fuPCR as diagnostic method for the detection of rare fungal pathogens, such as Trichosporon, Cryptococcus and Fusarium

Abstract

Fungal respiratory tract colonisation is a common finding in patients with hematologic neoplasms due to immunosuppression inherent in the diseases and exacerbated by therapy. This greatly increases the risk of fungal infections of the lungs, which is associated with significant mortality. Therefore, reliable diagnostic methods with rapidly available results are needed to administer adequate antifungal therapy. We have established an improved method for fungal DNA extraction and amplification that allows simultaneous detection of fungal families based on a set of multiplexed real time PCR reactions (fuPCR). We analysed respiratory rinses and blood of 94 patients with haematological systemic diseases by fuPCR and compared it with the results of culture and serological diagnostic methods. 40 healthy subjects served as controls. Regarding Candida species, the highest prevalence resulted from microbiological culture of respiratory rinses and from detection of antibodies in blood serum in patients (61% and 47%, respectively) and in the control group (29% and 51%, respectively). Detection of other pathogenic yeasts, such as Cryptococcus and Trichosporon, and moulds, such as Fusarium, was only possible in patients by fuPCR from both respiratory rinses and whole blood and serum. These fungal species were found statistically significantly more frequent in respiratory rinses collected from patients after myeloablative therapy for stem cell transplantation compared to samples collected before treatment (p<<0.05i>). The results show that fuPCR is a valuable complement to culturing and its inclusion in routine mycological diagnostics might be helpful for early detection of pathophysiologically relevant respiratory colonisation for patients with hematologic neoplasms. Lay Abstract We validated a set of PCR reactions (fuPCR) for use in routine diagnostic. In contrast to culture and serological methods, only by fuPCR pathogenic yeasts (Cryptococcus and Trichosporon) and moulds (Aspergillus and Fusarium) were detected in respiratory rinses and blood of haematological patients.

JMM Profile: Trichosporon yeasts: from superficial pathogen to threat for haematological-neutropenic patients

“…Some studies have illustrated the use of in-house developed pan-fungal PCR assays on body fluids and human tissues affected by this pathogen. Although promising, these studies have used the amplification of non-discriminatory DNA targets for Trichosporon species [e.g., internal transcribed (ITS) spacer region of the rDNA gene], generating misidentification between genera and species [15][16][17][18]. Although there is a clear need for better diagnostic tests, in the current guideline for the diagnosis and management of Trichosporon infections, the use of direct DNA detection and identification of this genus from clinical samples by using ITS-directed pan-fungal PCR is 'moderately recommended' [13].…”