{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"indexed":{"date-parts":[[2026,8,18]],"date-time":"2026-08-18T03:01:45Z","timestamp":1787022105986,"version":"build-2736575974"},"reference-count":31,"publisher":"Public Library of Science (PLoS)","issue":"6","license":[{"start":{"date-parts":[[2022,6,14]],"date-time":"2022-06-14T00:00:00Z","timestamp":1655164800000},"content-version":"vor","delay-in-days":0,"URL":"http:\/\/creativecommons.org\/licenses\/by\/4.0\/"}],"funder":[{"DOI":"10.13039\/501100001807","name":"Funda\u00e7\u00e3o de Amparo \u00e0 Pesquisa do Estado de S\u00e3o Paulo","doi-asserted-by":"publisher","award":["2017\/03022-6"],"award-info":[{"award-number":["2017\/03022-6"]}],"id":[{"id":"10.13039\/501100001807","id-type":"DOI","asserted-by":"publisher"}]},{"DOI":"10.13039\/501100001807","name":"Funda\u00e7\u00e3o de Amparo \u00e0 Pesquisa do Estado de S\u00e3o Paulo","doi-asserted-by":"publisher","award":["2018\/22718-4"],"award-info":[{"award-number":["2018\/22718-4"]}],"id":[{"id":"10.13039\/501100001807","id-type":"DOI","asserted-by":"publisher"}]}],"content-domain":{"domain":["www.plosone.org"],"crossmark-restriction":false},"short-container-title":["PLoS ONE"],"abstract":"<jats:p>\n                    Meningitis caused by\n                    <jats:italic>Streptococcus pneumoniae<\/jats:italic>\n                    is still a disease of great impact on Public health, which requires immediate diagnosis and treatment. However, the culture of clinical specimens is often negative and antibiotic susceptibility testing (AST) must be performed with isolated strains. Multiplex real-time polymerase chain reaction (qPCR) has high sensitivity and specificity, produces faster results to identify the pathogen, and it can also be an important tool to identify resistance antibiotic genes earlier than AST, especially in the absence of an isolated strain. This study developed a multiplex qPCR assay, using SYBR Green as a nonspecific dye, to detect antibiotic resistance genes to predict pneumococcal susceptibility\/resistance in cerebrospinal fluid (CSF) samples from meningitis patients. From 2017 to 2020, CSF samples were cultured and analyzed by qPCR to detect the main three bacteria causing meningitis. Isolated and reference strains were applied in SYBR Green qPCR multiplex to detect\n                    <jats:italic>pbp2b<\/jats:italic>\n                    ,\n                    <jats:italic>ermB<\/jats:italic>\n                    , and\n                    <jats:italic>mef<\/jats:italic>\n                    genes, and the results were compared with the AST. Pneumococcal-positive CSF samples (\n                    <jats:italic>lytA<\/jats:italic>\n                    -positive gene) without isolated strains were also tested to evaluate the antimicrobial susceptibility profile in the region from 2014 to 2020. From the received 873 CSF samples; 263 were cultivated, 149 were\n                    <jats:italic>lytA<\/jats:italic>\n                    -positive in the qPCR, and 25 produced viable isolated pneumococci strains, which were evaluated by AST. Melting temperature for each gene and the acceptance criteria were determined (\n                    <jats:italic>pbp2b<\/jats:italic>\n                    : 78.24\u201379.86;\n                    <jats:italic>ermB<\/jats:italic>\n                    : 80.88\u201382.56;\n                    <jats:italic>mef<\/jats:italic>\n                    : 74.85\u201376.34 \u00baC). A total of 48\/51 strains presented a genetic profile in agreement with the AST results. Resistant strains to erythromycin and clindamycin were\n                    <jats:italic>ermB<\/jats:italic>\n                    -positive, and two were also\n                    <jats:italic>mef<\/jats:italic>\n                    -positive, indicating both resistance mechanisms were present. In the retrospective study of the genetic profile of resistance, 82\n                    <jats:italic>lytA<\/jats:italic>\n                    -positive CSF samples plus 4 strains were applied in the SYBR Green qPCR multiplex: 51% of samples presented the wild genotype (\n                    <jats:italic>pbp2b<\/jats:italic>\n                    positive and\n                    <jats:italic>ermB<\/jats:italic>\n                    \/\n                    <jats:italic>mef<\/jats:italic>\n                    negative); 15% were negative for all the three evaluated, indicating pneumococci resistant to penicillin; and 17% represented the multidrug-resistant pneumococci (\n                    <jats:italic>pbp2b<\/jats:italic>\n                    negative and\n                    <jats:italic>ermB<\/jats:italic>\n                    positive or\n                    <jats:italic>pbp2b<\/jats:italic>\n                    negative and\n                    <jats:italic>ermB<\/jats:italic>\n                    and\n                    <jats:italic>mef<\/jats:italic>\n                    positive). Therefore, SYBR Green qPCR multiplex proved to be a reliable tool to identify resistance genes in\n                    <jats:italic>S<\/jats:italic>\n                    .\n                    <jats:italic>pneumoniae<\/jats:italic>\n                    and would be less expensive than multiplex qPCR using specific probes. This could be easily introduced into the routine of diagnostic laboratories and provide a strong presumption of pneumococcal resistance, especially in the absence of isolated strains.\n                  <\/jats:p>","DOI":"10.1371\/journal.pone.0269895","type":"journal-article","created":{"date-parts":[[2022,6,14]],"date-time":"2022-06-14T13:58:04Z","timestamp":1655215084000},"page":"e0269895","update-policy":"https:\/\/doi.org\/10.1371\/journal.pone.corrections_policy","source":"Crossref","is-referenced-by-count":5,"title":["Multiplex real-time PCR using SYBR Green: Unspecific intercalating dye to detect antimicrobial resistance genes of Streptococcus pneumoniae in cerebrospinal fluid"],"prefix":"10.1371","volume":"17","author":[{"given":"Mariana Brena","family":"Souza","sequence":"first","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Maria Cec\u00edlia","family":"Cergole-Novella","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Delma Aparecida","family":"Molinari","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Daniela Rodrigues","family":"Colpas","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-0602-4623","authenticated-orcid":true,"given":"Andr\u00e9ia Moreira dos Santos","family":"Carmo","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Vilma dos Santos Menezes Gaiotto","family":"Daros","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0003-0334-0572","authenticated-orcid":true,"given":"Ivana Barros de","family":"Campos","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]}],"member":"340","published-online":{"date-parts":[[2022,6,14]]},"reference":[{"issue":"9693","key":"pone.0269895.ref001","doi-asserted-by":"crossref","first-page":"893","DOI":"10.1016\/S0140-6736(09)61204-6","article-title":"Burden of disease caused by Streptococcus pneumoniae in children younger than 5 years: global estimates","volume":"374","author":"KL O\u2019Brien","year":"2009","journal-title":"Lancet"},{"key":"pone.0269895.ref002","unstructured":"Brasil. Secretaria de Vigil\u00e2ncia em Sa\u00fade. Boletim Epidemiol\u00f3gico. Meningite bacteriana n\u00e3o especificada no Brasil 2007\u20132016: desafio para a vigil\u00e2ncia das meningites. Minist\u00e9rio da Sa\u00fade, v. 50, n. 03, jan. 2019. < http:\/\/portalarquivos2.saude.gov.br\/images\/pdf\/2019\/fevereiro\/01\/2018-038.pdf>. Access in 09\/10\/2020."