Molecular phylogenetic analysis of paranasal sinus tissue-associated fungal pathogens acquired from COVID-19 patients

Objectives: During the second wave of the COVID-19 pandemic, a rapid increase in cases of mucormycosis (MM), or black fungus-associated infection, has been reported throughout India. Many of the immunocompromised individuals with substantial use of steroids, oxygen therapy, and patients with comorbidities such as diabetes mellitus, hypertension, ischemic heart disease, and pneumonia were at high risk of getting MM as a serious co-infection along with COVID-19. Even after the hype around MM and other fungal pathogens in COVID-19 patients, they still largely remain neglected and least studied infections. Materials and Methods: Here, we report the internal transcribed spacer ( ITS )-5.8S ribosomal DNA (rDNA) region-based molecular phylogenetic characterization of ten pure fungal cultures isolated from paranasal sinus tissue samples of COVID-19 patients clinically and radiologically suspected of invasive fungal infection. From all the fungal isolates, whole genomic DNA was extracted, polymerase chain reaction (PCR) amplified, and subjected to sequencing with primers specific for the ITS gene. Results: All ten sequences generated were above 550 base pairs in length, and these quality-checked sequences were deposited in the National Center for Biotechnology Information GeneBank. Molecular phylogenetic analysis revealed that the ten fungal sequences (accession numbers OR140558–OR140567) generated during the current study belonged to four broad clusters of Rhizopus delemar, Rhizopus microsporus, Flavodon spp., and Aspergillus flavus . Conclusion: We suggest that ITS-5.8S rDNA region-based molecular identification tools, such as PCR and Sanger sequencing, are suitable for early diagnosis and identification of fungal pathogens in most of the immunocompromised patients during the viral epidemic and/or pandemic situations. Increased awareness amongst clinicians and public health providers about including these molecular techniques for early and accurate diagnosis of invasive fungal infections is required.