Real-time PCR and DNA sequencing for detection and identification of Trichophyton rubrum as a cause of culture negative chronic granulomatous dermatophytosis.

A 31-year-old patient presented with a diagnosis of granulomatous dermatophytosis based on the clinical aspect of the lesions and the rare presence of hyphae on direct microscopic examination of clinical material. A chronic evolution and progression of the disease, its resistance to a wide range of antifungal agents, the occasional presence of hyphae on direct examination but consistently negative cultures over a 5-year period prompted the use of amplification-based DNA analyses of several successive swab samples or skin biopsies. DNA was extracted using a combination of two semi-automated DNA isolation methods (FastPrep preparation and NucliSENS lysis magnetic extraction method). Identification relied both on sequence analysis of amplicons after SYBR Green real-time PCR of the panfungal internal transcribed spacer 1 (ITS1) genetic target, as well as the unique amplicon melting curve profile of positive samples. Accordingly, Trichophyton rubrum was unambiguously identified in several clinical samples collected over a 7-month period. This case illustrates the contribution of DNA-based assays applied directly to sample biopsies for identifying causative agents in cases in which fungal pathogens are highly suspected but culture are repeatedly negative. It also pinpoints the benefit of combining semi-automated DNA preparation methods, analysis of ITS1 amplicon melting curve profiles and sequence analysis on repeated skin biopsy samples for unambiguous identification of the causative fungal species.

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.

Rhodotorula psychrophila sp. nov., Rhodotorula psychrophenolica sp. nov. and Rhodotorula glacialis sp. nov., novel psychrophilic basidiomycetous yeast species isolated from alpine environments.

Three novel psychrophilic species of the genus Rhodotorula are described. Rhodotorula psychrophila sp. nov. (type strain PB19(T)=CBS 10440(T)=DSM 18768(T)), Rhodotorula psychrophenolica sp. nov. (type strain AG21(T)=CBS 10438(T)=DSM 18767(T)) and Rhodotorula glacialis sp. nov. (type strain A19(T)=CBS 10436(T)=DSM 18766(T)) were isolated from soil collected from an alpine railway area, from mud in the thawing zone of a glacier foot and from glacier cryoconite, respectively. All three species have been assigned to the genus Rhodotorula on the basis of molecular sequence data and physiological and morphological properties. Rhodotorula psychrophila is not able to grow at temperatures above 15 degrees C. Rhodotorula psychrophenolica and Rhodotorula glacialis degrade high concentrations of phenol (up to 12.5 and 5 mM, respectively) as the sole carbon source at 10 degrees C. Sequence analyses of the 26S rDNA D1/D2 regions indicated that the novel species are phylogenetically related and belong to a clade that includes other psychrophilic yeasts.

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.

Quantitative TaqMan PCR for detection of Pneumocystis jiroveci.

We developed a quantitative real-time PCR assay for detection and quantification of Pneumocystis jiroveci in bronchoalveolar lavage (BAL) specimens based on primers and probe targeting the gene encoding beta-tubulin. The assay was able to detect 50 DNA copies per ml of a standard plasmid containing the target sequence. The intra- and interassay coefficients of variation were 0.46%-4.27% and 0.05-2.00% over 5 log(10) values. Fifty-seven controls of human, viruses, bacteria and fungi DNA samples were amplified and found negative. Fifty-three BAL samples sent to the laboratory for diagnosis of pneumocystosis were prospectively investigated by real-time PCR and direct microscopic examinations (DME) using Giemsa stain and direct immunofluorescence. All PCR negative samples were negative by microscopy. Among the 24 (45%) BAL found PCR positive, 8 were positive by microscopy (35%). The copy numbers of the target gene were between 4.4 x 10(3) and 2.8 x 10(6) per ml for the microscopically positive samples and between 8 and 9.2 x 10(3) per ml for the microscopically negative samples. In conclusion, we developed a rapid, sensitive and specific real time PCR for the diagnosis and quantification of Pneumocystis jiroveci in BAL samples.

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.

Low-temperature biodegradation of high amounts of phenol by Rhodococcus spp. and basidiomycetous yeasts.

Four cold-adapted microbial strains able to degrade high amounts of phenol were isolated from hydrocarbon-contaminated alpine soils. Two of the strains were bacteria identified as Rhodococcus spp., and two strains were basidiomycetous yeasts. One of the yeasts was identified as Trichosporon dulcitum, while the second yeast strain belonged to the Urediniomycetes and probably represents a novel species. This strain was not able to grow at temperatures above 20 degrees C, while the other three strains were cold-tolerant and could grow at temperatures ranging from 1-25 degrees C (T. dulcitum) or 1-30 degrees C (rhodococci). The yeast strains were characterized by a substantially lower optimum temperature for growth and biodegradation compared to the bacteria. The urediniomycete strain degraded 5 mM phenol at 1 degrees C faster than the two bacteria at 10 degrees C. The optimum temperature for phenol degradation was 10 degrees C (novel yeast species), 20 degrees C (T. dulcitum), or 30 degrees C (rhodococci). Using fed-batch cultivation in mineral medium with phenol as the sole carbon source, high amounts of phenol were degraded at 10 degrees C. Both rhodococci degraded up to 12.5 mM phenol, while the two yeast strains even utilized as much as 15 mM phenol.

One-tube cell lysis and DNA extraction procedure for PCR-based detection of Mycobacterium ulcerans in aquatic insects, molluscs and fish.

The purpose of this study was to develop a simple procedure for cell lysis and DNA extraction for direct detection of Mycobacterium ulcerans in aquatic insects, gills and intestinal contents of fish, molluscs and human tissue samples using a nested PCR method specific for the insertion sequence IS2404. The simultaneous action of sodium N-lauroyl sarcosine, guanidinium isothiocyanate, chloroform and Tris-saturated phenol on mycobacteria, followed by a DNA purification method using mini-columns fitted with silica-cellulose membranes was successfully employed to extract DNA from cultured bacteria, environmental and human tissue samples. All specimens were collected from Buruli ulcer endemic regions. M. ulcerans DNA was detected in 11 of 57 aquatic insects, one of six molluscs and three of 15 fish, supporting the hypothesis that the fauna of major Buruli ulcer endemic foci in swampy terrain of tropical and subtropical regions can be a source of M. ulcerans infection.