JCM Accepts, published online ahead of print on 14 May 2008

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J. Clin. Microbiol. doi:10.1128/JCM.02331-07
Copyright (c) 2008, American Society for Microbiology and/or the Listed Authors/Institutions. All Rights Reserved.

A Real-Time PCR Method for Detection of Zygomycetes

D. Jane Hata, Seanne P. Buckwalter, Bobbi S. Pritt, Glenn D. Roberts, and Nancy L. Wengenack^*

Division of Clinical Microbiology, Mayo Clinic, Rochester, Minnesota 55905

^* To whom correspondence should be addressed. Email: wengenack.nancy{at}mayo.edu.

Zygomycete infections can be devastating in immunocompromised hosts. Difficulties in the histopathologic differentiation of this class from other filamentous fungi (eg., Aspergillus spp., Fusarium spp.) may lead to delays in diagnosis and initiation of appropriate treatment thereby significantly affecting patient outcome. A real-time PCR assay was developed to detect the zygomycete genera Absidia, Apophysomyces, Cunninghamella, Mucor, Rhizopus, and Saksenaea from culture and tissue samples. Primers and FRET hybridization probes were designed to detect a 167-bp conserved region of the multi-copy zygomycete cytochrome b gene. A plasmid containing target sequence from Mucor racemosus was constructed as a positive control. The analytical sensitivity of the assay is 10 targets/µl and a specificity panel consisting of other filamentous fungi, yeast, and bacteria demonstrated no cross-reactivity in the assay. The clinical sensitivity and specificity of the assay from culture isolates was 100% (39/39) and 92% (59/64) respectively. Sensitivity and specificity from a limited number of fresh tissue specimens was 100% (2/2). The sensitivity from formalin-fixed, paraffin-embedded tissues was 56% (35/62), and the specificity was 100% (19/19). The speed, sensitivity, and specificity of the PCR assay indicate that it is useful for the rapid and accurate diagnosis of zygomycete infection.

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