Speciation of fungi using real time PCR with molecular beacons: Can we solve the enigma of diagnosis of invasive fungal disease?

BACKGROUND

Invasive fungal diseases (IFDs) are difficult to diagnose and associated with high mortality rates, especially in the immunosuppressed. Species of and are the cause of majority of invasive fungal disease however IFDs are also caused by , Zygomycetes, , etc. Early detection is crucial for appropriate antifungal therapy. Blood cultures usually fail to isolate filamentous fungi, while detection of circulating beta-d-glucan or galactomannan antigens show variable sensitivity and specificity. There is a need of reliable, sensitive and specific diagnostic tests for IFDs.

METHODS

A real-time Polymerase Chain Reaction (PCR) assay with a universal primer/molecular beacon system was developed for detecting and speciating most of the pathogenic fungi implicated in IFD. A single-reaction assay was designed targeting a carefully selected region of the ITS2 and ITS5 subunits of the fungal rDNA gene along with four molecular beacons capable of differential hybridization to the amplicons of different species. This generated a signature set of melting temperatures using the standard strains. The assay was tested on clinical specimens from patients with suspected invasive fungal disease.

RESULTS

The assay was tested on 72 clinical samples and 72 healthy controls. Of these, 22 clinical samples (6/8 proven; 13/29 probable; 3/35 possible IFD, classified by the EORTC/MSG criteria) were positive by PCR and generated a set of melting temperatures enabling identification of the causative fungus. The assay was negative in all healthy controls.

CONCLUSION

The molecular beacon assay is a promising tool providing a rapid method for detection and monitoring of invasive fungal disease in immunosuppressed patients.