Molecular Assay Detects Pathogenic Fungi

A real-time polymerase chain reaction (PCR) assay detects and differentiates dimorphic fungi from culture isolates and directly from clinical specimens.

Primers and fluorescence resonance energy transfer-hybridization probes were designed to identify specific genes of Blastomyces dermatitidis and Histoplasma capsulatum, two pathogenic fungi that may cause severe mycoses.

Scientists at the Mayo Clinic (Rochester, MN, USA) collected over a 12-month period, 797 clinical specimens from patients with suspected fungal infection that were analyzed immediately. Primers and fluorescence resonance energy transfer (FRET) hybridization probes were designed to target a 174-base pair (bp) region of the dimorphism-regulating histidine kinase (DRK-1) gene of B. dermatitidis and a 192-bp region of the glyceraldehyde-3-phosphate dehydrogenase (GAPDH) gene of H. capsulatum. Respiratory specimens, such as bronchial washings, bronchoalveolar fluid and pleural fluid, were also cultured.

The real-time PCR assay was developed on the LightCycler 2.0 instrument (Roche Applied Sciences, Indianapolis, IN, USA). The analytical sensitivity was determined to be 100 copies/µL for B. dermatitidis and H. capsulatum. From culture isolates, the assay demonstrated 100% specificity and 100% sensitivity for B. dermatitidis and 100% specificity and 94% sensitivity for H. capsulatum. Direct detection from the 797 clinical specimens demonstrated specificities and sensitivities of 99% and 86% for B. dermatitidis and 100% and 73% for H. capsulatum compared with the results for culture. There was no cross reactivity with other pathogenic fungi and the assay could detect and differentiate B. dermatitidis and H. capsulatum directly from clinical specimens within about four hours from specimen receipt.

Although microbiologic culture is a standard method for detecting these pathogens, their recovery may require days to weeks, and the manipulation of cultures presents a significant safety hazard to laboratory personnel. The authors concluded that the real-time PCR assay offers a rapid, sensitive, and specific method for the detection and differentiation of B. dermatitidis and H. capsulatum from culture isolates and clinical specimens. An additional advantage of the real-time PCR assay is increased safety for laboratory personnel by reducing the need to manipulate cultures of specimens that are positive by the PCR assay. The study was published in September 2011, in the Journal of Clinical Microbiology.

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