{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"indexed":{"date-parts":[[2026,7,30]],"date-time":"2026-07-30T13:36:08Z","timestamp":1785418568865,"version":"3.56.0"},"reference-count":39,"publisher":"American Society for Microbiology","issue":"11","license":[{"start":{"date-parts":[[2025,11,4]],"date-time":"2025-11-04T00:00:00Z","timestamp":1762214400000},"content-version":"vor","delay-in-days":0,"URL":"https:\/\/creativecommons.org\/licenses\/by\/4.0\/"},{"start":{"date-parts":[[2025,11,4]],"date-time":"2025-11-04T00:00:00Z","timestamp":1762214400000},"content-version":"tdm","delay-in-days":0,"URL":"https:\/\/journals.asm.org\/non-commercial-tdm-license"}],"funder":[{"DOI":"10.13039\/501100001807","name":"S\u00e3o Paulo Research Foundation","doi-asserted-by":"publisher","award":["2018\/04144-0"],"award-info":[{"award-number":["2018\/04144-0"]}],"id":[{"id":"10.13039\/501100001807","id-type":"DOI","asserted-by":"publisher"}]},{"DOI":"10.13039\/501100001807","name":"S\u00e3o Paulo Research Foundation","doi-asserted-by":"publisher","award":["2022\/02861-2"],"award-info":[{"award-number":["2022\/02861-2"]}],"id":[{"id":"10.13039\/501100001807","id-type":"DOI","asserted-by":"publisher"}]},{"DOI":"10.13039\/501100002322","name":"Coordena\u00e7\u00e3o de Aperfei\u00e7oamento de Pessoal de N\u00edvel Superior","doi-asserted-by":"publisher","award":["88887.753154\/2022-00"],"award-info":[{"award-number":["88887.753154\/2022-00"]}],"id":[{"id":"10.13039\/501100002322","id-type":"DOI","asserted-by":"publisher"}]}],"content-domain":{"domain":["journals.asm.org"],"crossmark-restriction":true},"short-container-title":["Microbiol Spectr"],"published-print":{"date-parts":[[2025,11,4]]},"abstract":"ABSTRACT<\/jats:title>\n \n \n \n Enteroaggregative\n Escherichia coli<\/jats:italic>\n (EAEC) is one of the main\n diarrhea causative agents. This\n E. coli<\/jats:italic>\n pathotype is\n defined by an aggregative adherence pattern observed in HEp-2 cells.\n Molecular diagnosis is still a challenge due to EAEC genetic heterogeneity.\n Different publications described primers for\n aggR<\/jats:italic>\n and\n aatA<\/jats:italic>\n detection to characterize EAEC. Recently,\n afpR<\/jats:italic>\n has been suggested as EAEC molecular marker of\n some strains, demonstrating the existence of two subpopulations within this\n pathotype: typical and atypical EAEC, respectively, comprising\n aatA<\/jats:italic>\n +\/\n aggR<\/jats:italic>\n + and\n aatA<\/jats:italic>\n +\/\n afpR<\/jats:italic>\n +. Studies performed by our\n group, however, detected conflicting results using different primer pairs\n for\n aggR<\/jats:italic>\n . Therefore, this study mainly aimed to analyze the\n variation of\n aggR<\/jats:italic>\n sequences and its implication in EAEC\n characterization, defining consensus primers. As a consequence, a\n triplex-PCR targeting\n aggR<\/jats:italic>\n ,\n aatA,<\/jats:italic>\n and\n afpR<\/jats:italic>\n is proposed as a one-step molecular diagnosis for\n both typical and atypical EAEC. Previously reported primer sequences used\n for\n aggR, aatA<\/jats:italic>\n , and\n afpR<\/jats:italic>\n detection, and\n their annealing sites were analyzed using sequence databases. The best\n primers for each target were selected, and a triplex-PCR was standardized\n