Mario Pasquetti

Evaluation of the antifungal susceptibility of Malassezia pachydermatis to clotrimazole, miconazole and thiabendazole using a modified CLSI M27-A3 microdilution method.

BACKGROUND In this study, we evaluated the antifungal susceptibility of Malassezia pachydermatis to clotrimazole (CTZ), miconazole (MCZ), and thiabendazole (TBD), azole derivatives employed in aural formulations labeled for treatment of canine otitis. METHODS The procedure for in vitro testing was based on the indications of the Clinical and Laboratory Standards Institute (CLSI) M27-A3 microdilution method. A lipid-enriched medium was employed to enhance the yeast growth (Christensens urea broth, with 0.1% Tween 80 and 0.5% Tween 40 as the lipid sources), while the inoculums size corresponded to approximately 1-5 × 10(5) yeast cells/mL. Microplates were incubated at 37°C and read 48 h after inoculation. Azole MICs inhibiting fungal growth were the lowest drug concentrations that showed an optical density of ≤ 50% of the (drug-free) growth control, as assessed by spectrophotometer (630 nm filter). RESULTS All isolates were inhibited by the three azoles, with different minimum inhibitory concentration (MIC) values. Most isolates were inhibited by drug concentrations of 2-8 (CTZ), 1-4 (MCZ), or 16-32 (TBD) μg/mL. These results are partially in agreement with the findings of previous studies, in which substantially higher/lower MICs were occasionally reported. This is likely because of the different methodologies employed. Such discrepancies may not apply to clinical situations, where the compounds are applied topically. CONCLUSION AND CLINICAL IMPORTANCE The concept that clinical failure is linked to increased MICs is debatable, because significantly higher concentrations (in most cases at least 1,000 × the MIC) of the antifungals that were included in our study are routinely used in formulated products.

Development and validation of a microsatellite marker-based method for tracing infections by Microsporum canis

BACKGROUND Microsporum canis is a dermatophyte fungus harbored by cats and dogs and is frequently transmitted to humans. Molecular tools able to discriminate fungal isolates at the strain level would prove extremely useful for confirming the route of infection, thus contributing to optimization of prophylaxis and hygienic regimens. OBJECTIVE To develop and validate a microsatellite marker-based method for use in tracking infections by M. canis. METHODS Primers were designed against sequences flanking the microsatellites individuated by a BLAST search using the nucleotide sequence information assembled by the M. canis CBS 113480 genome project. The PCR conditions were standardized and fragment analysis was performed using a genetic analyzer. The resolving power of the markers was investigated on 26 unrelated M. canis strains while the reproducibility of the technique and the stability of the markers were evaluated on a single strain subcultured in time as well as on 36 strains isolated from nine outbreak episodes. RESULTS Eight markers were recognized as being the most polymorphic within the set of M. canis strains isolated from unrelated distant hosts, with a total of 22 multilocus genotypes, which corresponded to a genotypic diversity of 97%. Repeated tests on subcultures of M. canis reference strain CBS 113480 always yielded the same results. Identical multilocus genotypes were obtained for all the isolates from each outbreak episode. CONCLUSION The high resolving power and reproducibility of the markers that were identified support the potential of these tools to detect sources and routes of infection by M. canis.

Use of western blot to study Microsporum canis antigenic proteins in canine dermatophytosis

Western blotting was used to describe the Microsporum canis proteins with antigenic activity in dogs with dermatophytosis. Electrophoretic separation of whole fungal strain extract cultured from a cat was performed under denaturing conditions. The proteins were blotted onto nitrocellulose and probed with sera collected from 22 dogs with dermatophytosis (18 M. canis, 3 M. gypseum, 1 Trichophyton mentagrophytes; group A), 20 dogs with skin diseases other than dermatophytosis, and 22 dogs with no clinical cutaneous signs (group B, n = 42). Nine principal IgG‐binding proteins with apparent molecular weights of 180, 144, 130, 120, 102, 96, 80, 68, and 48 kD were visualised on group A blots. For these proteins, serological cross‐reactivity with different strains of M. canis may be indirectly confirmed, whereas additional proteins were found to react with sera from individual dogs. The proteins visualised in this study may represent diagnostic markers of dermatophyte infection. The proteins should be further evaluated for their role in the cellular immune response of dogs with dermatophytosis.

