genetics and physiology of fungi
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biomed.cas.cz/mbu/lab112/
Improving determinations tools in Aspergillus


Epidemiology, diversity and taxonomy of dermatophytes
Dermatophytes are the most successful human pathogenic fungi
Dermatophytes are a group of ecologically, morphologically and phylogenetically related microscopic fungi comprising the most important causal agents of superficial fungal infections of humans and animals. Dermatophytoses belong to the world’s most common infectious diseases with prevalence between 20% and 25% (Havlickova, et al., 2008). High financial costs are associated worldwide with treatment, diagnosis, prevention and sickness leave of some patients (Drake, et al., 1996, Gupta, 1998).
For effective treatment and prevention of the spread of diseases, it is important to correctly determine the causal agents at the species level, which allows prescription of targeted therapy and, at the same time, identification of the source of infection. The spectrum of infectious agents of dermatophytoses shows large geographic differences and dynamically changes over time. Correct identification is also an indispensable prerequisite for monitoring changes in the frequency of individual species, helps to evaluate the results of preventive measures and interventions, and is a basic requirement for the preparation of epidemiological studies. In addition, instable taxonomy of dermatophytes, problematic species concept and phenotypic identification limit comparability of recent epidemiological data with studies from the past.
In the past, a large number of molecular genetic methods were published capable of identifying dermatophytes to a greater or lesser degree of accuracy. ITS rDNA sequencing has high discriminatory power for differentiation of dermatophyte species and is considered as the gold standard for molecular genetic identification. In addition, the sequences are available for all accepted species and are accessible in a curated database (http://www.cbs.knaw.nl/dermatophytes/). Some PCR fingerprinting methods are also a suitable and cheaper alternative to sequencing, and are also able to reliably identify dermatophytes. PCR fingerprinting with the primer M13-core has been used by our group to determine the majority of the isolates in a pilot study on epidemiology of dermatophytes in the Czech Republic. The method produced species-specific patterns for all 15 identified species (verified also by ITS rDNA sequencing). Reliability of the method was demonstrated by the detection of two hitherto undescribed species during this study, that were initially identified on the basis of unique PCR fingerprinting.
AIM 1: The aim of this epidemiological surveillance study of dermatophytosis in the Czech Republic is to determine all major etiological agents of dermatophytosis and their incidence in the Czech population. Isolates will be identified by molecular methods to uncover cryptic and new species, and also to reliably identify atypical isolates that are commonly found in dermatophytes. The long-term and large-scale observation will allow us to identify major risk factors associated with infection caused by particular species, seasonal and regional variability in the species spectrum. These data will be used to improve the diagnosis, prevention and empiric as well as agent-specific treatment.
AIM 2: A microsatellite typing scheme will be developed for the newly emerging pathogen A. benhamiae that causes epidemic zoonotic infections in Central Europe and Japan. Such a scheme should be a tool for epidemiological monitoring of this newly spreading infection, but may also find application in occupational medicine (infection in pet shop workers or laboratory technicians working with rodents and clinical samples).
AIM 3: The population structure of A. benhamiae will be assessed to answer the following questions: Are there any differences in genotype composition between isolates of human and animal origin? Are particular genotypes specifically associated with a geographical origin? Are any animal genotypes increasingly transmitted to humans (increased virulence) and were these genotypes responsible for infections in humans and animals in the past (before the beginning of the outbreak)?
AIM 5: Medical history data collected in this project and treatment outcome data will be used for improvement of empiric and agent-specific therapy. Guidelines reflecting the current as well as long-term epidemiological situation will be published each year and also posted on a free website with approximately a one-year delay. Gender, age and several supplementary medical history questions are sufficient for the estimation of the best empiric therapy.
AIM 6: Antifungal susceptibility of dermatophyte strains (identified by molecular methods) to common topical and oral antifungals will be assessed using the broth microdilution method (CLSI methodology). Emphasis will be placed on the species complexes where the correct species identification is difficult without molecular methods and on rare species.
AIM 7: A new manual will be developed for identification of medically important dermatophytes that occur in the European region (approximately 20-25 species). The species represented by isolates identified by molecular methods (obtained from this project and foreign culture collections) will be characterized by morphology (growth on standard media and micromorphology), growth in temperature gradient (Fig. 3), standard physiological and biochemical tests (T1-T7 agar media, urease test, hair perforation test, etc.).
AIM 8: New species will be described using a polyphasic approach involving DNA sequence data from several loci (ITS rDNA, β-tubulin, actin and RNA polymerase II gene), micro- and macromorphology, physiology and mating experiments. Susceptibility to common antifungal agents will be tested.