and validated. Database alignment of\n aggR<\/jats:italic>\n revealed numerous\n variant regions along its sequence, impairing the annealing of most reported\n primers, while significant variations in the annealing sites of\n aatA<\/jats:italic>\n and\n afpR<\/jats:italic>\n primers were not\n observed. Broad-range primers for\n aggR<\/jats:italic>\n were determined and\n combined with\n aatA<\/jats:italic>\n and\n afpR<\/jats:italic>\n , resulting in\n a triplex-PCR. Our triplex-PCR targeting\n aggR, aatA<\/jats:italic>\n , and\n afpR<\/jats:italic>\n successfully detected only EAEC, covering all\n aggR<\/jats:italic>\n variants, promoting a broad and specific detection\n of this pathotype.\n <\/jats:p>\n \n IMPORTANCE<\/jats:title>\n \n Phenotypical and genotypical characterization of enteroaggregative\n Escherichia coli<\/jats:italic>\n (EAEC) is either time-consuming or\n of variable sensitivity. Therefore, developing an accurate molecular\n diagnostic method for this\n E. coli<\/jats:italic>\n pathotype is\n relevant for public health. This study analyzed the primers currently\n used to detect typical EAEC-related genes (\n aatA<\/jats:italic>\n and\n aggR<\/jats:italic>\n ), and\n afpR<\/jats:italic>\n as a marker for\n atypical EAEC. After an extensive literature review, it was observed\n that the\n aatA<\/jats:italic>\n primers are suitable for its broad\n detection. On the other hand, none of the\n aggR<\/jats:italic>\n primer\n pairs described were able to detect all variants of the\n aggR<\/jats:italic>\n gene. To address this issue, universal primers\n for\n aggR<\/jats:italic>\n were proposed to improve the detection of all\n aggR<\/jats:italic>\n variants. Considering the demand for a fast\n and accurate molecular approach for EAEC diagnosis, a triplex-PCR assay\n was developed, validated, and optimized using the universal\n aggR<\/jats:italic>\n primers in combination with primers for\n aatA<\/jats:italic>\n and\n afpR<\/jats:italic>\n , detecting both\n typical and atypical EAEC.\n <\/jats:p>\n <\/jats:sec>\n <\/jats:sec>","DOI":"10.1128\/spectrum.01441-25","type":"journal-article","created":{"date-parts":[[2025,9,24]],"date-time":"2025-09-24T13:00:43Z","timestamp":1758718843000},"update-policy":"https:\/\/doi.org\/10.1128\/asmj-crossmark-policy-page","source":"Crossref","is-referenced-by-count":1,"title":["Importance of\n aggR<\/i>\n sequence variants detection for accurate molecular diagnosis of enteroaggregative\n Escherichia coli<\/i>"],"prefix":"10.1128","volume":"13","author":[{"ORCID":"https:\/\/orcid.org\/0000-0002-8617-6765","authenticated-orcid":false,"given":"Alejandra M. G.","family":"Del Carpio","sequence":"first","affiliation":[{"id":[{"id":"https:\/\/ror.org\/01whwkf30","id-type":"ROR","asserted-by":"publisher"}],"name":"Laborat\u00f3rio de Bacteriologia, Instituto Butantan","place":["S\u00e3o Paulo, Brazil"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-6218-1009","authenticated-orcid":true,"given":"Claudia A.","family":"Freire","sequence":"additional","affiliation":[{"id":[{"id":"https:\/\/ror.org\/01whwkf30","id-type":"ROR","asserted-by":"publisher"}],"name":"Laborat\u00f3rio de Bacteriologia, Instituto Butantan","place":["S\u00e3o Paulo, Brazil"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-4525-8705","authenticated-orcid":true,"given":"T\u00e2nia