Abstract:Background: Molecular techniques are appealing for the detection of human pathogens, but the detection of fungal pathogens is particularly challenging. Fungi have cell walls that impede the efficient lysis of organisms and liberation of DNA, which can lead to false-negative results. Conversely, some human pathogens are ubiquitous environmental saprophytes that can contaminate PCR reagents and cause false-positive results. Aims and methods: In a cross-sectional study in Tehran, Iran from January 2017 to August 2018 we aimed to evaluate the incidence of fungal infections in 420 specimens from patients suspected of fungal disease and determined the clinical application of nested PCR for the diagnosis of fungal infections. Direct examination with 10% potassium hydroxide, culture on mycological media and nested polymerase chain reaction (PCR) by amplifying the conserved sequences of internal transcribed spacer 1 (ITS1) ribosomal DNA were performed in this study. Results: 79 (18.8 %) out of 420 specimens were positive by direct examination, including 64 (81%) dermatophytes and 15 (19 %) Aspergillus spp. 52 (12.3%) had positive culture including 40 (76.9 %) dermatophytes and 12 (23.1) Aspergillus spp. 76/79 isolates were detected by nested-PCR (96.2%). Conclusions: Our results showed that the incidence of fungal infections in the study population was relatively high and dermatophytes were the most common cause of fungal infections. Nested PCR had a high sensitivity and may be useful as a reliable screening method, especially in immunosuppressed and immunocompromised patients.