gms | German Medical Science

Objectives: The rise in the incidence of fungal infections and the expanding spectrum of fungal pathogens make early and broad detection as well as accurate identification of fungal pathogens essential. While species- or genus-specific PCR assays assume a certain infection, panfungal real-time PCR enables the unspecific detection and quantification of any fungal DNA present in a clinical sample. In a panfungal PCR assay, universal primers should target sequences specific for all fungal species to guarantee the detection of all fungal taxa. For species identification, PCR amplicons have subsequently to be sequenced and phylogenetically analysed. Thus, in addition to improving turn-around time of microbiological identification, panfungal PCR can provide results which in some cases lead to potentially surprising diagnoses, as enabling diagnosis of also uncommon and rare fungal infections.

Methods: In this study a new panfungal HybProbe real-time PCR assay was designed and analytically as well as clinically evaluated. The panfungal real-time PCR assay targets the complete fungal ITS2 region using the LightCycler instrument (Roche®). Due to the broad-range feature of the primers and probe set, the assay allows for the detection of any fungal pathogen. Fungal DNA can be detected by a positive amplification curve at 640 nm in the LightCycler instrument (Roche^®). Species are identified by subsequent phylogenetic sequence analysis (BLAST).

After having developed the PCR assay 401 clinical samples derived from patients with clinically suspected invasive or superficial fungal infection were investigated. Results were compared to conventional methods (culture, KOH and histology) and clinical signs of infection and other molecular techniques.

Results: The samples consisted of 90 BALs and 6 bronchial secretions, 52 tissue samples, 66 samples of various sterile fluids, four paraffin tissue sections, 60 EDTA blood samples and 133 dermatological samples (90 nail samples and 43 skin scrapings). Out of these 401 samples 206 showed concordant positive or negative results. 20 samples showed positive results only by the panfungal PCR thus allowing for diagnosis which would have been missed if only culture would have been used. Especially, the use of PCR in blood and tissue samples showed better results than culture. However, there were cases when PCR detected airborne contamination (e.g. Cladosporium sp.) or colonization (e.g. Candida spp. in respiratory samples) In sum, fungal pathogens were properly identified by the panfungal assay.

Conclusion: Results showed that the new assay improved the early diagnosis of fungal infections. The molecular approach helped to identify the species of culture negative but histologically positive samples. The assay was able to reduce time to detection and identification from two weeks down to two days. It was further able to successfully detect rare emerging pathogens, particularly in specimens from invasive infections. Its evident benefits make it a valuable tool especially where accurate and fast detection is necessary, such as the emergency setting, or where culture does not provide a clear or no result. As fungi due to colonization or airborne contamination can be detected as well as the infecting agent, results have to be interpreted in context with conventional methods and clinical data for reliable diagnosis.