A novel comprehensive set of fungal Real time PCR assays (fuPCR) for the detection of fungi in immunocompromised haematological patients—A pilot study

Abstract

Fungal infections are recognized in an increasing number of patients with immunological deficits and are associated with high rates of mortality (Brown et al., 2012a). In this pilot-study, a rapid Real time PCR (fuPCR) was designed for the detection and differentiation of fungal pathogens in clinical specimens of haematological patients. The fuPCR, targeting the internal transcribed spacer region 2 (ITS2) of rDNA region, is comprised of seven multiplex reactions, which were shown to be specific and sensitive for a comprehensive spectrum of clinically relevant fungal species. This was validated by testing respective fungal DNAs in each fuPCR reaction and 28 respiratory samples of fungal pneumonia-proven patients. Clinical sample sets of throat swab, EDTA-blood and blood sera from 50 patients with severe haematological malignancies, including haematopoietic stem cell transfer (HSCT), and samples from 30 healthy individuals were then analysed. In a first step, 198 samples of immunosuppressed patients were solely examined by fuPCR; and 50.8% (33/65) respiratory swabs, 4.8% (3/63) EDTA blood samples and 1.4% (1/70) blood serum samples were tested positive. In a second step, 56 respiratory samples of immunosuppressed patients and 30 of healthy individuals were simultaneously analysed by fuPCR and standard cultivation techniques. By both methods 30.4% (17/56) swabs of the immunocompromised patients were tested positive, 37.5% (21/56) were tested negative and 32.1% (18/56) were tested fuPCR positive and culture negative. In analysing the blood samples of the immunocompromised patients 5.4% (3/56) EDTA blood samples and 16.1% (9/56) sera samples were tested fuPCR-positive, whereas all samples of 30 healthy individuals with no signs of immunological deficits were tested negative by fuPCR.38.9% (14/36) of the fungi detected in respiratory samples of the immunosuppressed patients, belonged to Candida spp., 47.2% (17/36) to Saccharomyces spp., 5.6% (2/36) to Cladosporium spp. and 8.3% (3/36) to Alternaria spp., whereas cultivation only identified Candida spp. (10/17) and Saccharomyces spp. (7/17).In this pilot study a novel fuPCR assay was developed and validated for the simultaneous and comprehensive detection of fungal pathogens in clinical respiratory specimens of haematological patients. Future work will focus on the validation of the blood-stream detected fungi in pathogenicity of these patients.