Molecular assays versus mycological methods for diagnosis of rhino orbital mucormycosis: analysis of 120 fresh clinical specimens from COVID-19 patients

Molecular assays versus mycological methods for diagnosis of rhino orbital mucormycosis: analysis of 120 fresh clinical specimens from COVID-19 patients | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article Molecular assays versus mycological methods for diagnosis of rhino orbital mucormycosis: analysis of 120 fresh clinical specimens from COVID-19 patients Sajedeh Soltani, Mahzad Erami, Kazem Ahmadikia, Shima Aboutalebian, and 4 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-4618013/v1 This work is licensed under a CC BY 4.0 License Status: Published Journal Publication published 05 Mar, 2025 Read the published version in Mycopathologia → Version 1 posted 5 You are reading this latest preprint version Abstract Mucormycosis, a fungal emergency, poses a serious threat to both COVID-19 and non-COVID-19 individuals due to its invasive nature, rapid progression, and high rates of morbidity and mortality which highlights the crucial need for its timely detection and management. Here, we investigated the utility of Mucorales-specific real-time PCR (rt-PCR) assays for the detection of mucormycosis from clinical specimens and compared with conventional methods and duplex PCR. Methods : Both SYBR Green and TaqMan rt-PCR methodologies were optimized using Mucorales-specific oligonucleotides to amplify the conserved 18S rDNA targets. DNAs extracted from 120 rhino sinus specimens, which all were collected from COVID-19 patients upon suspicion of invasive fungal infections, were used for molecular diagnosis. The results of both rt PCR assays were compared with the result of direct microscopy, culture, and duplex Mucorales-specific PCR assay. Results : SYBR Green rt-PCR detected Mucorales in 51 out of 120 (91.67% of K0H-positive samples), yielding a unique Tm pattern (80.24 ± 0.70°C), whereas TaqMan-probe PCR and culture methods detected it in 34 (73.84%) and 24 samples, respectively. The SYBR Green-based PCR was also more sensitive/specific than the duplex PCR technique. The lower sensitivity in probe-based PCR can be influenced by various factors such as probe degeneracy, which can lead to false-negative results. Conclusion : SYBR Green-based PCR showed superiority over duplex PCR, culture, and TaqMan-probe PCR in terms of cost-effectiveness, rapidness, and sensitivity for the diagnosis of mucormycosis. As there is no serological test, SYBR Green-based PCR can be used as an affirmatory test to rule in or rule out mucormycosis, particularly in cases with atypical hyphae or septate and non-septate hyphae suggestive of mixed infections in direct examination but negative culture. mucormycosis diagnosis real-time PCR COVID-19 degenerate probe Full Text Cite Share Download PDF Status: Published Journal Publication published 05 Mar, 2025 Read the published version in Mycopathologia → Version 1 posted Reviewers agreed at journal 27 Jun, 2024 Reviewers invited by journal 26 Jun, 2024 Editor invited by journal 25 Jun, 2024 Editor assigned by journal 22 Jun, 2024 First submitted to journal 21 Jun, 2024 You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. 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Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-4618013","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":319562477,"identity":"2540b9ef-8c42-47dd-ace0-777c4f307b5d","order_by":0,"name":"Sajedeh Soltani","email":"","orcid":"","institution":"Isfahan University of Medical Sciences","correspondingAuthor":false,"prefix":"","firstName":"Sajedeh","middleName":"","lastName":"Soltani","suffix":""},{"id":319562478,"identity":"01fad83b-7f71-4c81-b951-7713e2420794","order_by":1,"name":"Mahzad Erami","email":"","orcid":"","institution":"Kashan University of Medical 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Here, we investigated the utility of Mucorales-specific real-time PCR (rt-PCR) assays for the detection of mucormycosis from clinical specimens and compared with conventional methods and duplex PCR.\u003c/p\u003e \u003cp\u003e \u003cb\u003eMethods\u003c/b\u003e: Both SYBR Green and TaqMan rt-PCR methodologies were optimized using Mucorales-specific oligonucleotides to amplify the conserved 18S rDNA targets. DNAs extracted from 120 rhino sinus specimens, which all were collected from COVID-19 patients upon suspicion of invasive fungal infections, were used for molecular diagnosis. The results of both rt PCR assays were compared with the result of direct microscopy, culture, and duplex Mucorales-specific PCR assay.\u003c/p\u003e \u003cp\u003e \u003cb\u003eResults\u003c/b\u003e: SYBR Green rt-PCR detected Mucorales in 51 out of 120 (91.67% of K0H-positive samples), yielding a unique Tm pattern (80.24\u0026thinsp;\u0026plusmn;\u0026thinsp;0.70\u0026deg;C), whereas TaqMan-probe PCR and culture methods detected it in 34 (73.84%) and 24 samples, respectively. The SYBR Green-based PCR was also more sensitive/specific than the duplex PCR technique. The lower sensitivity in probe-based PCR can be influenced by various factors such as probe degeneracy, which can lead to false-negative results.\u003c/p\u003e \u003cp\u003e \u003cb\u003eConclusion\u003c/b\u003e: SYBR Green-based PCR showed superiority over duplex PCR, culture, and TaqMan-probe PCR in terms of cost-effectiveness, rapidness, and sensitivity for the diagnosis of mucormycosis. 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