Molecular Assays Versus Mycological Methods for Diagnosis of Rhino Orbital Mucormycosis: Analysis of 120 Clinical Specimens from COVID-19 Patients

Overview

Journal Mycopathologia

Specialties Microbiology
Pharmacology

Date 2025 Mar 5

PMID 40045088

Authors

Sajedeh Soltani

Mahzad Erami

Kazem Ahmadikia

Shima Aboutalebian

Faezeh Rouhi

Mojtaba Fakhrehi

Reza Mohammadi Manesh

Hossein Mirhendi

Affiliations

Soon will be listed here.

Abstract

Background: 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. This underscores the crucial need for timely detection and management. In this study, we investigated the utility of real-time PCR (RT-qPCR) assays for detecting Mucorales in clinical specimens, and assessed the performance of both SYBR Green and TaqMan probe RT-qPCR in amplifying Mucorales-specific 18S rDNA genes. We conducted accuracy analyses using direct examination with KOH as a standard for the laboratory diagnosis of mucormycosis. Additionally, we compared the results with culture and duplex PCR.

Patients/methods: Both SYBR Green and TaqMan RT-qPCR 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-qPCR assays were compared with the result of direct microscopy, culture, and duplex Mucorales-specific PCR assay.

Results: SYBR Green real-time PCR produced a distinct melting temperature (Tm) pattern (80.24 ± 0.70 °C) and detected Mucorales in 51 out of 120 clinical samples. When compared to direct examination with KOH, the standard method for diagnosing mucormycosis, SYBR Green PCR demonstrated a sensitivity of 91.67% (95% confidence interval (CI): 86.7-96.5%) and a specificity of 90.28% (95% CI: 84.9-95.5%). In contrast, TaqMan-probe PCR identified Mucorales in 34 out of 120 samples, with a sensitivity of 64.58% (95% CI: 56-73.1%) and a specificity of 95.83% (95% CI: 92.26-99.39%).

Conclusion: SYBR Green-based PCR can be used as a reliable confirmatory test for diagnosing mucormycosis, particularly in cases with atypical hyphae, mixed infections (featuring both septate and non-septate hyphae), or when the direct examination is positive but culture results are negative. The lower sensitivity of the TaqMan-probe PCR may be attributed to factors such as using a degenerate probe, which can lead to false-negative results.

References

Hayden R, Qian X, Procop G, Roberts G, Lloyd R . In situ hybridization for the identification of filamentous fungi in tissue section. Diagn Mol Pathol. 2002; 11(2):119-26. DOI: 10.1097/00019606-200206000-00009. View

Aboutalebian S, Erami M, Ahsaniarani A, Momen-Heravi M, Sharif A, Hadipour M . Diagnosis of mucormycosis using a simple duplex PCR assay: Analysis of 160 clinical samples from COVID-19 patients. Med Mycol. 2023; 61(9). DOI: 10.1093/mmy/myad091. View

Erami M, Aboutalebian S, Hashemi Hezaveh S, Daie Ghazvini R, Momen-Heravi M, Jafari Y . Microbial and clinical epidemiology of invasive fungal rhinosinusitis in hospitalized COVID-19 patients, the divergent causative agents. Med Mycol. 2023; 61(3). DOI: 10.1093/mmy/myad020. View

U S Rao V, Arakeri G, Madikeri G, Shah A, Oeppen R, Brennan P . COVID-19 associated mucormycosis (CAM) in India: a formidable challenge. Br J Oral Maxillofac Surg. 2021; 59(9):1095-1098. PMC: 8239211. DOI: 10.1016/j.bjoms.2021.06.013. View

Cornely O, Arikan-Akdagli S, Dannaoui E, Groll A, Lagrou K, Chakrabarti A . ESCMID and ECMM joint clinical guidelines for the diagnosis and management of mucormycosis 2013. Clin Microbiol Infect. 2014; 20 Suppl 3:5-26. DOI: 10.1111/1469-0691.12371. View

Garg D, Muthu V, Sehgal I, Ramachandran R, Kaur H, Bhalla A . Coronavirus Disease (Covid-19) Associated Mucormycosis (CAM): Case Report and Systematic Review of Literature. Mycopathologia. 2021; 186(2):289-298. PMC: 7862973. DOI: 10.1007/s11046-021-00528-2. View

Badiee P, Arastefar A, Jafarian H . Comparison of histopathological analysis, culture and polymerase chain reaction assays to detect mucormycosis in biopsy and blood specimens. Iran J Microbiol. 2015; 5(4):406-10. PMC: 4385169. View

Hammond S, Bialek R, Milner D, Petschnigg E, Baden L, Marty F . Molecular methods to improve diagnosis and identification of mucormycosis. J Clin Microbiol. 2011; 49(6):2151-3. PMC: 3122746. DOI: 10.1128/JCM.00256-11. View

Rickerts V, Just-Nubling G, Konrad F, Kern J, Lambrecht E, Bohme A . Diagnosis of invasive aspergillosis and mucormycosis in immunocompromised patients by seminested PCR assay of tissue samples. Eur J Clin Microbiol Infect Dis. 2006; 25(1):8-13. DOI: 10.1007/s10096-005-0078-7. View

10.

