{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"indexed":{"date-parts":[[2026,10,8]],"date-time":"2026-10-08T14:23:28Z","timestamp":1791469408937,"version":"4.3.4"},"reference-count":45,"publisher":"Wiley","issue":"9","license":[{"start":{"date-parts":[[2023,6,25]],"date-time":"2023-06-25T00:00:00Z","timestamp":1687651200000},"content-version":"vor","delay-in-days":0,"URL":"http:\/\/onlinelibrary.wiley.com\/termsAndConditions#vor"}],"funder":[{"DOI":"10.13039\/501100001411","name":"Indian Council of Medical Research","doi-asserted-by":"publisher","award":["Myco\/1\/2020\u2010ECD\/II"],"award-info":[{"award-number":["Myco\/1\/2020\u2010ECD\/II"]}],"id":[{"id":"10.13039\/501100001411","id-type":"DOI","asserted-by":"publisher"}]}],"content-domain":{"domain":["onlinelibrary.wiley.com"],"crossmark-restriction":true},"short-container-title":["Mycoses"],"published-print":{"date-parts":[[2023,9]]},"abstract":"<jats:title>Abstract<\/jats:title><jats:sec><jats:title>Background<\/jats:title><jats:p>To compare the performance of conventional, semi\u2010nested and real\u2010time panfungal ITS PCRs for diagnosing fungal keratitis (FK) and develop genus\u2010specific real\u2010time PCR for the most common aetiology of FK.<\/jats:p><\/jats:sec><jats:sec><jats:title>Methods<\/jats:title><jats:p>This multicentric study includes 232 corneal samples from suspected FK patients from four centres across India between November 2019 through August 2021. A total of 87 corneal buttons were included for the comparison of conventional, semi\u2010nested and real\u2010time ITS PCRs, of which 68 were from confirmed FK patients. Of these 87 samples, 44 (microscopy and culture positive for <jats:italic>Aspergillus<\/jats:italic> sp. and\/or <jats:italic>Fusarium<\/jats:italic> sp.) were used for the standardisation of genus\u2010specific real\u2010time primers\/probes. Subsequently, the best method showing highest sensitivity and specificity was validated in 188 samples.<\/jats:p><\/jats:sec><jats:sec><jats:title>Results<\/jats:title><jats:p>On Bayesian comparison, conventional ITS2 PCR showed best performance (sensitivity and specificity of 55.88% and 100%, respectively). Since, real\u2010time ITS2 PCR was also considerably efficient (sensitivity and specificity of 51.47% and 84.21%, respectively) in comparison with the conventional PCR but faster, cost\u2010effective, and less labor\u2010intensive, ITS\u20102 real\u2010time PCR is a suitable method that can be applied along with culture and microscopy. During validation, real\u2010time PCR with genus\u2010specific primers showed 61.76% and 91.18% sensitivity with specificity of 98.05% and 79.22%, respectively, for <jats:italic>Aspergillus<\/jats:italic> sp. and <jats:italic>Fusarium<\/jats:italic> sp. <jats:italic>Aspergillus<\/jats:italic> probe, <jats:italic>Fusarium<\/jats:italic> probe and duplex PCR showed sensitivity of 52.94%, 50% and 54.41% with specificity of 92.86%, 82.47% and 75%, respectively. No cross\u2010reactivity of genus\u2010specific PCRs was observed during standardisation.<\/jats:p><\/jats:sec><jats:sec><jats:title>Conclusions<\/jats:title><jats:p>ITS\u20102 real\u2010time PCR can be applied as an adjunct with conventional methods for the diagnosis of FK. The genus\u2010specific duplex real\u2010time PCRs are rapid which reduces the turnaround time (TAT) avoiding the need for sequencing.<\/jats:p><\/jats:sec>","DOI":"10.1111\/myc.13618","type":"journal-article","created":{"date-parts":[[2023,6,26]],"date-time":"2023-06-26T03:49:30Z","timestamp":1687751370000},"page":"801-809","update-policy":"https:\/\/doi.org\/10.1002\/crossmark_policy","source":"Crossref","is-referenced-by-count":11,"title":["Development of single\u2010tube real\u2010time <scp>PCR<\/scp> assay for the rapid detection of <i>Aspergillus<\/i> and <i>Fusarium<\/i>\u2014The two most common causative agents in fungal keratitis"],"prefix":"10.1111","volume":"66","author":[{"given":"Yamini","family":"Tawde","sequence":"first","affiliation":[{"name":"Department of Medical Microbiology PGIMER  Chandigarh India"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Sourav","family":"Das","sequence":"additional","affiliation":[{"name":"Department of Medical Microbiology PGIMER  Chandigarh India"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Amit","family":"Gupta","sequence":"additional","affiliation":[{"name":"Advance Eye Center PGIMER  Chandigarh India"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0001-9979-264X","authenticated-orcid":false,"given":"Savitri","family":"Sharma","sequence":"additional","affiliation":[{"name":"L.V. 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