Proteolytic activity of clinical Candida albicans isolates in relation to genotype and strain source.

Proteolytic activity is regarded as one of the most important virulence factors of Candida albicans. Several authors recently demonstrated that some karyotypes and genotypes harbouring a group I self-splicing intron (CaLSU) located in the gene encoding the large rRNA subunit showed a high level of proteinase production. The aim of this study was to investigate the correlation between the level of proteinase production and the presence of the CaLSU intron in C. albicans isolates originating from the blood and respiratory tracts (sputum/pharyngeal swabs) of patients with and without oropharyngeal candidosis. The results revealed statistically significant differences in genotype distribution and the level of proteinase production between the C. albicans isolates obtained from blood and from the respiratory tract. Genotype A, without the intron, was prevalent in all groups of strains and its prevalence was higher among isolates from blood (75%) and from patients with candidosis (80%) compared with strains from colonisation (as opposed to infection) (57.8%). Isolates from blood produced significantly less proteinase than isolates from the respiratory tract (p<0.02), and this difference should be attributed to lower proteinase production of genotypes B and C from blood compared with genotypes B and C from the respiratory tract (p<0.01). The higher proteinase production of genotype B than of genotype A was found among respiratory tract isolates only. The presented data indicate that the association between proteinase production and the CaLSU intron depends on the strains' population. Further study is needed on well-defined groups of clinical isolates to elucidate whether the observed diversity in proteinase production plays a role in the selection of strains inducing bloodstream infections.

Fungal cultures of different parts of the upper and lower airways in chronic rhinosinusitis.

The relation between fungi, upper and lower airways in chronic rhinosinusitis (CRS) patients are not clear yet. So the aim of this study was to identify the different cultured fungi in various sub-sites of the nasal cavity and lower airways in adult (CRS) patients and to correlate the cultured fungi to the associated cellular inflammatory changes. In the outpatient clinic a control group of 10 normal subjects was subjected to total nasal lavages to validate our mycological culture technique. Twenty-five adult CRS patients were enrolled in this prospective study. Under general anaesthesia before functional endoscopic sinus surgery (FESS) operation 50 nasal vestibular swabs, 25 bronchoalveolar lavages (BALs), 50 middle meatal lavages (MMLs) and 50 nasal cavity lavages (NCLs) were obtained in the operating room. These samples were processed for fungal culture and eosinophilic cellular counts. The intraoperative pathological specimens were examined using Haematoxylin and Eosin (H&E) and Gomori methanamine silver (GMS) staining. In the normal control group total nasal lavages showed 100% positive fungal cultures. In the CRS patient group the BALs showed positive fungal cultures in 28%. Nasal vestibule cultures were positive in 8%. Positive middle meatal cultures were obtained in 44% of the 25 CRS patients. Two cases (8%) with maxillary fungal ball showed a positive maxillary sinus culture but a negative middle meatal culture. Nasal cavity lavages were positive in 36%. Middle meatal eosinophilia was identified in 33.6% of the positive middle meatal fungal culture. Following the deShazo's criteria of diagnosis of allergic fungal rhinosinusits (AFRS), only 16% of the subjects in this study fulfilled the criteria. No correlation existed between fungal culture, cellular and other clinical parameters. Also no correlation existed between upper and lower airway positive cultures. In conclusion fungi seemed to be present in different percentages and types in different sub sites of the airways but without associated eosinophilia. There were no significant correlations between the fungal culture and clinical parameters of CRS nor were there significant correlations between fungal culture and objective lower airway involvement.

Zygomycosis in the immunocompromised patient: a case report.

Zygomycosis is a highly aggressive infection observed in immunocompromised patients, such as those with haematological malignancies. The sites most frequently involved are the sinuses and the lungs. New diagnostic tools and new antifungal treatments are essential in order to diagnose early and treat efficiently infections due to moulds. We report a case of sinusitis due to Absidia corymbifera occurring during chemotherapy-induced bone marrow aplasia in a patient with acute leukaemia. The sinusitis was successfully treated with AmBisome, and surgical debridement.

