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

Abstract

BackgroundMucormycosis, 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/MethodsBoth 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.ResultsSYBR Green real-time PCR produced a distinct melting temperature (Tm) pattern (80.24 +/- 0.70 degrees 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).ConclusionSYBR 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.