Background: Diagnosis of candidaemia typically relies upon blood cultures, which often require several days for correct species identification. Diagnosis of invasive fungal infection (IFI) relies upon a consensus of clinical and laboratory criteria with certainty ranging from definite to probable or possible. Because definite diagnosis requires visualization of the organism in tissue, the majority of patients with IFI fall into the probable or possible categories. We investigated the hypothesis that a single molecular assay based upon broad PCR amplification followed by mass spectrometry could be used to identify a wide variety of fungi and yeast. Methods: Four broad-range PCR primers were selected within the 5.8S and 26S rRNAs for detection of fungi such that the product base compositions could be used to directly identify species or narrow possibilities to a range of species. Additional twelve primer pairs were designed to identify specific groups by virtue of product amplification and identify species by virtue of product base compositions. Automated PCR/Electrospray ionization mass spectrometry and data analysis provide base composition signatures that identify clinically-relevant species of fungi and yeast. Results: An assay has been defined to target a range of fungi and yeast that consists of 16 primer pairs, allowing 6 samples to be run on a 96-well plate. The assay broadly detects fungi and yeast, and identifies members of Candida spp. (speciating C. albicans, C. tropicalis, C. glabrata, C. parapsilosis, and C. krusei), Aspergillus spp., Cryptococcus neoformans, Mucorales, Coccidiodes immitis/posadasii, Ajellomyces spp. and, Fusarium spp. A collection of clinically-relevant isolates consisting of 25 Aspergillus spp. isolates, 58 Candida spp. isolates, 13 Cryptococcus spp. isolates, 10 Mucor / Rhizopus isolates, 5 Saccharomycete isolates and 9 Sordariomycete isolates have been tested and correctly identified with automated data analysis. Assay limits of detection were determined using quantitated C albicans and C. glabrata isolates obtained from ATCC. Both species were detected at 30-60 genome copies/PCR. Conclusion: PCR/ESI-MS is a novel approach to fungal diagnostics with potential for rapid identification of a broad variety of fungal or yeast pathogens in a single assay.
Full conference title:
19th European Congress of Clinical Microbiology and Infectious Diseases
- ECCMID 19th (2009)