Fungal infections diagnosis - Past, present and future

Abstract

Despite the scientific advances observed in the recent decades and the emergence of new methodologies, the diagnosis of systemic fungal infections persists as a problematic issue. Fungal cultivation, the standard method that allows a proven diagnosis, has numerous disadvantages, as low sensitivity (only 50% of the patients present positive fungal cultures), and long growth time. These are factors that delay the patient's treatment and, consequently, lead to higher hospital costs. To improve the accuracy and quickness of fungal infections diagnosis, several new methodologies attempt to be implemented in clinical microbiology laboratories. Most of these innovative methods are independent of pathogen isolation, which means that the diagnosis goes from being considered proven to probable. In spite of the advantage of being culture-independent, the majority of the methods lack standardization. PCR-based methods are becoming more and more commonly used, which has earned them an important place in hospital laboratories. This can be perceived now, as PCR-based methodologies have proved to be an essential tool fighting against the COVID-19 pandemic. This review aims to go through the main steps of the diagnosis for systemic fungal infection, from diagnostic classifications, through methodologies considered as "gold standard", to the molecular methods currently used, and finally mentioning some of the more futuristic approaches.

Keywords: Fungal infections diagnosis; Gold standard methodologies; PCR-based methodologies; Probable diagnosis; Proven diagnosis.

Fig. 1

Systemic fungal infection diagnosis framework. When a patient does not respond to the antibiotic treatment (A), a systemic fungal infection should be included in the differential diagnosis (B–C). After evaluating the 3 parameters defined by the EORTC and MSG-ERC (D) (host factors, clinical manifestations and mycological evidence), and if there is a strong evidence for a systemic fungal infection, mycological evidences should be assessed (E). The main goal is that the method used to identify the pathogenic fungi delivers fast and accurate results, which leads to a better therapeutic plan and lower hospital costs (F). Abbreviations: EORTC, European Organization for Research and Treatment of Cancer/Invasive Fungal Infections Cooperative Group; MSG-ERC, Mycoses Study Group – Education & Research Consortium; NMR, Nuclear Magnetic Resonance; FISH, Fluorescence in situ hybridization; CAGT, Candida albicans Germ Tube Antibody Assay; MALDI-TOF, Matrix-Assisted Laser Desorption/Ionization – Time of Flight.

Fig. 2

PCR-based methods framework for fungal infections diagnosis and the possible outcomes. ^aQuantitative real-time PCR using intercalation dyes may require melting curve analysis of amplicons. ^bProbe-based qPCR uses probes with a specific fluorescence to distinguish different amplicons, being ideal for multiplex situations. Abbreviations: PCR, Polymerase Chain Reaction; FISH, Fluorescence in situ Hybridization; RFLP, restriction fragment length polymorphism.