Enhanced Molecular Identification of Aspergillus and Mucorales in Clinical Samples: A Comparative Analysis of Molecular and Culture Techniques

This study compared the diagnostic performance of conventional culture-based methods with internal transcribed spacer (ITS)-based polymerase chain reaction–restriction fragment length polymorphism (PCR-RFLP) for the identification of clinically important molds. Sixty-seven clinical specimens were processed using both fungal culture and PCR-RFLP targeting the ITS region with MwoI digestion. Atypical patterns were further assessed through in-silico analysis and confirmed with Mucorales-specific primers. Statistical analysis was performed using Chi-square and Cohen’s Kappa tests. Fungal growth was detected by culture in seven samples, whereas ITS-PCR-RFLP identified fungal DNA in twelve samples (17.9%). Molecular analysis revealed Aspergillus flavus, Aspergillus fumigatus, Aspergillus ochraceus, Aspergillus nidulans, Aspergillus amstelodami, Aspergillus terreus, and Rhizopus arrhizus, including mixed infections that culture failed to detect. The incidental identification of R. arrhizus by novel MwoI digestion patterns, subsequently confirmed with specific primers, demonstrated the broader applicability of this method. Although Chi-square testing showed a significant association (p < 0.05) between methods, Cohen’s Kappa indicated poor concordance (κ = 0.058), reflecting discordant results. These findings indicate that ITS-PCR-RFLP provides higher sensitivity than conventional culture for detecting Aspergillus and Mucorales, enabling species-level identification and co-infection detection. Incorporating molecular diagnostics into clinical workflows can improve diagnostic accuracy and facilitate timely antifungal treatment.