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Invasive fungal infections (IFIs) have risen dramatically in recent years among high risk immunocompromised patients.Rapid detection of fungal pathogens is crucial to timely and accurate antifungal therapy. Two multiplex polymerasechain reaction (PCR) assays were developed to detect major fungal species that cause invasive infections and identifyresistant species. Genus specific primers for Candida, Aspergillus, Fusarium and species specific primers for Candidaglabrata, Candida krusei and Aspergillus terreus which are known to be clinically resistant species, were designed fromthe internal transcribed spacer (ITS) regions of ribosomal ribonucleic acid (rRNA) gene complex. Both assays wereperformed simultaneously to promote rapid detection of fungal isolates based on distinct amplicon sizes. Inclusion of theuniversal fungal primers ITS 1 and ITS 4 in the genus specific assay produced a second amplicon for each isolate whichserved to confirm the detection of a fungal target. The limit of detection for the genus specific assay was 1 nanogram(ng) deoxyribonucleic acid (DNA) for Aspergillus fumigatus and Candida albicans, 0.1 ng DNA for Fusarium solani, whilethe species-specific assay detected 0.1 ng DNA of A. terreus and 10 picogram (pg) DNA of C. krusei and C. glabrata. Themultiplex PCR assays, apart from universal detection of any fungal target, are able to detect clinically important fungiand differentiate resistant species rapidly and accurately, which can contribute to timely implementation of effectiveantifungal regime.

Dual Panel Multiplex PCR Assay for Rapid Detection of Medically Important Fungi and Resistant Species of Candida and Aspergillus