Hu","year":"2019","journal-title":"Front Cell Infect Microbiol"},{"issue":"3","key":"pone.0253402.ref021","doi-asserted-by":"crossref","first-page":"368","DOI":"10.1099\/jmm.0.000917","article-title":"Detection and identification of bacterial pathogens directly from sputum samples by pyrosequencing","volume":"68","author":"J Lyu","year":"2019","journal-title":"J Med Microbiol"},{"issue":"3\u20134","key":"pone.0253402.ref022","doi-asserted-by":"crossref","first-page":"55","DOI":"10.2739\/kurumemedj.55.55","article-title":"Evaluation of a real-time PCR assay for the diagnosis of Pneumocystis pneumonia","volume":"55","author":"K. Etoh","year":"2008","journal-title":"Kurume Med J"},{"issue":"11","key":"pone.0253402.ref023","doi-asserted-by":"crossref","first-page":"1487","DOI":"10.1038\/aps.2010.115","article-title":"Selection of reliable reference genes for gene expression study in nasopharyngeal carcinoma","volume":"31","author":"Y Guo","year":"2010","journal-title":"Acta Pharmacol Sin"},{"issue":"6","key":"pone.0253402.ref024","doi-asserted-by":"crossref","first-page":"733","DOI":"10.3346\/jkms.2015.30.6.733","article-title":"Acceptability of sputum specimens for diagnosing pulmonary tuberculosis","volume":"30","author":"YJ Lee","year":"2015","journal-title":"J Korean Med Sci"},{"issue":"11","key":"pone.0253402.ref025","doi-asserted-by":"crossref","DOI":"10.3390\/v9110324","article-title":"Clinical Relevance of Total HIV DNA in Peripheral Blood Mononuclear Cell Compartments as a Biomarker of HIV-Associated Neurocognitive Disorders (HAND)","volume":"9","author":"V Ruhanya","year":"2017","journal-title":"Viruses"},{"issue":"5","key":"pone.0253402.ref026","doi-asserted-by":"crossref","first-page":"1303","DOI":"10.4269\/ajtmh.17-0214","article-title":"Detection of Histoplasma capsulatum in Organic Fertilizers by Hc100 Nested Polymerase Chain Reaction and Its Correlation with the Physicochemical and Microbiological Characteristics of the Samples","volume":"98","author":"LF Gomez","year":"2018","journal-title":"Am J Trop Med Hyg"},{"key":"pone.0253402.ref027","doi-asserted-by":"crossref","first-page":"92","DOI":"10.1016\/j.ijfoodmicro.2016.12.015","article-title":"Development of quantitative real-time PCR for detection and enumeration of Enterobacteriaceae","volume":"246","author":"H Takahashi","year":"2017","journal-title":"Int J Food Microbiol"},{"issue":"6","key":"pone.0253402.ref028","doi-asserted-by":"crossref","first-page":"683","DOI":"10.5858\/arpa.2017-0573-RA","article-title":"Diagnosis of Infectious Diseases in the Lower Respiratory Tract: A Cytopathologist\u2019s Perspective","volume":"143","author":"RJ Baldassarri","year":"2019","journal-title":"Arch Pathol Lab Med"}],"container-title":["PLOS ONE"],"original-title":[],"language":"en","link":[{"URL":"https:\/\/dx.plos.org\/10.1371\/journal.pone.0253402","content-type":"unspecified","content-version":"vor","intended-application":"similarity-checking"}],"deposited":{"date-parts":[[2021,6,17]],"date-time":"2021-06-17T13:38:30Z","timestamp":1623937110000},"score":1,"resource":{"primary":{"URL":"https:\/\/dx.plos.org\/10.1371\/journal.pone.0253402"}},"subtitle":[],"editor":[{"given":"Ruslan","family":"Kalendar","sequence":"first","affiliation":[],"role":[{"vocabulary":"crossref","role":"editor"}]}],"short-title":[],"issued":{"date-parts":[[2021,6,17]]},"references-count":28,"journal-issue":{"issue":"6","published-online":{"date-parts":[[2021,6,17]]}},"URL":"https:\/\/doi.org\/10.1371\/journal.pone.0253402","relation":{},"ISSN":["1932-6203"],"issn-type":[{"value":"1932-6203","type":"electronic"}],"subject":[],"published":{"date-parts":[[2021,6,17]]}}}