E. Campisi

Application of PCR to Distinguish Common Species of Dermatophytes

ABSTRACT This report describes the application of PCR fingerprinting for the identification of species and varieties of common dermatophytes and related fungi utilizing as a single primer the simple repetitive oligonucleotide (GACA)4. The primer was able to amplify all the strains, producing species-specific profiles for Microsporum canis, Microsporum gypseum, Trichophyton rubrum, Trichophyton ajelloi, andEpidermophyton floccosum. Intraspecific variability was not observed for these species. Instead, three different profiles were observed in the Trichophyton mentagrophytes group.

Detection of Candida dubliniensis in oropharyngeal samples from human immunodeficiency virus infected and non-infected patients and in a yeast culture collection

The incidence of Candida dubliniensis in oropharyngeal swabs of 132 human immunodeficiency virus (HIV)‐positive and 89 HIV‐negative patients was determined. The samples were plated onto CHROMagar Candida medium and 82 strains, presumptively identified as C. albicans or C. dubliniensis, were further investigated (temperature test, chlamydoconidia production, specific primer PCR). In addition, 487 collection strains (isolated from clinical samples and previously identified as C. albicans on the basis of a positive germ tube test) were screened in order to identify C. dubliniensis isolates. Two C. dubliniensis strains were isolated from two HIV‐positive patients without oral candidiasis. Candida dubliniensis was not isolated from 89 HIV‐negative patients nor was it identified among the collection strains.

PCR Fingerprinting for Identification of Common Species of Dermatophytes

Classical identification of dermatophytes relies on morphological and physiological characteristics. The search for these characteristics often makes their identification long and laborious. In addition, phenotypic features can frequently vary from strain to strain or the organism can become devoid of distinguishing features. In the last few years, genotypic approaches to identification have proven to be useful in solving taxonomic problems regarding dermatophytes. In fact, genotypic differences are considered more stable and more precise than phenotypic characteristics (2, 3). In one of our preceding studies (1), we demonstrated the possibility of identifying various species of common dermatophytes and related fungi by PCR fingerprinting utilizing the simple repetitive oligonucleotide (GACA)4 as a single primer (4). This primer appeared able to amplify all the strains that we tested and produced species-specific profiles for Microsporum canis, Microsporum gypseum, Trichophyton rubrum, Trichophyton ajelloi, and Epidermophyton floccosum, while intraspecific variability was not observed for these species. Three different profiles were observed in the Trichophyton mentagrophytes group (1). We felt that the capacity of this primer to produce species-specific profiles together with the simplicity of the method and the reproducibility of the results (when one strictly maintains experimental conditions) could be exploited for identifying colonies that do not present species-specific morphological characteristics and are not identifiable with the classical methods (1). In particular, PCR fingerprinting could be useful for identifying the following: (i) very young colonies, (ii) colonies which have lost the morphological characteristics typical of the species, and (iii) dead strains. To verify the first hypothesis, 12 strains from the Institut Pasteur collection (M. canis IP 2289-94 and IP 2145-93, T. mentagrophytes IP 1468-83 and IP 407-74, Trichophyton interdigitale IP 447-74 and IP 2190-93, T. rubrum IP 2360-96 and IP 2073-92, M. gypseum IP 2143-93 and IP 1463-83, and E. floccosum IP 1454-83 and IP 1559-84) were grown in Sabourauds dextrose agar (SDA) (Difco) at 25°C; after 3 days, a colony (mean diameter, 5 mm) was taken and transferred to an Eppendorf tube containing 40 μl of sterile distilled water; the mycelium was homogenized with a manual homogenizer (Micro-Grinder; International PBI) for 1 min. The Dynabeads DNA Direct System I (Dynal, Oslo, Norway) was used for the rapid DNA extraction. Briefly, 400 μl of Dynabeads was added to the homogenized mycelium, DNA was extracted as previously described (1), and the purity and quantity of the extract were determined spectrophotometrically (260 nm). Template DNA (25 ng) was amplified using the (GACA)4 primer, and PCR products were separated by electrophoresis and detected by ethidium bromide staining (1). To verify the second hypothesis, we studied eight strains which had lost their typical morphological characteristics but which had originally been identified as M. canis (two strains), T. rubrum (four strains), or T. interdigitale (two strains) and came from both the collection of the Institut Pasteur (M. canis IP 2144-93 and T. interdigitale IP 406-72 and IP 2189-93) and our departments collection (five clinical isolates: one M. canis strain and four T. rubrum strains). When cultivated on SDA, these strains quickly developed white cottony-looking colonies that had none of the morphological characteristics (macroscopic and microscopic) of their species. Some aerial mycelium was taken from these colonies, DNA was extracted, and the PCR fingerprinting was done as described above. We also studied 12 strains which upon subculture had not shown any growth. These old cultures had been grown 2 years previously and were preserved at room temperature in cork-closed SDA tubes. Except for two clinical isolates of T. interdigitale, which came from our departments collection, these nonviable strains came from the Institut Pasteur (M. canis IP 2145-93 and IP 1687-87, T. mentagrophytes IP 1468-83 and IP 401-69, T. mentagrophytes var. granulosum IP 1711-88 and IP 1182-79, T. interdigitale IP 102-77 and IP 447-74, and E. floccosum IP 1454-83 and IP 1559-84). The DNA extracted from young colonies, the DNA extracted from strains that had lost their typical morphological characteristics, and the DNA extracted from nonviable strains were compared with the DNA extracted from cultures presenting typical morphological characteristics. This last DNA had been preserved at −20°C for 30 months after use in our previous study (1). The electrophoretic profiles of all the strains were perfectly superimposable on those of their own species which presented typical morphological characteristics. In conclusion, the primer (GACA)4 has produced species-specific profiles from fungi that are not identifiable with the classical techniques, and this further confirms that PCR fingerprinting could be of great help in the mycology laboratory in solving the many problems inherent in the identification of dermatophytes. Although results with our technique proved to be extremely encouraging, we must caution that our results are restricted to a few species and strains of dermatophytes. Further investigation of a larger number of isolates could shed light upon possible interspecific relations.