Infection by Microsporum canis in Paediatric Patients: A Veterinary Perspective

Microsporum canis is a dermatophyte fungus of which cats and dogs are recognized as the natural hosts. M. canis is also easily transmitted to humans, causing lesions to the glabrous skin (tinea corporis) and to the head (tinea capitis). The present study describes some cases of infection with M. canis in children from a veterinary perspective, highlighting some important features of this clinical entity (e.g., the necessity to identify the animal source of infection with appropriate diagnostic tests; the fact that infected cats may present with no or atypical dermatological signs; and the importance of the environment as a fungal reserve).

Trichophyton verrucosum infection in livestock in the Chitral district of Pakistan

INTRODUCTION Trichophyton verrucosum belongs to the dermatophyte fungi, closely related organisms that cause skin infections in animals and humans. T. verrucosum infection has been reported in livestock and people in different countries from all continents. Human cases have been reported in different areas of Pakistan, but there is little information about the animal source of the fungus. METHODOLOGY Dermatological specimens collected in the Chitral district of Pakistan for a study on mange in livestock were retrospectively analyzed for the presence of T. verrucosum. In total, 5,873 animals (1,087 cows, 2,033 goats, and 2,753 sheep) were screened for evidence of dermatological lesions during two surveys performed in the summer and winter seasons. Skin scrapings collected from animals with lesions were analyzed by direct microscopic examination after digestion in sodium hydroxide and a real-time polymerase chain reaction (PCR) targeting pathogenic Trichophyton species. RESULTS At microscopy, samples from 18 cows (1.6%), 3 sheep (0.1%), and 4 goats (0.2%) were positive for fungal elements consistent with T. verrucosum. PCR confirmed the microscopy results. The prevalence was lower than that reported in other countries in intensive breeding farms. Results agree with the literature regarding factors affecting T. verrucosum diffusion, i.e., infection was more prevalent in cattle, especially in younger animals during the winter season. CONCLUSIONS This study reports, for the first time, the presence of T. verrucosum in livestock in Pakistan. A better knowledge of the animal role in the spread of this fungus may allow the adoption of more efficient control measures and prophylaxis.

Evaluation of In Vitro Synergistic Interaction of Miconazole and Polymyxin B Against Clinical Strains of Malassezia pachydermatis

Malassezia pachydermatis is a yeast that is frequently involved as a secondary/perpetuating factor in canine otitis externa, along with numerous species of bacteria. As a result, otitis is generally treated with topical therapies using combinations of antifungal, antimicrobial and anti-inflammatory agents. The evaluation of the combined effect of drugs included in commercially available products is therefore important. In vitro synergy of polymyxin B and miconazole has been demonstrated against one strain type of M. pachydermatis. This study was aimed at investigating the in vitro interactions of these two agents against five clinical strains of this yeast. A chequerboard broth microdilution method was employed. We observed a synergistic action against all strains tested (fractional inhibitory concentration index comprised between 0.2 and 0.4). Our results indicate a strong therapeutic value for the association of these antimicrobial agents in the treatment of canine otitis externa caused by M. pachydermatis.

Methodological Issues in Antifungal Susceptibility Testing of Malassezia pachydermatis

Reference methods for antifungal susceptibility testing of yeasts have been developed by the Clinical and Laboratory Standards Institute (CLSI) and the European Committee on Antibiotic Susceptibility Testing (EUCAST). These methods are intended to test the main pathogenic yeasts that cause invasive infections, namely Candida spp. and Cryptococcus neoformans, while testing other yeast species introduces several additional problems in standardization not addressed by these reference procedures. As a consequence, a number of procedures have been employed in the literature to test the antifungal susceptibility of Malassezia pachydermatis. This has resulted in conflicting results. The aim of the present study is to review the procedures and the technical parameters (growth media, inoculum preparation, temperature and length of incubation, method of reading) employed for susceptibility testing of M. pachydermatis, and when possible, to propose recommendations for or against their use. Such information may be useful for the future development of a reference assay.

Scrotal granulomatous aspergillosis in a dromedary camel (Camelus dromedarius)

BackgroundThis report describes a case of primary subcutaneous aspergillosis in a 7-year-old neutered male dromedary camel (Camelus dromedarius).Case presentationThe animal developed a large nodular lesion in the right scrotum two years after surgical intervention for neutering. The mass had a firm consistency and was painful at palpation. Histopathology revealed dermal granulomatous inflammation with a necrotic centre, surrounded by plasma cells, macrophages, neutrophils, and sparse fungal hyphae characterised by parallel cell walls, distinct septa, and dichotomous branching. Fungal culture was not performed, but a panel of mono- and polyclonal antibodies specific for different fungal genera identified the hyphae as Aspergillus sp.ConclusionsThe occurrence of subcutaneous lesions is a rare manifestation of aspergillosis in animals, and this appears to be the first case reported in the dromedary camel.