Updating the Taxonomy of Dermatophytes of the BCCM/IHEM Collection According to the New Standard: A Phylogenetic Approach.

Recent taxonomical revisions based on multilocus gene sequencing have provided some clarifications to dermatophyte (Arthrodermataceae) family tree. These changes promoted us to investigate the impact of the changed nomenclature of the dermatophyte strains in the BCCM/IHEM fungal collection, which contains strains of all dermatophyte genera except for Ctenomyces. For 688 strains from this collection, both internal transcribed spacer region (ITS) and partial ß-tubulin (BT) sequences were aligned and a multilocus phylogenetic tree was constructed. The ITS + BT phylogentic tree was able to distinguish the genera Arthroderma, Lophophyton, Microsporum, Paraphyton, Nannizzia and Trichophyton with high certainty. Epidermophyton, which is widely considered as a well-defined genus with E. floccosum as the only representative, fell within the Nannizzia clade, whereas the phylogenetic analysis, based on the ITS region alone, differentiates Epidermophyton from Nannizzia as a separate genus. Re-identification and reclassification of many strains in the collection have had a profound impact on the composition of the BCCM/IHEM dermatophyte collection. The biggest change is the decline of prevalence of Arthroderma strains; starting with 103 strains, only 22 strains remain in the genus after reassessment. Most Arthroderma strains were reclassified into Trichophyton, with A. benhamiae and A. vanbreuseghemii leaving the genus. The amount of Microsporum strains also dropped significantly with most of these strains being reclassified into the genera Paraphyton and Nannizzia.

Nucleotide Sequence Database Comparison for Routine Dermatophyte Identification by Internal Transcribed Spacer 2 Genetic Region DNA Barcoding.

Conventional dermatophyte identification is based on morphological features. However, recent studies have proposed to use the nucleotide sequences of the rRNA internal transcribed spacer (ITS) region as an identification barcode of all fungi, including dermatophytes. Several nucleotide databases are available to compare sequences and thus identify isolates; however, these databases often contain mislabeled sequences that impair sequence-based identification. We evaluated five of these databases on a clinical isolate panel. We selected 292 clinical dermatophyte strains that were prospectively subjected to an ITS2 nucleotide sequence analysis. Sequences were analyzed against the databases, and the results were compared to clusters obtained via DNA alignment of sequence segments. The DNA tree served as the identification standard throughout the study. According to the ITS2 sequence identification, the majority of strains (255/292) belonged to the genus Trichophyton, mainly T. rubrum complex (n = 184), T. interdigitale (n = 40), T. tonsurans (n = 26), and T. benhamiae (n = 5). Other genera included Microsporum (e.g., M. canis [n = 21], M. audouinii [n = 10], Nannizzia gypsea [n = 3], and Epidermophyton [n = 3]). Species-level identification of T. rubrum complex isolates was an issue. Overall, ITS DNA sequencing is a reliable tool to identify dermatophyte species given that a comprehensive and correctly labeled database is consulted. Since many inaccurate identification results exist in the DNA databases used for this study, reference databases must be verified frequently and amended in line with the current revisions of fungal taxonomy. Before describing a new species or adding a new DNA reference to the available databases, its position in the phylogenetic tree must be verified.

A molecular approach for the rapid, selective and sensitive detection of Exophiala jeanselmei in environmental samples: development and performance assessment of a real-time PCR assay.

Exophiala jeanselmei is an opportunistic pathogenic black yeast growing in humid environments such as water reservoirs of air-conditioning systems. Because this fungal contaminant could be vaporized into the air and subsequently cause health problems, its monitoring is recommended. Currently, this monitoring is based on culture and microscopic identification which are complex, sometimes ambiguous and time-demanding, i.e., up to 21 days. Therefore, molecular, culture-independent methods could be more advantageous for the monitoring of E. jeanselmei. In this study, we developed a SYBR®green real-time PCR assay based on the internal transcribed spacer 2 from the 18S ribosomal DNA complex for the specific detection of E. jeanselmei. The selectivity (100 %), PCR efficiency (95.5 %), dynamic range and repeatability of this qPCR assay were subsequently evaluated. The limit of detection for this qPCR assay was determined to be 1 copy of genomic DNA of E. jeanselmei. Finally, water samples collected from cooling reservoirs were analyzed using this qPCR assay to deliver a proof of concept for the molecular detection of E. jeanselmei in environmental samples. The results obtained by molecular analysis were compared with those of classical methods (i.e., culture and microscopic identification) used in routine analysis and were 100 % matching. This comparison demonstrated that this SYBR®green qPCR assay can be used as a molecular alternative for monitoring and routine investigation of samples contaminated by E. jeanselmei, while eliminating the need for culturing and thereby considerably decreasing the required analysis time to 2 days.