},{"issue":"4","key":"pone.0269895.ref003","doi-asserted-by":"crossref","first-page":"1553","DOI":"10.1128\/JCM.39.4.1553-1558.2001","article-title":"Simultaneous detection of Neisseria meningitidis, Haemophilus influenzae, and Streptococcus pneumoniae in suspected cases of meningitis and septicemia using real-time PCR","volume":"39","author":"CE Corless","year":"2001","journal-title":"J Clin Microbiol"},{"issue":"6","key":"pone.0269895.ref004","doi-asserted-by":"crossref","first-page":"e20675","DOI":"10.1371\/journal.pone.0020675","article-title":"Incorporation of real-time PCR into routine public health surveillance of culture negative bacterial meningitis in S\u00e3o Paulo, Brazil","volume":"6","author":"CT Sacchi","year":"2011","journal-title":"PLoS One"},{"issue":"4","key":"pone.0269895.ref005","doi-asserted-by":"crossref","first-page":"224","DOI":"10.1590\/0004-282x20190028","article-title":"Validation of multiplex PCR for the diagnosis of acute bacterial meningitis in culture negative cerebrospinal fluid","volume":"77","author":"SM de Almeida","year":"2019","journal-title":"Arq Neuropsiquiatr"},{"issue":"3","key":"pone.0269895.ref006","doi-asserted-by":"crossref","first-page":"395","DOI":"10.2217\/fmb.12.2","article-title":"Molecular mechanisms of \u03b2-lactam resistance in Streptococcus pneumoniae","volume":"7","author":"R Hakenbeck","year":"2012","journal-title":"Future Microbiol"},{"key":"pone.0269895.ref007","doi-asserted-by":"crossref","first-page":"26","DOI":"10.1186\/1471-2334-13-26","article-title":"Accuracy of real-time PCR, Gram stain and culture for Streptococcus pneumoniae, Neisseria meningitidis and Haemophilus influenzae meningitis diagnosis","volume":"13","author":"HM Wu","year":"2013","journal-title":"BMC Infect Dis"},{"key":"pone.0269895.ref008","unstructured":"World Health Organization & Centers for Disease Control and Prevention (U.S.). (2011). Laboratory methods for the diagnosis of meningitis caused by Neisseria meningitidis, Streptococcus pneumoniae, and Haemophilus influenzae: WHO manual, 2nd ed. World Health Organization. https:\/\/apps.who.int\/iris\/handle\/10665\/70765."},{"issue":"2","key":"pone.0269895.ref009","doi-asserted-by":"crossref","first-page":"e0147765","DOI":"10.1371\/journal.pone.0147765","article-title":"Development of Real-Time PCR Methods for the Detection of Bacterial Meningitis Pathogens without DNA Extraction","volume":"11","author":"J Vuong","year":"2016","journal-title":"PLoS One"},{"issue":"3","key":"pone.0269895.ref010","doi-asserted-by":"crossref","first-page":"188","DOI":"10.1016\/j.diagmicrobio.2018.10.008","article-title":"Triplex real-time PCR assay for the detection of Streptococcus pneumoniae, Neisseria meningitidis and Haemophilus influenzae directly from clinical specimens without extraction of DNA","volume":"93","author":"M Ouattara","year":"2019","journal-title":"Diagn Microbiol Infect Dis"},{"issue":"103","key":"pone.0269895.ref011","first-page":"16","article-title":"Improved Real Time PCR assay for diagnostic and epidemiological surveillance of bacterial meningitis","volume":"9","author":"MM Salgado","year":"2012","journal-title":"BEPA"},{"issue":"4","key":"pone.0269895.ref012","doi-asserted-by":"crossref","first-page":"453","DOI":"10.1016\/j.diagmicrobio.2011.08.017","article-title":"Quadriplex real-time polymerase chain reaction (lytA, mef, erm, pbp2b(wt)) for pneumococcal detection and assessment of antibiotic susceptibility","volume":"71","author":"V Srinivasan","year":"2011","journal-title":"Diagn Microbiol Infect Dis"},{"issue":"7","key":"pone.0269895.ref013","doi-asserted-by":"crossref","first-page":"1019","DOI":"10.1093\/bioinformatics\/btr065","article-title":"uMELT: prediction of high-resolution melting curves and dynamic melting profiles of PCR products in a rich web application","volume":"27","author":"Z Dwight","year":"2011","journal-title":"Bioinformatics"},{"key":"pone.0269895.ref014","unstructured":"Clinical and Laboratory Standards Institute (CLSI). Performance Standards for Antimicrobial Susceptibility Testing. 