Objectives: The majority of invasive fungal infections (IFIs) are attributable to human-pathogenic yeast species. Identification of pathogenic yeasts in developing countries are mainly performed by phenotypic and biochemical assays, which are time-consuming and prone to errors. False species identification might result in inefficient treatment choice and to inaccurate epidemiological data. To improve rapid species identification, a panel of three multiplex PCR assays (YEAST PANEL Multiplex PCR assay) targeting the most clinically important yeast pathogens was designed.

Methods: Desired sequences for primer design were retrieved from the NCBI database. Eight-hundred clinical and 400 CBS isolates, including major and less predominant clinical species of Candida, Trichosporon, Rhodotorula, Cryptococcus, and Geotrichum (Table 1) were subjected to PCR. Specificity and performance tests were performed to check for any cross-reactivity. The possibility of
compatibility of pure colonies instead of purified DNA was evaluated. Recommended workflow for the “YEAST PANEL Multiplex PCR Assay” is summarized in Figure 1.

Results: All targeted species were correctly identified (Figure 2). No cross-reactivity with closely- and distantly-related yeast pathogens, Aspergillus spp., and human DNA was observed. Utilization of pure colony testing showed distinct fragments for each species, eliminating the need for DNA extraction.

Conclusion: This assay targets 99% of the pathogenic yeast species, which could be supplemented with other phenotypic and biochemical assays. Due to high accuracy and encompassing the wide range of pathogenic yeasts included, this assay could be used for identification of pure cultures in routine laboratories and epidemiological studies. Additionally, compared to phenotypic assays, our assay is less expensive, more time-saving and highly specific.

1 / YEAST PANEL Multiplex PCR Assay for Identification of Invasive Yeast Pathogens: Novel Diagnostic Strategy, Useful for Developing Countries