Gabriella Pini

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.

Isolation of Trichosporon in a hematology ward

During mycologic monitoring of the air in a hematology ward, we found massive air contamination caused by Trichosporon asahii, both in the room where neutropenic patients were staying and the corridor immediately outside the room. This fungal species had never been isolated in previous samplings. The urine culture taken from one of the patients in this room, whose urinary catheter had been removed immediately prior to air sampling, resulted positive for T. asahii. Both macroscopic and microscopic morphologic observation was insufficient for confirming the hypothesis of a close relationship between the strains isolated from the patient, from the air in the room and corridor. Therefore, we used genomic typing with random amplified polymorphic DNA (RAPD). The five primers used, (GTG)5, (GACA)4, M13, OPE01, RC08, produced different patterns of polymerase chain reaction (PCR) products; the genomic profiles obtained with the same primer, however, resulted perfectly superimposable for all the strains. This result led us to conclude that the massive air contamination caused by T. asahii can have effectively been determined by the removal of the urinary catheter from the patient who presented an asymptomatic infection caused by this microorganism.

Invasive pulmonary aspergillosis in neutropenic patients and the influence of hospital renovation

To evaluate the effects of airborne Aspergillus contamination during and after the renovation work of a Florentine haematology unit, we conducted (November 2003–January 2005) a strict programme of environmental fungal surveillance. Air samples were taken from patients’ rooms, along the corridors inside the wards, along the corridor between wards and outside the building. The concentration of Aspergillus fumigatus was high along the corridor between the two haematology wards (2.98 CFU m−3), lower in the non‐neutropenic patients’ rooms and outside the hospital building (1.53 and 1.42 CFU m−3, respectively), very low in the neutropenic patients’ rooms (0.09 CFU m−3). During this period, three proven cases (A. fumigatus), two probable ones and two possible cases of invasive pulmonary aspergillosis were documented in 97 patients with acute leukaemia (7%). The three cases of proven aspergillosis coincided with the period of renovation work and with the period in which we have found the maximum concentration of A. fumigatus along the corridor. These data suggest a possible relationship between environmental fungal contamination and the incidence of invasive aspergillosis, and underline the importance of environmental surveillance.

Two years of a fungal aerobiocontamination survey in a Florentine haematology ward

The control of microbial air contamination in hospital wards has assumed great importance particularly for those hospital infections where an airborne infection route is hypothesised, such as aspergillosis. Invasive aspergillosis represents one of the most serious complications in immunocompromised patients. For some authors there is a direct association between this pathology and the concentrations of Aspergillus conidia in the air; in addition, reports of aspergillosis concurring during building construction have been frequent. In this study, two haematology wards were monitored for about 2 years in order to make both a qualitative and quantitative evaluation of fungal burden in the air, also in relation to major construction and demolition work taking place in the same building. Air samples were taken from the hospital rooms of neutropenic patients, in the corridors of their ward and outside the building. Total fungal concentration resulted higher outside (mean 572 Colony Forming Units/m3 of air), lower in the corridors (147CFU/m3) and even lower in the rooms (50 CFU/m3). In all the samples we found the development of at least one fungal colony. Cladosporium was the most frequently isolated genus (57%), in contrast to Aspergillus spp. (2%). The average concentration of Cladosporium spp. was 24 CFU/m3 in the rooms, 78 CFU/m3 in the corridors and 318 CFU/m3 outside. The average concentration of Aspergillus spp. was 1.2 CFU/m3 in the rooms, 3.5 CFU/m3 in the corridors, 5.6 CFU/m3 outside. Our observations show low concentrations of Aspergillus fumigatus and A. flavus in all the environments examined and particularly in the rooms (0.09 and 0.10 CFU/m3 respectively); this observation could explain the absence of cases of invasive aspergillosis during the period of air monitoring in the two haematology wards.

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.

Extracellular phospholipase activity of "Malassezia" strains isolated from individuals with and without dermatological disease

BACKGROUND The Malassezia genus includes mainly lipophilic yeasts belonging to the cutaneous microbiota of man and other mammals. Some Malassezia species have been associated with various dermatological diseases. The factors permitting the transformation of yeasts of the Malassezia genus from a commensal organism to a pathogenic agent are still little known but the production of various enzymes such as lipase, phospholipase and lipoxygenase could contribute to the pathogenic activity of these yeasts. AIMS Here we have determined and compared the extracellular phospholipase activity of sixty human isolates of Malassezia so as to relate this feature to the species of Malassezia and to the origin (from dermatological diseases or not) of the strains examined. METHODS Phospholipase production was determined using the semi-quantitative egg-yolk plate method. RESULTS AND CONCLUSIONS Malassezia obtusa, Malassezia slooffiae, Malassezia globosa, Malassezia restricta had difficulty developing in the chosen culture medium so that it was not possible to measure phospholipasic activity. Malassezia pachydermatis showed the highest phospholipase activity. Twenty-nine Malassezia sympodialis strains produced phospholipase; the isolates from patients with pityriasis versicolor had significantly higher phospholipasic activity than those isolated from healthy individuals. This observation suggests that the phospholipasic activity of Malassezia may play a role in the onset of skin lesions, especially in the case of pityriasis versicolor.

Comparative evaluation of Sensititre® YeastOne vs. the NCCLS M27A protocol and E-test for antifungal susceptibility testing of yeasts

A recently developed microdilution method (Sensititre® YeastOne) may represent a valid alternative to the National Committee for Clinical Laboratory Standards (NCCLS) method for routine testing. The Medical Mycology Committee of the Associazione Microbiologi Clinici Italiani (AMCLI) decided to evaluate its reproducibility and reliability compared with the NCCLS M27A protocol and the E‐test. Nineteen strains each of Candida albicans and Ca. parapsilosis, isolated from systemic infections, were tested against amphotericin B, flucytosine, ketoconazole, itraconazole, and fluconazole. All the participating laboratories tested the YeastOne panels, while the E‐test and the NCCLS method were performed by two laboratories each. Interlaboratory reproducibility showed a good correlation (from 95% for amphotericin B to 92.5% for flucytosine). The agreement between NCCLS and YeastOne ranged from 95 (ketoconazole and itraconazole) to 100% (amphotericin B and flucytosine), whereas the agreement between E‐test and YeastOne ranged from 72.5 (fluconazole) to 100% (amphotericin B and flucytosine). The Sensititre® YeastOne panels appear to be an excellent alternative to both the E‐test and the NCCLS protocol for antifungal susceptibility testing.