28th ed. CLSI supplement M100. Wayne, PA, USA: Clinical and Laboratory Standards Institute; 2018."},{"key":"pone.0269895.ref015","doi-asserted-by":"crossref","first-page":"26","DOI":"10.1186\/s12941-021-00432-z","article-title":"Antimicrobial resistance profile and multidrug resistance patterns of Streptococcus pneumoniae isolates from patients suspected of pneumococcal infections in Ethiopia","volume":"20","author":"B Sharew","year":"2021","journal-title":"Ann Clin Microbiol Antimicrob"},{"issue":"6","key":"pone.0269895.ref016","doi-asserted-by":"crossref","first-page":"711","DOI":"10.1093\/bioinformatics\/bti066","article-title":"Comparison of different melting temperature calculation methods for short DNA sequences","volume":"21","author":"A Panjkovich","year":"2005","journal-title":"Bioinformatics"},{"issue":"8","key":"pone.0269895.ref017","doi-asserted-by":"crossref","first-page":"e12433","DOI":"10.1371\/journal.pone.0012433","article-title":"A phenomenological model for predicting melting temperatures of DNA sequences","volume":"5","author":"G Khandelwal","year":"2010","journal-title":"PLoS One"},{"key":"pone.0269895.ref018","doi-asserted-by":"crossref","first-page":"101","DOI":"10.1186\/1471-2105-13-101","article-title":"MELTING, a flexible platform to predict the melting temperatures of nucleic acids","volume":"13","author":"M Dumousseau","year":"2012","journal-title":"BMC Bioinformatics"},{"key":"pone.0269895.ref019","volume-title":"Current Protocols in Molecular Biology","author":"FM Ausubel","year":"2003"},{"issue":"4","key":"pone.0269895.ref020","doi-asserted-by":"crossref","first-page":"1681","DOI":"10.1073\/pnas.95.4.1681","article-title":"Conformation sensitive gel electrophoresis for simple and accurate detection of mutations: Comparison with denaturing gradient gel electrophoresis and nucleotide sequencing","volume":"95","author":"J K\u00f6rkk\u00f6","year":"1998","journal-title":"Proceedings of the National Academy of Sciences"},{"issue":"4","key":"pone.0269895.ref021","doi-asserted-by":"crossref","first-page":"481","DOI":"10.1016\/j.ajme.2017.11.004","article-title":"Low cost detection of hepatitis C virus RNA in HCV infected patients by SYBR Green I real-time PCR","volume":"54","author":"DE Metawlly","year":"2018","journal-title":"Alexandria Journal of Medicine"},{"issue":"9","key":"pone.0269895.ref022","doi-asserted-by":"crossref","first-page":"1139","DOI":"10.1016\/j.jiph.2021.07.009","article-title":"Development and validation of an in-house, low-cost SARS-CoV-2 detection assay","volume":"14","author":"FS Alhamlan","year":"2021","journal-title":"J Infect Public Health"},{"issue":"1","key":"pone.0269895.ref023","doi-asserted-by":"crossref","first-page":"632","DOI":"10.1186\/s13104-018-3719-5","article-title":"Analysis of amino acid motif of penicillin-binding proteins 1a, 2b, and 2x in invasive Streptococcus pneumoniae nonsusceptible to penicillin isolated from pediatric patients in Casablanca, Morocco","volume":"11","author":"I Diawara","year":"2018","journal-title":"BMC Res Notes"},{"issue":"6","key":"pone.0269895.ref024","doi-asserted-by":"crossref","first-page":"420","DOI":"10.7883\/yoken.JJID.2019.015","article-title":"Erythromycin-Susceptible but Clindamycin-Resistant Phenotype of Clinical ermB-PCR-Positive Group B