Imbert S, Portejoie L, Pfister E, Tauzin B, Revers M, Uthurriague J . A Multiplex PCR and DNA-Sequencing Workflow on Serum for the Diagnosis and Species Identification for Invasive Aspergillosis and Mucormycosis. J Clin Microbiol. 2022; 61(1):e0140922. PMC: 9879116. DOI: 10.1128/jcm.01409-22. View

11.

Nagao K, Ota T, Tanikawa A, Takae Y, Mori T, Udagawa S . Genetic identification and detection of human pathogenic Rhizopus species, a major mucormycosis agent, by multiplex PCR based on internal transcribed spacer region of rRNA gene. J Dermatol Sci. 2005; 39(1):23-31. DOI: 10.1016/j.jdermsci.2005.01.010. View

12.

Hrncirova K, Lengerova M, Kocmanova I, Racil Z, Volfova P, Palousova D . Rapid detection and identification of mucormycetes from culture and tissue samples by use of high-resolution melt analysis. J Clin Microbiol. 2010; 48(9):3392-4. PMC: 2937726. DOI: 10.1128/JCM.01109-10. View

13.

Springer J, Goldenberger D, Schmidt F, Weisser M, Wehrle-Wieland E, Einsele H . Development and application of two independent real-time PCR assays to detect clinically relevant Mucorales species. J Med Microbiol. 2016; 65(3):227-234. DOI: 10.1099/jmm.0.000218. View

14.

Erami M, Mirhendi H, Momen-Heravi M, Hashemi Hezaveh S, Ahsaniarani A, Sabet S . A case of COVID-19-associated rhino-orbito-cerebral mucormycosis caused by with a review of the literature. Front Cell Infect Microbiol. 2022; 12:898477. PMC: 9615570. DOI: 10.3389/fcimb.2022.898477. View

15.

Aboutalebian S, Ahmadikia K, Fakhim H, Chabavizadeh J, Okhovat A, Nikaeen M . Direct Detection and Identification of the Most Common Bacteria and Fungi Causing Otitis Externa by a Stepwise Multiplex PCR. Front Cell Infect Microbiol. 2021; 11:644060. PMC: 8027314. DOI: 10.3389/fcimb.2021.644060. View

16.

Nayak N, Khan E, Panigrahi D . COVID-19 and Mucormycosis Coinfection: How Challenging It Is. Can J Infect Dis Med Microbiol. 2022; 2022:8617212. PMC: 9010177. DOI: 10.1155/2022/8617212. View

17.

Abdorahimi M, Pakdel F, Salehi M, Alcazar-Fuoli L, Jamal Hashemi S, Daie Ghazvini R . COVID-19 Associated Rhino-Orbital-Cerebral Mucormycosis: Clinical Features, Antifungal Susceptibility, Management and Outcome in a Tertiary Hospital in Iran. Mycopathologia. 2023; 188(5):783-792. DOI: 10.1007/s11046-023-00785-3. View

18.

Osman N, Anwar M, Singh B, Gupta G, Rabie A . A peek behind the curtain in the diagnosis and management of COVID‑19‑Associated Mucormycosis (CAM). J Egypt Public Health Assoc. 2023; 98(1):4. PMC: 9977480. DOI: 10.1186/s42506-022-00125-1. View

19.

Cornely O, Alastruey-Izquierdo A, Arenz D, Chen S, Dannaoui E, Hochhegger B . Global guideline for the diagnosis and management of mucormycosis: an initiative of the European Confederation of Medical Mycology in cooperation with the Mycoses Study Group Education and Research Consortium. Lancet Infect Dis. 2019; 19(12):e405-e421. PMC: 8559573. DOI: 10.1016/S1473-3099(19)30312-3. View

20.

Bergallo M, Tullio V, Roana J, Allizond V, Mandras N, Dapra V . A Rapid and Specific Real-Time PCR Assay for the Detection of Clinically Relevant Mucorales Species. Int J Mol Sci. 2022; 23(23). PMC: 9735628. DOI: 10.3390/ijms232315066. View