Use of magnetic beads to extract fungal DNA

Authors compare two methods of extracting DNA from different fungi: the classic method with phenol/chloroform (P/C) and that with magnetic beads. Both were tested on Candida albicans and Cryptococcus neoformans var. neoformans, belonging to the yeast group and Microsporum canis, M. gypseum, Trichophyton rubrum, T. interdigitale, T. ajelloi, Epidermophyton floccosum, belonging to the dermatophytes group. Extraction products underwent polymerase chain reaction (PCR) fingerprinting with the appropriate primers to point out any disagreement in the genomic profiles. After having determined that the genomic profiles obtained from the DNA extracted from the same strain with the two methods correspond perfectly, the authors concluded that the extraction method with magnetic beads from fungal cells is simpler and quicker than with P/C extraction, greatly facilitating the obtainment of fungal DNA.

Investigation in central Italy of the possible association between Cryptococcus neoformans var. Gattii and Eucalyptus camaldulensis

The authors present a worldwide review of isolations of Cryptococcus neoformans, var. neoformans and C. neoformans var. gattii from animals and vegetation, referring in particular to the already well-known association of the former variety with Eucalyptus camaldulensis. They then review the Italian situation relative to this association and their studies carried out in Central Italy: in Latina (Lazio), Pisa, Viareggio and Lake Massaciuccoli (Tuscany). From the 256 E. camaldulensis trees examined C. neoformans var. gattii was not isolated. An E. camaldulensis tree situated in the nature reserve on Lake Massaciuccoli proved to be positive for C. neoformans var. neoformans. This variety was isolated from the leaves, flowers, bark and the debris at the foot of the tree, suggesting that it had colonized the entire tree and that it was capable of developing not only on its usual habitat (bird guano, soil rich with guano) but also on Eucalyptus trees. The identity of the isolates was confirmed by their genomic profiles obtained by random amplification polymorphic DNA (RAPD) with the primer (GACA)4. The presence of a single genotype indicates a sole source of contamination, perhaps brought by a bird coming from a contaminated environment.