A. T.","family":"Gomes","sequence":"additional","affiliation":[{"id":[{"id":"https:\/\/ror.org\/02k5swt12","id-type":"ROR","asserted-by":"publisher"}],"name":"Departamento de Microbiologia, Imunologia e Parasitologia, Escola Paulista de Medicina, Universidade Federal de S\u00e3o Paulo","place":["S\u00e3o Paulo, Brazil"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0003-0218-9372","authenticated-orcid":true,"given":"Cecilia M.","family":"Abe","sequence":"additional","affiliation":[{"id":[{"id":"https:\/\/ror.org\/01whwkf30","id-type":"ROR","asserted-by":"publisher"}],"name":"Laborat\u00f3rio de Bacteriologia, Instituto Butantan","place":["S\u00e3o Paulo, Brazil"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0003-3470-5706","authenticated-orcid":true,"given":"Waldir P.","family":"Elias","sequence":"additional","affiliation":[{"id":[{"id":"https:\/\/ror.org\/01whwkf30","id-type":"ROR","asserted-by":"publisher"}],"name":"Laborat\u00f3rio de Bacteriologia, Instituto Butantan","place":["S\u00e3o Paulo, Brazil"]}],"role":[{"vocabulary":"crossref","role":"author"}]}],"member":"235","reference":[{"key":"e_1_3_4_2_2","doi-asserted-by":"publisher","DOI":"10.1128\/CMR.00112-13"},{"key":"e_1_3_4_3_2","doi-asserted-by":"publisher","DOI":"10.1093\/cid\/cis754"},{"key":"e_1_3_4_4_2","doi-asserted-by":"publisher","DOI":"10.1371\/journal.pntd.0005798"},{"key":"e_1_3_4_5_2","doi-asserted-by":"publisher","DOI":"10.1016\/j.bjm.2016.10.015"},{"key":"e_1_3_4_6_2","doi-asserted-by":"publisher","DOI":"10.1111\/j.1753-4887.2008.00082.x"},{"key":"e_1_3_4_7_2","doi-asserted-by":"publisher","DOI":"10.3390\/ijms25179238"},{"key":"e_1_3_4_8_2","doi-asserted-by":"publisher","DOI":"10.1371\/journal.pntd.0008274"},{"key":"e_1_3_4_9_2","doi-asserted-by":"publisher","DOI":"10.3390\/microorganisms10020302"},{"key":"e_1_3_4_10_2","doi-asserted-by":"publisher","DOI":"10.1097\/00006454-198709000-00008"},{"key":"e_1_3_4_11_2","doi-asserted-by":"publisher","DOI":"10.1016\/j.mimet.2014.07.030"},{"key":"e_1_3_4_12_2","doi-asserted-by":"publisher","DOI":"10.1093\/infdis\/jir757"},{"key":"e_1_3_4_13_2","doi-asserted-by":"publisher","DOI":"10.1590\/s0074-02762007005000116"},{"key":"e_1_3_4_14_2","doi-asserted-by":"publisher","DOI":"10.1128\/JCM.36.12.3609-3613.1998"},{"key":"e_1_3_4_15_2","doi-asserted-by":"publisher","DOI":"10.3389\/fcimb.2020.00200"},{"key":"e_1_3_4_16_2","doi-asserted-by":"publisher","DOI":"10.1093\/infdis\/161.6.1249"},{"key":"e_1_3_4_17_2","doi-asserted-by":"publisher","DOI":"10.1128\/JCM.41.5.2138-2140.2003"},{"key":"e_1_3_4_18_2","doi-asserted-by":"publisher","DOI":"10.3389\/fcimb.2022.997208"},{"key":"e_1_3_4_19_2","first-page":"211","article-title":"Investigation on enteroaggregative\n Escherichia coli infection by multiplex PCR","volume":"39","author":"Ratchtrachenchai OA","year":"1997","unstructured":"Ratchtrachenchai OA, Subpasu S, Ito K. 1997. Investigation on enteroaggregative Escherichia coli infection by multiplex PCR. Bull Dept Med Sci 39:211\u2013220.","journal-title":"Bull Dept Med