Identification of fungal pathogens in Formalin-fixed, Paraffin-embedded tissue samples by molecular methods
Introduction
Invasive fungal infections (IFIs) continue to be a serious threat in a growing number of patient groups, including cancer patients, organ transplant recipients, the critically ill, and other immunocompromised hosts (Brown et al. 2012).
The clinical diagnosis of IFIs continues to be a challenging task, especially in mould infections. For example, aspergillosis is among the most frequently missed diagnosis in critically ill patients (Tejerina et al. 2012). Furthermore, the identification of a causative mould is not achieved in about half of the affected individuals (Chamilos et al., 2006, Neofytos et al., 2009). This impairs the optimal use of prophylactic measures, optimal treatment strategies, and the development of diagnostic tests. This may in turn lead to adverse outcomes in patients with IFI.
The identification of fungal pathogens by histopathology is an important component in the understanding of the epidemiology of IFI. Cohort studies of patients with IFI proven by histopathology documented etiologic shifts such as an increase in hyalohyphomycosis, primarily invasive aspergillosis starting in the 1980's, and an increase of mucormycosis during the 1990's (Groll et al., 1996, Chamilos et al., 2006, Kume et al., 2011). The distinction between mucormycosis and aspergillosis, that is often achieved directly by hyphal morphology from histopathology samples, is of great clinical importance. The agents of mucormycosis are resistant to antifungals such as voriconazole and the echinocandins and surgery is suggested to play an important role in patient management (Cornely et al. 2014). Given the emergence of fungal pathogens with different therapeutic implications but similar histomorphology, the identification of causative agents to the species level is needed for improving patient care, especially in mould infections refractory to standard therapies (Nucci & Perfect 2008). However, cultures identify fungi only in 30–60 % of histopathology positive samples (Tarrand et al., 2003, Rickerts et al., 2007). In addition, discrepancies between histopathology and cultivated fungi in 20 % of cases highlight the necessity for the development of alternative fungal identification strategies in order to understand the aetiology of IFI (Sangoi et al., 2009, Lee et al., 2010).
The identification of fungal pathogens from Formalin-fixed, Paraffin-embedded (FFPE) tissue from patients with proven IFI by molecular tools is an attractive way to investigate the aetiology of IFI. Different techniques, based on the amplification of fungal DNA via PCR or the hybridization of probes to fungal RNA have been successfully applied on FFPE tissues samples. In this manuscript, selected aspects of the identification of fungi from FFPE tissue by molecular techniques reported previously are reviewed.
Section snippets
Amplification of fungal DNA from tissue samples by PCR
Tissue samples provide unique opportunities to establish the aetiology of IFI by molecular methods. Fungi identified in deep tissue samples mostly represent true invasive pathogens. This is in contrast to samples such as bronchoalveolar lavage, where colonization by fungi may be a frequent finding in some patient groups, reducing the specificity of diagnostic tests (Bretagne et al., 1995, Rickerts et al., 2002). In addition, tissue samples often contain a higher fungal load as compared to blood
Studies evaluating PCR to detect fungi from FFPE tissue vary in methods used and results
Fungal DNA was amplified by PCR in 15–90 % of FFPE tissue samples from patients with proven IFI. This heterogeneity may in part be explained by the samples studied. First, the presence of fungal elements in tissue was confirmed in some studies using fungal stains (Rickerts et al., 2011, Bernhardt et al., 2014). This may select for samples with higher fungal load, especially when the performance of molecular tests is restricted to samples with a threshold of fungal elements seen by microscopy.
Localization of specific fungal RNA-sequences within fungal elements in infected tissue by in situ hybridization
Since its introduction in the late 1980's, in situ hybridization has become a widely used method for the identification and quantification of phylogenetically defined microbes (DeLong et al. 1989). The basic steps of in situ hybridization include the fixation of microbe containing samples to allow for the uptake of synthetic DNA probes labelled with fluorescent molecules or other reporter systems into microbial target cells. The subsequent identification of the microorganisms is achieved by
Combined insights: fungal identification, evidence for causation and more?
The most reliable molecular strategy to evaluate the aetiology of IFI from FFPE tissue may need to combine different detection methods. This may include the sensitive identification of the DNA of typical fungal pathogens by specific PCR assays combined with broadrange PCR to detect rare infectious causes. In addition, hybridization based tests document the fungal presence in deep tissue in association with tissue damage as the hallmark of IFIs.
An approach combining PCR and hybridization based
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