Streptococci Isolates with IS1216E-Inserted ermB","volume":"72","author":"H Moroi","year":"2019","journal-title":"Jpn J Infect Dis"},{"issue":"6","key":"pone.0269895.ref025","doi-asserted-by":"crossref","first-page":"e67507","DOI":"10.1371\/journal.pone.0067507","article-title":"Serotype distribution and antimicrobial resistance of Streptococcus pneumoniae isolates causing invasive diseases from Shenzhen Children\u2019s Hospital","volume":"8","author":"X Ma","year":"2013","journal-title":"PLoS One"},{"issue":"23","key":"pone.0269895.ref026","doi-asserted-by":"crossref","first-page":"3207","DOI":"10.1016\/j.vaccine.2021.02.063","article-title":"Dynamics of antimicrobial resistance of Streptococcus pneumoniae following PCV10 introduction in Brazil: Nationwide surveillance from 2007 to 2019","volume":"39","author":"MC Brandileone","year":"2021","journal-title":"Vaccine"},{"issue":"5","key":"pone.0269895.ref027","doi-asserted-by":"crossref","first-page":"1595","DOI":"10.1128\/AAC.45.5.1595-1598.2001","article-title":"Serotype 19F multiresistant pneumococcal clone harboring two erythromycin resistance determinants (erm(B) and mef(A)) in South Africa","volume":"45","author":"L McGee","year":"2001","journal-title":"Antimicrob Agents Chemother"},{"issue":"6","key":"pone.0269895.ref028","doi-asserted-by":"crossref","first-page":"990","DOI":"10.1093\/cid\/cix446","article-title":"A Population-Based Assessment of the Impact of 7- and 13-Valent Pneumococcal Conjugate Vaccines on Macrolide-Resistant Invasive Pneumococcal Disease: Emergence and Decline of Streptococcus pneumoniae Serotype 19A (CC320) With Dual Macrolide Resistance Mechanisms","volume":"65","author":"MR Schroeder","year":"2017","journal-title":"Clin Infect Dis"},{"issue":"11","key":"pone.0269895.ref029","doi-asserted-by":"crossref","first-page":"e0208211","DOI":"10.1371\/journal.pone.0208211","article-title":"Expansion of the multidrug-resistant clonal complex 320 among invasive Streptococcus pneumoniae serotype 19A after the introduction of a ten-valent pneumococcal conjugate vaccine in Brazil","volume":"13","author":"AP Cassiolato","year":"2018","journal-title":"PLoS One"},{"key":"pone.0269895.ref030","doi-asserted-by":"crossref","first-page":"47","DOI":"10.1016\/j.ijid.2013.11.009","article-title":"Susceptibility profiles and correlation with pneumococcal serotypes soon after implementation of the 10-valent pneumococcal conjugate vaccine in Brazil","volume":"20","author":"M Mott","year":"2014","journal-title":"Int J Infect Dis"},{"issue":"10","key":"pone.0269895.ref031","first-page":"000635","article-title":"Genomic surveillance of invasive Streptococcus pneumoniae isolates in the period pre-PCV10 and post-PCV10 introduction in Brazil","volume":"7","author":"SCG Almeida","year":"2021","journal-title":"Microb Genom"}],"container-title":["PLOS ONE"],"original-title":[],"language":"en","link":[{"URL":"https:\/\/dx.plos.org\/10.1371\/journal.pone.0269895","content-type":"unspecified","content-version":"vor","intended-application":"similarity-checking"}],"deposited":{"date-parts":[[2022,6,14]],"date-time":"2022-06-14T13:58:32Z","timestamp":1655215112000},"score":1,"resource":{"primary":{"URL":"https:\/\/dx.plos.org\/10.1371\/journal.pone.0269895"}},"subtitle":[],"editor":[{"given":"Jose","family":"Melo-Cristino","sequence":"first","affiliation":[],"role":[{"vocabulary":"crossref","role":"editor"}]}],"short-title":[],"issued":{"date-parts":[[2022,6,14]]},"references-count":31,"journal-issue":{"issue":"6","published-online":{"date-parts":[[2022,6,14]]}},"URL":"https:\/\/doi.org\/10.1371\/journal.pone.0269895","relation":{},"ISSN":["1932-6203"],"issn-type":[{"value":"1932-6203","type":"electronic"}],"subject":[],"published":{"date-parts":[[2022,6,14]]}}}