{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"indexed":{"date-parts":[[2025,2,21]],"date-time":"2025-02-21T14:07:33Z","timestamp":1740146853288,"version":"3.37.3"},"reference-count":33,"publisher":"Public Library of Science (PLoS)","issue":"5","license":[{"start":{"date-parts":[[2021,5,11]],"date-time":"2021-05-11T00:00:00Z","timestamp":1620691200000},"content-version":"vor","delay-in-days":0,"URL":"http:\/\/creativecommons.org\/licenses\/by\/4.0\/"}],"funder":[{"DOI":"10.13039\/501100009880","name":"Regione Lazio","doi-asserted-by":"publisher","award":["DSPMI-INN"],"award-info":[{"award-number":["DSPMI-INN"]}],"id":[{"id":"10.13039\/501100009880","id-type":"DOI","asserted-by":"publisher"}]}],"content-domain":{"domain":["www.plosone.org"],"crossmark-restriction":false},"short-container-title":["PLoS ONE"],"abstract":"<jats:p><jats:italic>Chlamydia trachomatis<\/jats:italic>, the leading cause of bacterial sexually transmitted diseases in developed countries, with around 127 million new cases per year, is mainly responsible for urethritis and cervicitis in women, and urethritis and epididymitis in men. Most <jats:italic>C<\/jats:italic>. <jats:italic>trachomatis<\/jats:italic> infections remain asymptomatic (&gt;50%) and, hence, untreated, leading to severe reproductive complications in both women and men, like infertility. Therefore, the detection of <jats:italic>C<\/jats:italic>. <jats:italic>trachomatis<\/jats:italic> as well as the antimicrobial susceptibility testing becomes a priority, and, along the years, several methods have been recommended, like cell culture and direct immunofluorescence (DFA) on cell cultures. Herein, we described the application of In-Cell Western assay (ICW) via Odyssey CLx as a fast, more accessible, and high-throughput platform for the quantification of <jats:italic>C<\/jats:italic>. <jats:italic>trachomatis<\/jats:italic> and the screening of anti-chlamydial drugs. As a first step, we set up a standard curve by infecting cell monolayers with 2-fold serial dilutions of <jats:italic>C<\/jats:italic>. <jats:italic>trachomatis<\/jats:italic> Elementary Body (EB) suspension. Then, different unknown <jats:italic>C<\/jats:italic>. <jats:italic>trachomatis<\/jats:italic> EB suspensions were quantified and the chlamydial susceptibility testing to erythromycin was performed, using the DFA as comparison. Our results showed a very high concordance between these two assays, as evidenced by the enumeration of chlamydial IFUs as well as the determination of erythromycin Minimum Inhibitory Concentration (MIC). In conclusion, the ICW assay may be a promising candidate as an accurate and accessible methodology for <jats:italic>C<\/jats:italic>. <jats:italic>trachomatis<\/jats:italic> antimicrobial susceptibility testing.<\/jats:p>","DOI":"10.1371\/journal.pone.0251075","type":"journal-article","created":{"date-parts":[[2021,5,11]],"date-time":"2021-05-11T17:25:37Z","timestamp":1620753937000},"page":"e0251075","update-policy":"https:\/\/doi.org\/10.1371\/journal.pone.corrections_policy","source":"Crossref","is-referenced-by-count":0,"title":["In-cell western assay as a high-throughput approach for Chlamydia trachomatis quantification and susceptibility testing to antimicrobials"],"prefix":"10.1371","volume":"16","author":[{"ORCID":"https:\/\/orcid.org\/0000-0002-2730-8588","authenticated-orcid":true,"given":"Simone","family":"Filardo","sequence":"first","affiliation":[],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Marisa","family":"Di Pietro","sequence":"additional","affiliation":[],"role":[{"role":"author","vocabulary":"crossref"}]},{"ORCID":"https:\/\/orcid.org\/0000-0001-5560-1979","authenticated-orcid":true,"given":"Patrizio","family":"Pasqualetti","sequence":"additional","affiliation":[],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Martina","family":"Manera","sequence":"additional","affiliation":[],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Fabiana","family":"Diaco","sequence":"additional","affiliation":[],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Rosa","family":"Sessa","sequence":"additional","affiliation":[],"role":[{"role":"author","vocabulary":"crossref"}]}],"member":"340","published-online":{"date-parts":[[2021,5,11]]},"reference":[{"issue":"3","key":"pone.0251075.ref001","doi-asserted-by":"crossref","first-page":"233","DOI":"10.1016\/j.puhe.2011.11.015","article-title":"The global burden of neglected tropical diseases","volume":"126","author":"A Fenwick","year":"2012","journal-title":"Public Health"},{"key":"pone.0251075.ref002","unstructured":"Report on global sexually transmitted infection surveillance, 2018. 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Detection Strategy To Rapidly Evaluate the Efficacy of Antichlamydial Agents","volume":"61","author":"Y Zhang","year":"2017","journal-title":"Antimicrob Agents Chemother"},{"issue":"104","key":"pone.0251075.ref028","first-page":"51131","article-title":"Using Fluorescent Proteins to Visualize and Quantitate Chlamydia Vacuole Growth Dynamics in Living Cells","author":"M Zuck","year":"2015","journal-title":"J Vis Exp"},{"issue":"6","key":"pone.0251075.ref029","doi-asserted-by":"crossref","first-page":"e99197","DOI":"10.1371\/journal.pone.0099197","article-title":"Quantitative monitoring of the Chlamydia trachomatis developmental cycle using GFP-expressing bacteria, microscopy and flow cytometry","volume":"9","author":"F Vromman","year":"2014","journal-title":"PLoS One"},{"issue":"1\u20132","key":"pone.0251075.ref030","doi-asserted-by":"crossref","first-page":"84","DOI":"10.1016\/j.jim.2007.05.004","article-title":"A novel automated method for enumeration of Chlamydia trachomatis inclusion 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