Sci"},{"key":"e_1_3_4_20_2","doi-asserted-by":"publisher","DOI":"10.32604\/biocell.2006.30.301"},{"key":"e_1_3_4_21_2","doi-asserted-by":"publisher","DOI":"10.11150\/kansenshogakuzasshi1970.70.569"},{"key":"e_1_3_4_22_2","doi-asserted-by":"publisher","DOI":"10.1128\/IAI.67.6.2692-2699.1999"},{"key":"e_1_3_4_23_2","doi-asserted-by":"publisher","DOI":"10.1074\/jbc.M306413200"},{"key":"e_1_3_4_24_2","doi-asserted-by":"publisher","DOI":"10.1128\/jcm.33.3.701-705.1995"},{"key":"e_1_3_4_25_2","doi-asserted-by":"publisher","DOI":"10.1038\/s41426-018-0209-8"},{"key":"e_1_3_4_26_2","doi-asserted-by":"publisher","DOI":"10.1080\/21505594.2021.2007645"},{"key":"e_1_3_4_27_2","doi-asserted-by":"publisher","DOI":"10.3389\/fcimb.2020.00144"},{"key":"e_1_3_4_28_2","doi-asserted-by":"publisher","DOI":"10.1186\/s12866-016-0827-z"},{"key":"e_1_3_4_29_2","doi-asserted-by":"publisher","DOI":"10.1111\/1348-0421.12173"},{"key":"e_1_3_4_30_2","doi-asserted-by":"publisher","DOI":"10.4269\/ajtmh.2009.80.294"},{"key":"e_1_3_4_31_2","doi-asserted-by":"publisher","DOI":"10.1128\/ecosalplus.esp-0011-2024"},{"key":"e_1_3_4_32_2","doi-asserted-by":"publisher","DOI":"10.1128\/iai.63.4.1318-1328.1995"},{"key":"e_1_3_4_33_2","doi-asserted-by":"publisher","DOI":"10.1186\/1471-2180-10-57"},{"key":"e_1_3_4_34_2","doi-asserted-by":"publisher","DOI":"10.1111\/j.1574-695X.2007.00308.x"},{"key":"e_1_3_4_35_2","doi-asserted-by":"publisher","DOI":"10.1016\/S2214-109X(24)00078-0"},{"key":"e_1_3_4_36_2","doi-asserted-by":"publisher","DOI":"10.1371\/journal.pntd.0008613"},{"key":"e_1_3_4_37_2","doi-asserted-by":"publisher","DOI":"10.1128\/IAI.68.6.3280-3285.2000"},{"key":"e_1_3_4_38_2","doi-asserted-by":"publisher","DOI":"10.1017\/s0950268800001072"},{"key":"e_1_3_4_39_2","doi-asserted-by":"publisher","DOI":"10.1128\/jcm.35.10.2546-2550.1997"},{"key":"e_1_3_4_40_2","doi-asserted-by":"publisher","DOI":"10.3389\/fmicb.2019.01791"}],"container-title":["Microbiology Spectrum"],"original-title":[],"language":"en","link":[{"URL":"https:\/\/journals.asm.org\/doi\/pdf\/10.1128\/spectrum.01441-25","content-type":"application\/pdf","content-version":"vor","intended-application":"text-mining"},{"URL":"https:\/\/journals.asm.org\/doi\/pdf\/10.1128\/spectrum.01441-25","content-type":"unspecified","content-version":"vor","intended-application":"similarity-checking"}],"deposited":{"date-parts":[[2025,11,4]],"date-time":"2025-11-04T14:06:16Z","timestamp":1762265176000},"score":1,"resource":{"primary":{"URL":"https:\/\/journals.asm.org\/doi\/10.1128\/spectrum.01441-25"}},"subtitle":[],"editor":[{"given":"Catherine M.","family":"Logue","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"editor"}]}],"short-title":[],"issued":{"date-parts":[[2025,11,4]]},"references-count":39,"journal-issue":{"issue":"11","published-print":{"date-parts":[[2025,11,4]]}},"alternative-id":["10.1128\/spectrum.01441-25"],"URL":"https:\/\/doi.org\/10.1128\/spectrum.01441-25","relation":{},"ISSN":["2165-0497"],"issn-type":[{"value":"2165-0497","type":"electronic"}],"subject":[],"published":{"date-parts":[[2025,11,4]]},"assertion":[{"value":"2025-05-08","order":0,"name":"received","label":"Received","group":{"name":"publication_history","label":"Publication History"}},{"value":"2025-09-04","order":2,"name":"accepted","label":"Accepted","group":{"name":"publication_history","label":"Publication History"}},{"value":"2025-09-24","order":3,"name":"published","label":"Published","group":{"name":"publication_history","label":"Publication History"}}],"article-number":"e01441-25"}}