MOLECULAR PROFILING REVEALS A DIVERSE ORAL MYCOBIOME IN HEAD AND NECK CANCER PATIENTS FROM PAKISTAN

Head and neck cancer (HNC) is a prevalent malignancy in Pakistan, with treatments like radiotherapy and chemotherapy increasing the risk of oral fungal infections. This study aimed to characterize the diversity of oral yeast species in HNC patients in Pakistan using a combination of phenotypic and molecular diagnostic techniques. Saliva samples were collected from 110 HNC patients at KIRAN Hospital and 100 healthy controls. Yeasts were isolated on culture media and initially screened using two chromogenic media: Brilliance Candida Agar (BCA) and CHROMagar™ Candida Plus (CCP). Molecular identification was performed by amplifying the Internal Transcribed Spacer (ITS) region, followed by Restriction Fragment Length Polymorphism (RFLP) analysis with the MspI enzyme and DNA sequencing for definitive speciation. Phylogenetic analysis and statistical modeling were conducted to understand species relationships and risk factors. A total of 126 yeast isolates were recovered from HNC patients. Chromogenic media identified up to six Candida species, with CCP showing superior differentiation. Molecular methods revealed a greater diversity of 11 yeast species. DNA sequencing confirmed the presence of common pathogens like Candida albicans, C. tropicalis, and Rhodotorula mucilaginosa, as well as uncommon species such as Cyberlindnera fabianii (reported for the first time in Pakistan in this context) and Candida gattii. PCR-RFLP was useful for common species but could not differentiate C. albicans from C. dubliniensis or species lacking an MspI restriction site. Statistical analysis confirmed that HNC patients had a significantly higher risk (OR: 19.94-174.48, p