Anti-Malassezia furfur antibodies in the population

Summary. The authors carried out research into anti‐Malassezia precipitating antibodies in the population, specifying the distribution by age and sex. A total of 868 serum specimens from subjects of both sexes, aged between 0 and 80 years, were studied. An immunoelectroprecipitation reaction was used using a M. furfur culture filtrate as antigen. No antibodies were found in children under 11 years, whereas they were present after that age and reached maximum frequency in subjects between 31 and 40 years of age. In subjects up to 50 years of age they were more frequent in women, but in subjects over the age of 50 years they were more frequent in men. Globally, antibodies were found in 31% (270/868) of the subjects examined. The presence of antibodies correlates with data from the literature regarding the isolation of this yeast in relation to age.

Prevalence of Toxocara canis eggs in dog faeces from public places of Florence, Italy

SummaryTo determine whether canine faecal contamination may represent a source of environmental contamination with Toxocara canis eggs within the urban area of Florence, a total number of 754 dog faeces were collected in 7 public places and examined by routine floatation technique during one-year period. The total prevalence of intestinal nematode eggs was 8.6 %. Trichuris vulpis (4.6 %) eggs were the most prevalent followed by T. canis (3.6 %) and Ancylostomidae (1.7 %) eggs. Mixed infections included T. canis/T. vulpis (0.7 %), Ancylostomidae/T. canis (0.4 %), and Ancylosto-midae/T. vulpis (0.3 %). Total prevalence of intestinal nematode eggs was significantly higher in spring than in winter (OR = 2.06). Our results indicate a low prevalence of T. canis eggs suggesting that dog faeces left on soil are unlikely to cause high environmental contamination with T. canis eggs in the town of Florence.

Enzymatic characterization of clinical and environmental Cryptococcus neoformans strains isolated in Italy

BACKGROUND Cryptococcus neoformans is an encapsulated yeast causing mainly opportunistic infections. The virulence factors involved in cryptococcosis pathogenesis include the presence and the size of the polysaccharide capsule, the production of melanin by phenoloxidase, the growth at 37°C and the enzyme secretion like proteinase, phospholipase and urease. Many other enzymes are secreted by C. neoformans but their role in the fungus virulence is not yet known. AIMS In order to investigate this topic, we compared the phospholipase production between strains from patients and from bird droppings, and we examined its relationship to phenoloxidase production. We further characterized the strains by determining the activity of 19 different extracellular enzymes. METHODS Two hundred and five Italian C. neoformans clinical isolates and 32 environmental isolates were tested. Phenoloxidase production was determined by the development of brown colonies on Staibs agar. Extracellular phospholipase activity was performed using the semiquantitative egg-yolk plate method. API ZYM commercial kit was used to observe the production and the activity of 19 different extracellular enzymes. RESULTS Statistical analysis of the results showed a significantly higher phospholipase activity in the clinical isolates than in the environmental isolates. No significant difference about the phenoloxidase production between both groups was found. Regarding the 19 extracellular enzymes tested using the API ZYM commercial kit, acid phosphatase showed the highest enzymatic activity in both groups. Concerning the enzyme α-glucosidase, the clinical isolates presented a significantly higher positivity percentage than the environmental isolates. A hundred percent positivity in the enzyme leucine arylamidase production was observed in both groups, but the clinical isolates metabolized a significantly greater amount of substrate. CONCLUSIONS The higher phospholipase production in the clinical isolates group confirms the possible role of this enzyme in the cryptococcosis pathogenesis. The extracellular activities of the enzymes acid phosphatase, α-glucosidase and leucine arylamidase, tested by means of the API ZYM commercial kit, appear to be very interesting. Many studies indicate that these enzymes are involved in the virulence of bacteria and parasites; our results suggest their possible role as virulence factors in Cryptococcus infections too.

Scanning electron microscopic pattern of recurrent vaginitis by Candida albicans in the mouse

Summary: The scanning electron microscopic pattern of the development of yeasts of Candida albicans on the vaginal mucosa of mice in oestral phase, was studied in four consecutive infections performed at 30 d intervals. The findings were practically identical following the 1st and 2nd infections, evidencing a progressive mycelial development starting from 12 to 72 h after inoculation with C. albicans yeasts. In the 3rd and 4th infections the mycelial tufts appeared extremely reduced in number and size, although there was an acceleration in the early evolutive phases and the filamentation process had already started 6 h after infection. It can be concluded that, after successive infections with C. albicans, mouse vaginal mucous membrane develops a local state of resistance evidenced by the decrease in mycelial tufts. The possible causes of the early onset of the filamentation process are discussed.