In vitro activities of voriconazole, fluconazole, itraconazole and amphotericin B against non Candida albicans yeast isolates.

Antifungal susceptibility testing was performed on 197 yeast isolates from the BCCM/IHEM biomedical fungi and yeasts collection (Belgian Co-ordinated Collections of Micro-organisms / IPH-Mycology) to study the in vitro activity of voriconazole against fluconazole, itraconazole and amphotericin B. MICs of the four antifungal agents were determined by an adapted NCCLS M27-A microdilution reference method. MIC readings were visually and spectrophotometrically determined. Optical density data were used for calculation of the MIC endpoints. For amphotericin B, the MIC endpoint was defined as the minimal antifungal concentration that exerts 90% inhibition, compared to the control growth. The azoles endpoints were determined at 50% inhibition of growth. The MIC distribution of voriconazole susceptibilities showed that 193 isolates had a MIC < or = 2 microg/ml and 185 a MIC < or = 1 microg/ml. Cross-tabulation of voriconazole, fluconazole, and itraconazole MICs indicated that voriconazole MICs raised with fluconazole and itraconazole MICs. The in vitro data obtained in this study suggest that voriconazole may also be effective treating yeast infection in patients infected with fluconazole or itraconazole resistant isolates.

Biodegradation of phenol and phenol-related compounds by psychrophilic and cold-tolerant alpine yeasts.

We characterized 32 cold-adapted, psychrophilic and cold-tolerant, yeast strains isolated from alpine habitats with regard to their taxonomy, growth temperature profile, and ability to degrade phenol and 18 phenol-related mono-aromatic compounds at 10 degrees C. Twenty of the strains were identified by sequencing of the ribosomal ITS region as seven species of the basidiomycota: Cryptococcus terreus (three strains), Cryptococcus terricola (one strain), Rhodosporidium lusitaniae (two strains), Rhodotorula creatinivora (10 strains), Rhodotorula ingeniosa (one strain), Mastigobasidium intermedium (one strain), and Sporobolomyces roseus (two strains). Twelve strains sharing closely related ITS sequences could not be identified to the species level; according to their ITS sequence they are included in the Microbotryomycetidae. These 12 strains were psychrophilic (no growth at temperatures above 20 degrees C); one-third of these strains did not grow above 15 degrees C. None of the 32 strains utilized any of the highly volatile mono-aromatic compounds (benzene, toluene, ethylbenzene, nitrobenzene, o-xylene, m-xylene, and p-xylene) as the sole carbon source. Non/low volatile aromatic compounds were degraded in the following order: phenol>hydroquinone>resorcinol>benzoate>catechol>salicylate>>p-cresol>m-cresol. o-Cresol, guaiacol, p-nitrophenol, or p-nitrotoluene were not utilized for growth. R. creatinivora strains degraded up to seven compounds, whereas C. terricola and S. roseus strains degraded only two compounds. The toxicity of the compounds was determined via growth inhibition in the presence of toxicants and nutrients at 10 degrees C. R. creatinivora strains were characterized by higher IC50 values than other species, S. roseus was the most sensitive species. The most toxic compounds were the xylene isomers, ethylbenzene, p-nitrophenol, and m-cresol. There was a relation between the chemical structure of the compounds and their toxicity, whereas a relation between the toxicity of the compounds and the ability of the yeasts strains to utilize these compounds for growth was only detected in some cases.

Molecular epidemiology of airway colonisation by Aspergillus fumigatus in cystic fibrosis patients.

A total of 109 sequential and multiple Aspergillus fumigatus isolates corresponding to 41 samples from seven cystic fibrosis (CF) patients was typed by random amplification of polymorphic DNA (RAPD) with the primer NS3 from the fungal ribosomal gene 18S subunit, and by sequence-specific DNA primer (SSDP) analysis. RAPD typing of the isolates revealed 10 different genotypes, whereas nine genotypes were identified by SSDP. Combination of the two typing methods permitted the differentiation of 25 overall genotypes. The colonisation typing patterns differed greatly between patients colonised for <1 year by A. fumigatus and long-term colonised patients. Two of three recently colonised patients presented a large number of types even in the same sample, unlike the chronically colonised patients, who harboured a limited number of genotypes. In the latter, the occurrence of a dominant genotype, usually the overall genotype 2, tended to reflect to the duration of colonisation. Moreover, anti-catalase antibodies to A. fumigatus appeared in most cases to be in response to genotype 2. These findings suggest that some strains of A. fumigatus may be selected during prolonged colonisation of the airways in CF patients.

