{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"indexed":{"date-parts":[[2026,10,18]],"date-time":"2026-10-18T04:47:51Z","timestamp":1792298871848,"version":"4.4.0"},"reference-count":37,"publisher":"Frontiers Media SA","license":[{"start":{"date-parts":[[2021,3,25]],"date-time":"2021-03-25T00:00:00Z","timestamp":1616630400000},"content-version":"vor","delay-in-days":0,"URL":"https:\/\/creativecommons.org\/licenses\/by\/4.0\/"}],"content-domain":{"domain":["frontiersin.org"],"crossmark-restriction":true},"short-container-title":["Front. Cell. Infect. Microbiol."],"abstract":"<jats:sec><jats:title>Background<\/jats:title><jats:p>Considering the importance of differential diagnosis of infectious otitis externa (OE), a stepwise PCR-based assay using universal and genus- or species-specific primers for the detection\/identification of the most prevalent bacterial and fungal OE was developed and evaluated on the ear aspiration specimens of clinically suspected patients.<\/jats:p><\/jats:sec><jats:sec><jats:title>Methods and Materials<\/jats:title><jats:p>A total of 120 ear aspiration specimens with otomycosis suspicion were subjected to manual DNA extraction using phenol\u2013chloroform extraction after tissue digestion with a lysis buffer. The multiplex PCR was initially performed using pan-fungal and bacterial homemade primers.<jats:italic>Pseudomonas<\/jats:italic>and<jats:italic>Staphylococcus<\/jats:italic>specific primers were simultaneously used in one reaction mixture to identify the bacterial genera. Furthermore, for the identification of fungal agents,<jats:italic>Candida<\/jats:italic>species-specific multiplex primers targeting the most clinically important<jats:italic>Candida<\/jats:italic>species causing OE (<jats:italic>i.e<\/jats:italic>.,<jats:italic>C. albicans<\/jats:italic>,<jats:italic>C. parapsilosis<\/jats:italic>, and<jats:italic>C. auris<\/jats:italic>), as well as<jats:italic>Aspergillus<\/jats:italic>related multiplex PCR identifying the most prevalent<jats:italic>Aspergillus<\/jats:italic>species were used in two separate reaction mixtures. All the results of multiplex PCR were interpreted based on the amplicon size.<\/jats:p><\/jats:sec><jats:sec><jats:title>Results<\/jats:title><jats:p>The overall multiplex PCR-based detection rate of bacterial (n = 88; 73.3%) and fungal (n = 97; 81%) OE was documented to be 100% along with and complete consistency with the results of direct examination and Giemsa staining. Double amplicon bands of bacterial and fungal pathogens were evidenced in 76 specimens (63.3%). Moreover, the positivity rate of pan-fungal PCR was higher than that of the culture result. Out of 88 pan-bacterial positive PCR specimens, 66 and 47 ones were positive for<jats:italic>Staphylococcus<\/jats:italic>and<jats:italic>Pseudomonas<\/jats:italic>, respectively. In addition, 30 samples exhibited mixed infection of both, and five specimens remained negative. Out of 97 pan-fungal positive PCR specimens, 67 and 51 ones contained<jats:italic>Candida<\/jats:italic>and<jats:italic>Aspergillus<\/jats:italic>species, respectively. It should be noted that dual amplicon bands of<jats:italic>Candida<\/jats:italic>and<jats:italic>Aspergillus<\/jats:italic>-related multiplex PCR were yielded in 30 specimens.<\/jats:p><\/jats:sec><jats:sec><jats:title>Conclusion<\/jats:title><jats:p>The stepwise multiplex PCR assay proved to be more sensitive, more rapid, as well as less cumbersome in detection and identification of fungal and bacterial OE, compared to culture.<\/jats:p><\/jats:sec>","DOI":"10.3389\/fcimb.2021.644060","type":"journal-article","created":{"date-parts":[[2021,3,25]],"date-time":"2021-03-25T06:49:41Z","timestamp":1616654981000},"update-policy":"https:\/\/doi.org\/10.3389\/crossmark-policy","source":"Crossref","is-referenced-by-count":57,"title":["Direct Detection and Identification of the Most Common Bacteria and Fungi Causing Otitis Externa by a Stepwise Multiplex PCR"],"prefix":"10.3389","volume":"11","author":[{"given":"Shima","family":"Aboutalebian","sequence":"first","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Kazem","family":"Ahmadikia","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Hamed","family":"Fakhim","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Javaher","family":"Chabavizadeh","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Ahmadreza","family":"Okhovat","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Mahnaz","family":"Nikaeen","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Hossein","family":"Mirhendi","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]}],"member":"1965","published-online":{"date-parts":[[2021,3,25]]},"reference":[{"key":"B1","doi-asserted-by":"publisher","first-page":"101","DOI":"10.1111\/myc.12886","article-title":"Candida auris otomycosis in Iran and review of recent literature","volume":"62","author":"Abastabar","year":"2019","journal-title":"Mycoses"},{"key":"B2","doi-asserted-by":"publisher","first-page":"918","DOI":"10.1099\/jmm.0.000985","article-title":"Molecular epidemiology of otomycosis in Isfahan revealed a large diversity in causative agents","volume":"68","author":"Aboutalebian","year":"2019","journal-title":"J. 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