The Pan-AC assay: a single-reaction real-time PCR test for quantitative detection of a broad range of Aspergillus and Candida species

Abstract

In view of the growing incidence and the high mortality of invasive aspergillosis and candidiasis, adequate diagnostic techniques permitting timely onset of treatment are of paramount importance. More than 90 % of all invasive fungal infections in immunocompromised individuals can be attributed to Candida and Aspergillus species. To date, standardized techniques permitting rapid, sensitive and, no less importantly, economic screening for the clinically most relevant fungi are lacking. In the present report, a real-time quantitative PCR assay, developed for the detection of the most common pathogenic Candida and Aspergillus species, is described. The single-reaction PCR assay targets a judiciously selected region of the 28S subunit of the fungal rDNA gene. The unique design of the universal primer/probe system, including a pan-Aspergillus and pan-Candida (Pan-AC) hydrolysis probe, facilitates the detection of numerous Aspergillus species (e.g. Aspergillus fumigatus, Aspergillus flavus, Aspergillus niger, Aspergillus terreus, Aspergillus versicolor and Aspergillus nidulans) and Candida species (e.g. Candida albicans, Candida glabrata, Candida krusei, Candida tropicalis, Candida parapsilosis, Candida kefyr, Candida guilliermondii, Candida lusitaniae and Candida dubliniensis). The assay permits highly reproducible detection of 10 fg fungal DNA, which corresponds to a fraction of a fungal genome, and facilitates accurate quantification of fungal load across a range of at least five logs. Upon standardization of the technique using cultured fungal strains, the applicability in the clinical setting was assessed by investigating a series of clinical specimens from patients with documented fungal infections (n=17). The Pan-AC assay provides an attractive and economic approach to the screening and monitoring of invasive aspergillosis and candidiasis, which is readily applicable to routine clinical diagnosis.