Susceptibility testing of sequential isolates of Aspergillus fumigatus recovered from treated patients.

Two-hundred sequential Aspergillus fumigatus isolates recovered from 26 immunocompromised patients with invasive aspergillosis or bronchial colonization were tested for their in vitro susceptibility to posaconazole, itraconazole, voriconazole, terbinafine and amphotericin B. Twenty-one patients were treated with amphotericin B and/or itraconazole. Antifungal susceptibilities of the isolates recovered before treatment were not significantly different from those of isolates recovered after the onset of antifungal therapy. The highest MICs were 0.125, 0.5, 0.5, 1 and 1 microg ml(-1) for posaconazole, itraconazole, voriconazole, terbinafine and amphotericin B, respectively. It is concluded that the emergence of resistance in A. fumigatus during antifungal therapy with amphotericin B or itraconazole is an uncommon phenomenon.

Hypersensitivity pneumonitis due to molds in a saxophone player.

This 48-year-old patient was evaluated for an interstitial pneumonia. An open-lung biopsy showed a pattern of nonspecific interstitial pneumonia. The CT scan appearance, showing mosaic ground-glass opacities in the ventilated parts of the lung, the centrolobular predominance of inflammation on the lung sections, and the presence of a lymphocytic alveolitis at BAL suggested a hypersensitivity pneumonitis. The patient was a white-collar worker and had no contact with pets, birds, drugs, or molds at home. He used to play the saxophone as a hobby. Two molds, Ulocladium botrytis and Phoma sp, were detected in the saxophone. Precipitating antibodies to these molds were present in his serum. An additional study confirmed the frequent colonization of saxophones with potentially pathogenic molds, such as Fusarium sp, Penicillium sp, and Cladosporium sp. Respiratory physicians should be aware of the risk of hypersensitivity pneumonitis in saxophone or perhaps other wind instrument players.

Disseminated Scedosporium apiospermum infection in a cystic fibrosis patient after double-lung transplantation.

Scedosporium apiospermum is a saprophytic ubiquitous filamentous fungus. It can cause a wide spectrum of diseases, from localized to invasive infections. S apiospermum has been described as one of the major fungal agents of chronic colonization of airways in cystic fibrosis (CF) patients. Invasive infections due to S apiospermum are only rarely reported in CF after lung transplantation. A 26-year-old woman with CF and chronic bronchial colonization by S apiospermum developed bilateral chorioretinitis and subcutaneous nodules 4 weeks after double-lung transplantation (LTx). Isolates of S apiospermum from sputum samples before and after LTx and from vitreal fluid were typed by random amplification of polymorphic DNA (RAPD). The patient was treated with voriconazole (VRC). The patient improved with VRC given orally for 6 months. Two days after VRC discontinuation, she developed sub-acute meningitis (isolation of S apiospermum from the cerebrospinal fluid). She was again given VRC, but died 23 days later from uncontrolled fungal infection. Molecular typing of clinical isolates of S apiospermum performed by RAPD demonstrated that all isolates belonged to the same genotype. S apiospermum is a frequent, but late colonizing fungal agent in CF patients. In the case of LTx, these patients can develop invasive infection due to the colonizing strain, as confirmed by molecular typing.

Manganese superoxide dismutase based phylogeny of pathogenic fungi.

