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

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.