PCR-RFLP as a useful tool for diagnosis of invasive mycoses in a healthcare facility in the North of Brazil

Abstract

Background: The incidence of invasive mycoses is increasing worldwide. Polymerase Chain Reaction (PCR)-based methods have been used as alternatives to conventional microscopy and culture with the additional benefit of greater accuracy. The aim of this study was to evaluate the usefulness of PCR amplification of ITS5-NL4 and subsequent analysis of products by Restriction Fragment Length Polymorphism (PCR-RFLP) as a routine tool to detect and identify fungi involved in invasive mycoses in a healthcare center in the North of Brazil. PCR-RFLP was applied to identification of 10 reference strains and 90 cultures of agents of invasive mycoses. In addition, the new approach was applied to detect fungal agents in 120 biological samples (blood, cerebrospinal fluid and bone marrow) from patients with suspicion of systemic mycoses. PCR-RFLP results were compared with the ones obtained with conventional methods (culture, microscopy, and biochemical testing).

Results: The assay carried out with the reference strains (Candida albicans, Candida parapsilosis, Candida tropicalis, Candida krusei, Candida guilliermondii, Cryptococcus neoformans, Cryptococcus gattii and Histoplasma capsulatum) demonstrated that the RFLP profiles were correctly predicted by the in silico investigation and allowed unequivocal identification of all chosen reference strains. The investigated PCR-RFLP also identified correctly 90 cultures of agents of invasive mycoses 2.5 times faster than the conventional assays. By using this assays in the routine of diagnosis of invasive mycosis it was observed that the PCR was found to be highly accurate with 100% and the RFLP profiles allowed identification of the etiological agents: C. neoformans (n = 3) and C. gattii (n = 1) in CSF samples, H. capsulatum (n = 1) in bone marrow and C. albicans (n = 2) in blood cultures. The detection and identification by PCR-RFLP were found to be between two to ten times faster than the conventional assays.

Conclusion: The results demonstrated that PCR-RFLP is a valuable tool for the identification of invasive mycoses that can be implemented in hospital laboratories, allowing for a high number of analyses per day.