S1.2c Diagnosis of fungal infections in animals: Combining the old and the new to maximize results
Authors: Peano Andrea 
Published: September 2022

Abstract

S1.2 Emerging and Expanding Endemic Mycoses, September 21, 2022, 11:00 AM - 12:30 PM
There is a broad spectrum of fungal infections involving companion, zootechnical and wild animals. Some fungi are distributed worldwide and act as opportunistic pathogens. Others, such as the dimorphic fungi Blastomyces dermatitidis and Sporothrix brasiliensis, are primary pathogens with a more defined geographical distribution. Dermatophytes cause less severe diseases limited to the skin. However, they are relevant since they are widely diffused. Moreover, some dermatophytes are transmitted from animals to humans; therefore, these infections represent a public health problem.

In recent years, opportunistic fungal infections (e.g., Aspergillosis, Candidiosis, Cryptococcosis) in human medicine have increased. The main reason is the rise of people with immunosuppression of various origins (AIDS, chemotherapy, immunosuppressive therapies in organ transplant) (Kozel and Wickes, 2014. Cold Spring Harb Perspect Med, 4: a019299). Moreover, the spectrum of fungi causing infections is expanding, which constitutes an identification challenge for even the most experienced mycologists. To achieve an even earlier and more precise diagnosis, new methods for the detection of fungal elements in tissue samples (e.g., PCR based techniques, serological tests) and fungal identification (e.g., matrix assisted laser desorption/ionisation time-of-flight analyzer technology) are now available in adjunction to traditional methods (microscopic examination of clinical samples, histopathology, and culture). Cases of opportunistic deep mycosis are more rarely reported in animals because the situations leading to immunosuppression in human patients are not mirrored in veterinary medicine. However, there is an increasing interest in these cases involving animals. Thus, new diagnostic procedures are being applied more and more to animal infections (Elad and Segal, 2018. Front Microbiol, 9:1303).

Direct microscopy retains its importance as a quick and inexpensive tool to ‘intercept’ a fungal infection. It also allows observing the cellular population involved in the immune response and finding other pathogens. It is helpful to interpret the results of more advanced tests (culture, PCR). The sensitivity of microscopic exams varies with the individual agent, source and quality of the specimen, and the skills and experience of the laboratorian. Diagnosis of invasive fungal infection by direct microscopy and histopathology may require the use of biopsies of deep tissues, which may pose a risk for the patient. Often it does not allow fungal identification.

Fungal culture can yield the specific etiological agent if positive, which allows antifungal susceptibility testing (AST). It may take many days to achieve a result. Identification of less common fungi requires a high level of expertise and equipment.

A widely employed identification method is PCR + sequencing of the ITS region (other DNA regions used are: LSU, SSU, β-Tubulin, and Calmodulin). Data generated from an unknown fungus can be used to search public databases, such as GenBank, using the web-based BLASTn algorithm. Database searches must be performed with caution owing to the public nature of the database and the high frequency of erroneous deposits. The suggestion is to employ verified, published, recent sequences.

The most popular non-nucleic acid sequence-based molecular diagnostic assay for fungi is Matrix Assisted Laser Desorption Ionization Time of Flight (MALDI-TOF). The technique generates spectra that are screened against a library of reference spectra, which correspond to individual species. The strength of MALDI-TOF technology lies in the rapid sample analysis (minutes) and the absence of any downstream data manipulation. Weaknesses of this system include the need for an existing library to compare generated spectra to and potential variability in results of unknown fungi if they are not grown under conditions similar to reference spectra.

Thanks to the improvement of the identification methods in veterinary medicine, it has been possible to describe new cryptic species responsible for specific diseases, e.g., the species included in the Aspergillus viridinutans complex, agents of the sino-orbital Aspergillosis in cats) (Talbot and Barrs, 2017. Med Mycol, 56 [1]: 1:12). Another example is represented by the recently described dermatophyte species within the T. benhamiae-complex (Čmoková et al. 2020, Fungal Diver, 104 [1]: 333-387; Peano et al. 2022, Vet Dermatol, Online ahead of print).

PCR-based methods targeting specific fungi are now used to detect several fungal pathogens directly from clinical samples. Real-time PCR uses fluorescent dyes to enhance specificity through either a nonspecific DNA binding dye, SYBR green, or a specific fluorescently labeled probe directed to a target sequence. Since one (or more, in the case of multiplex PCR) specific pathogen is targeted, it is possible to work on ‘contaminated’ samples. These techniques are very ‘clinical-friendly’ since they are presented as ‘panels’ (e.g., PCR panel for ‘seizure episodes in cats’ to detect the main agents responsible for neurologic infections, Cryptococcus, Toxoplasma, Neospora).

The use of serological tests (e.g., the search for wall fungal components, such Beta-Glucan) may be a precious tool to diagnose and monitor the therapy response in a variety of diseases (e.g., disseminated Aspergillosis in dogs; avian Aspergillosis) (Burco et al., 2012. Avian Dis, 56 [1]: 183-191). New diagnostic tools likely will reveal animal infection cases that the traditional methods would have missed.