{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"indexed":{"date-parts":[[2026,7,30]],"date-time":"2026-07-30T00:58:44Z","timestamp":1785373124056,"version":"3.55.0"},"reference-count":47,"publisher":"Wiley","issue":"14","license":[{"start":{"date-parts":[[2026,6,4]],"date-time":"2026-06-04T00:00:00Z","timestamp":1780531200000},"content-version":"vor","delay-in-days":0,"URL":"http:\/\/creativecommons.org\/licenses\/by-nc-nd\/4.0\/"},{"start":{"date-parts":[[2026,6,4]],"date-time":"2026-06-04T00:00:00Z","timestamp":1780531200000},"content-version":"tdm","delay-in-days":0,"URL":"http:\/\/doi.wiley.com\/10.1002\/tdm_license_1.1"}],"content-domain":{"domain":["onlinelibrary.wiley.com"],"crossmark-restriction":true},"short-container-title":["Clinical Laboratory Analysis"],"published-print":{"date-parts":[[2026,7]]},"abstract":"<jats:title>ABSTRACT<\/jats:title>\n                  <jats:sec>\n                    <jats:title>Background<\/jats:title>\n                    <jats:p>\n                      Bloodstream infections (BSIs) caused by multidrug\u2010resistant bacteria (MDR) are a leading cause of mortality and morbidity among hospitalized patients. Extended\u2010Spectrum Beta\u2010Lactamase (ESBL), carbapenem\u2010resistant\n                      <jats:italic>Enterobacterales<\/jats:italic>\n                      (CRE), and colistin resistance are critically important antibiotic\u2010resistant bacteria. The rapid identification of these critical antibiotic resistance genes from positive blood cultures is essential for accelerating the diagnostic turnaround time, enabling early therapy, and supporting effective antibiotic stewardship programs.\n                    <\/jats:p>\n                  <\/jats:sec>\n                  <jats:sec>\n                    <jats:title>Methods<\/jats:title>\n                    <jats:p>\n                      Recombinase Polymerase Amplification (RPA) integrated with an in\u2010house Lateral Flow Assay (LFA) for duplex detection of\n                      <jats:italic>bla<\/jats:italic>\n                      <jats:sub>CTX\u2010M<\/jats:sub>\n                      \/\n                      <jats:italic>mcr\u20101<\/jats:italic>\n                      and\n                      <jats:italic>bla<\/jats:italic>\n                      <jats:sub>oxa\u201048\u2010like<\/jats:sub>\n                      \/\n                      <jats:italic>bla<\/jats:italic>\n                      <jats:sub>NDM<\/jats:sub>\n                      was established in this study. 5\u2032 modified primers compatible with those immobilized antibodies on LFA were designed. The primer concentrations, temperature, and time were optimized at 0.2\u20130.6\u2009\u03bcM, 36\u00b0C\u201339\u00b0C, and 20\u201330\u2009min, respectively. The duplex RPA amplicons were diluted in buffer and visually detected on in\u2010house lateral flow strips within 10\u201315\u2009min. The limit of detection (LOD), specificity, and method validation were evaluated.\n                    <\/jats:p>\n                  <\/jats:sec>\n                  <jats:sec>\n                    <jats:title>Results<\/jats:title>\n                    <jats:p>\n                      The optimal primer concentrations of\n                      <jats:italic>bla<\/jats:italic>\n                      <jats:sub>CTX\u2010M<\/jats:sub>\n                      \/\n                      <jats:italic>mcr\u20101<\/jats:italic>\n                      (0.35\u2009\u03bcM\/0.35\u2009\u03bcM) and\n                      <jats:italic>bla<\/jats:italic>\n                      <jats:sub>oxa\u201048\u2010like<\/jats:sub>\n                      \/\n                      <jats:italic>bla<\/jats:italic>\n                      <jats:sub>NDM<\/jats:sub>\n                      (0.2\u2009\u03bcM\/0.6\u2009\u03bcM) provided balanced signal intensities for all target genes at 