Molecular diagnosis of rhino-orbito-cerebral mucormycosis from fresh tissue samples

Purpose. We aimed to evaluate a PCR-based technique for the diagnosis of mucormycosis and the identification of fungi from fresh tissue specimens in patients with rhino-orbito-cerebral-mucormycosis (ROCM). Methodology. Fifty cases of ROCM were included in the study. Conventional identification was performed using microscopy and culture. Molecular diagnosis was performed by amplifying the ribosomal DNA using pan-fungal ITS primers and seminested Mucorales-specific primers of the 18S region. The amplified products were sequenced to identify the agents. The utility of PCR-RFLP of the 18S region of rDNA was evaluated to identify the Mucorales. Results. The ROCM cases were diagnosed by the demonstration of aseptate ribbon-like hyphae in biopsy specimens collected from the patients. Isolation was possible in 24 (48 %) samples. The ITS2 PCR confirmed mucormycosis in 27 cases (54 %; CI 59.4-68.2). By comparison, Mucorales-specific PCR was able to amplify DNA and the sequence enabled the identification of Mucorales speciesin all the patients. PCR-RFLP of the 18S region of rDNA could only identify the agent to genus level. Conclusion. The molecular technique was able to identify Mucorales species in 26 (42 %) cases that were negative by culture. Mucorales-specific semi-nested PCR targeting the 18S region is a better technique than ITS2 PCR for diagnosis. PCR-RFLP of the 18S region helps in identification to genus level.