Superoxide dismutases (SODs), which provide protection against oxidative stress, exhibit an essential role for fungal cell survival, especially during host invasion. Here, 20 partial SOD sequences from 19 pathogenic fungi were determined and aligned with 43 homologous fungal sequences from databases. All sequences encoded tetrameric manganese (Mn)-containing SODs showing predicted cytosolic or mitochondrial subcellular localization. Numerous fungi possessed both cytosolic and mitochondrial MnSODs in addition to the mainly cytosolic copper/zinc isozyme. Moreover, MnSOD sequence variability was higher than SSU rRNA and similar to ITS rRNA, suggesting MnSOD could be used to identify closely related fungal species. MnSOD- and SSU rRNA-based phylogenetic trees were constructed and compared. Despite a more complex topology of the MnSOD tree, due to several gene duplication events, all the classic fungal classes and orders were recovered. A salient point was the existence of two paralogous cytosolic and mitochondrial MnSODs in some Ascomycota. A hypothetical evolutionary scenario with an early gene duplication of the "mitochondrial" gene is proposed.

Epidemiology of human sporotrichosis investigated by amplified fragment length polymorphism.

Amplified fragment length polymorphism (AFLP) was used to analyze the genetic diversity of Peruvian strains of Sporothrix schenckii and to compare them to a panel of non-Peruvian strains. AFLP analysis suggests that the Peruvian strains can be divided into two homogeneous clusters with no reference to geographical origin or the clinical form of sporotrichosis. The strains from abroad present heterogeneous profiles, with the Bolivian strain and the Colombian strains related to one of the Peruvian population. Sequencing of internal transcribed spacer 2, used to examine the relationships over a longer distance, confirmed the division of Peruvian strains into two populations that can be identified on the basis of a single but specific sequence divergence. This paper introduces automated AFLP analysis as a valuable tool for further investigation of the epidemiology and ecology of S. schenckii.

In vitro interaction of micafungin with conventional and new antifungals against clinical isolates of Trichosporon, Sporobolomyces and Rhodotorula.

OBJECTIVES: The infections caused by basidiomycetous yeasts are often difficult to resolve. Combined therapy might be useful in those severe cases where a monotherapy was ineffective. The aim of this study was to evaluate the in vitro activity of combinations of micafungin with amphotericin B or fluconazole, itraconazole, voriconazole and ravuconazole against isolates of Trichosporon, Rhodotorula and Sporobolomyces. METHODS: Twenty-seven clinical isolates were tested, i.e. 10 of Trichosporon asahii, two of Trichosporon mucoides, five of Sporobolomyces salmonicolor and 10 of Rhodotorula glutinis. Drug interactions were assessed by the chequerboard technique using the NCCLS microdilution method (M27-A2). The fractional inhibitory concentration index (FICI) was used to classify drug interactions. Results were interpreted as follows: synergy (FICI < or =0.5), no interaction (FICI >0.5 and < or =4.0), or antagonism (FICI >4.0). RESULTS: Micafungin combined with amphotericin B showed the highest percentage of synergic interactions (78%) followed by micafungin/ravuconazole and micafungin/itraconazole (48% for each), and micafungin/fluconazole and micafungin/voriconazole (34% for each). Antagonism was not observed in any case. CONCLUSIONS: Some of the combinations tested, especially micafungin/amphotericin B, have potential for the treatment of basidiomycetous yeast infections.

In vitro interactions of micafungin with other antifungal drugs against clinical isolates of four species of Cryptococcus.

The combination of micafungin (MFG) with amphotericin B (AMB), fluconazole, itraconazole, voriconazole, or ravuconazole was evaluated against 37 strains of four species of Cryptococcus by the checkerboard method. Antagonism was never seen. Synergy was observed for some isolates for each combination and was most frequent with MFG-AMB.

In vitro antifungal susceptibilities of uncommon basidiomycetous yeasts.

The in vitro activities of eight antifungal drugs against 50 isolates of basidiomycetous yeasts were determined by a microdilution method. In general fluconazole and micafungin were inactive. Terbinafine was active only against Sporobolomyces salmonicolor. The activities of the other antifungals were variable and depended on the species tested. The new triazoles showed the lowest MICs, but amphotericin B and itraconazole were the only drugs active against Cryptococcus albidus.