37\u00b0C\u201338\u00b0C within 25\u2009min. The LOD across incubation time of 0\u20138\u2009h was determined as 10\n                      <jats:sup>7<\/jats:sup>\n                      \u201310\n                      <jats:sup>2<\/jats:sup>\n                      \u2009CFU\/mL. No cross\u2010reactivity was observed with other common Gram\u2010negative pathogens. Sensitivity and specificity of duplex RPA\u2010LFA for\n                      <jats:italic>bla<\/jats:italic>\n                      <jats:sub>CTX\u2010M<\/jats:sub>\n                      ,\n                      <jats:italic>mcr\u20101<\/jats:italic>\n                      , and\n                      <jats:italic>bla<\/jats:italic>\n                      <jats:sub>oxa\u201048\u2010like<\/jats:sub>\n                      \/\n                      <jats:italic>bla<\/jats:italic>\n                      <jats:sub>NDM<\/jats:sub>\n                      detection were 85.71%\u201398.28% and 93.62%\u2013100%. The accuracy was 93.33%\u201396.13%.\n                    <\/jats:p>\n                  <\/jats:sec>\n                  <jats:sec>\n                    <jats:title>Conclusion<\/jats:title>\n                    <jats:p>This developed duplex RPA\u2010LFA assay is well\u2010suited for rapid screening in positive blood cultures, providing a cost\u2010effective tool for the treatment, surveillance, and control of MDR in critical infections.<\/jats:p>\n                  <\/jats:sec>","DOI":"10.1002\/jcla.70278","type":"journal-article","created":{"date-parts":[[2026,6,4]],"date-time":"2026-06-04T10:51:52Z","timestamp":1780570312000},"update-policy":"https:\/\/doi.org\/10.1002\/crossmark_policy","source":"Crossref","is-referenced-by-count":0,"title":["Duplex Recombinase Polymerase Amplification\u2010Lateral Flow Assay for Rapid Visual Detection of Critical Antibiotic Resistance Genes in\n                    <scp>\n                      <i>Escherichia coli<\/i>\n                    <\/scp>\n                    and\n                    <scp>\n                      <i>Klebsiella pneumoniae<\/i>\n                    <\/scp>\n                    From Positive Blood Cultures"],"prefix":"10.1002","volume":"40","author":[{"ORCID":"https:\/\/orcid.org\/0000-0001-9426-5568","authenticated-orcid":false,"given":"Variya","family":"Nemidkanam","sequence":"first","affiliation":[{"name":"Department of Transfusion Medicine and Clinical Microbiology, Faculty of Allied Health Sciences, Center of Excellence for Innovative Diagnosis of Antimicrobial Resistance Chulalongkorn University  Bangkok Thailand"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0009-0001-2682-0811","authenticated-orcid":false,"given":"Pattamaporn","family":"Deechart","sequence":"additional","affiliation":[{"name":"Department of Transfusion Medicine and Clinical Microbiology, Faculty of Allied Health Sciences, Center of Excellence for Innovative Diagnosis of Antimicrobial Resistance Chulalongkorn University  Bangkok Thailand"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-0717-5466","authenticated-orcid":false,"given":"Nuntaree","family":"Chaichanawongsaroj","sequence":"additional","affiliation":[{"name":"Department of Transfusion Medicine and Clinical Microbiology, Faculty of Allied Health Sciences, Center of Excellence for Innovative Diagnosis of Antimicrobial Resistance Chulalongkorn University  Bangkok Thailand"}],"role":[{"vocabulary":"crossref","role":"author"}]}],"member":"311","published-online":{"date-parts":[[2026,6,4]]},"reference":[{"key":"e_1_2_11_2_1","doi-asserted-by":"publisher","DOI":"10.1093\/cid\/ciaa210"},{"key":"e_1_2_11_3_1","doi-asserted-by":"publisher","DOI":"10.1016\/j.jinf.2018.03.005"},{"key":"e_1_2_11_4_1","doi-asserted-by":"publisher","DOI":"10.1371\/journal.pone.0251594"},{"key":"e_1_2_11_5_1","unstructured":"B.Bayraktar S.Pelit M. 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