Development and performance assessment of a qualitative SYBR® green real-time PCR assay for the detection of Aspergillus versicolor in indoor air.

Currently, contamination of indoor environment by fungi and molds is considered as a public health problem. The monitoring of indoor airborne fungal contamination is a common tool to help understanding the link between fungi in houses and respiratory problems. Classical analytical monitoring methods, based on cultivation and microscopic identification, depend on the growth of the fungi. Consequently, they are biased by difficulties to grow some species on certain culture media and under certain conditions or by noncultivable or dead fungi that can consequently not be identified. However, they could have an impact on human health as they might be allergenic. Since molecular methods do not require a culture step, they seem an excellent alternative for the monitoring of indoor fungal contaminations. As a case study, we developed a SYBR® green real-time PCR-based assay for the specific detection and identification of Aspergillus versicolor, which is frequently observed in indoor environment and known to be allergenic. The developed primers amplify a short region of the internal transcribed spacer 1 from the 18S ribosomal DNA complex. Subsequently, the performance of this quantitative polymerase chain reaction (qPCR) method was assessed using specific criteria, including an evaluation of the selectivity, PCR efficiency, dynamic range, and repeatability. The limit of detection was determined to be 1 or 2 copies of genomic DNA of A. versicolor. In order to demonstrate that this SYBR® green qPCR assay is a valuable alternative for monitoring indoor fungal contamination with A. versicolor, environmental samples collected in contaminated houses were analyzed and the results were compared to the ones obtained with the traditional methods.

Fast and accurate identification of dermatophytes by matrix-assisted laser desorption ionization-time of flight mass spectrometry: validation in the clinical laboratory.

The performance of a matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS) workflow using an extensive reference database for dermatophyte identification was evaluated on 176 clinical strains. Using a direct-deposit procedure after 3 incubation days yielded 40% correct identification. Both increasing incubation time and using an extraction procedure resulted in 100% correct identification.

Matrix-assisted laser desorption ionization time-of-flight mass spectrometry: revolutionizing clinical laboratory diagnosis of mould infections.

The clinical diagnosis of mould infections currently involves complex species identification based on morphological criteria, which is often prone to error. Employing an extensive mould species reference spectral library (up to 2832 reference spectra, corresponding to 708 strains from 347 species), we assessed the extent to which matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF MS) enhanced the accuracy of species identification. MALDI-TOF MS data were validated against morphology-based and DNA sequence-based results with 262 clinical isolates collected over a 4-month period in 2013. The implementation of MALDI-TOF MS resulted in a dramatic improvement in mould identification at the species level (from 78.2% to 98.1%) and a marked reduction in the misidentification rate (from 9.8% to 1.2%). We then compared the mould identification results obtained before (i.e. 2011) and after (i.e. 2013) the implementation of MALDI-TOF MS in routine identification procedures, which showed an improvement from 64.57% to 100%. Reassessment of a set of isolates from 2011 with this procedure, including MALDI-TOF MS, yielded an increase in species diversity from 16 to 42 species. Finally, application of this procedure during a 16-month period (2012-2013) enabled the identification of 1094 of 1107 (98.8%) clinical mould isolates corresponding to 107 distinct species. MALDI-TOF MS-based mould species identification may soon challenge traditional techniques in the clinical laboratory, as patient prognosis is largely contingent on rapid and accurate diagnosis.

The role of indoor pollution in the development and maintenance of chronic airway inflammation in children.

Air pollution is one of the great problems of this century and it plays an important role in the increasing prevalence of chronic inflammatory problems in the upper airway in children. Since their lungs and immune system are not fully developed when exposure begins, newborns and children appear to be more sensitive to the effects of both outdoor and indoor air pollution. Furthermore, children spend most of their time indoors and are exposed more often to pollutants in indoor air. The link between health problems, chemical products and allergens (the latter mainly from cats and mites) has been extensively studied. Other important indoor contaminants are fungi, which are often present in damp buildings and can cause severe respiratory disease by producing spores, allergens, volatile irritant compounds and toxins. A proper identification of mould contamination of this kind is vital for correct diagnosis, treatment and the prevention of health problems, and improvements have been observed after the removal or cleaning of the contaminated materials and improvements to the ventilation of buildings. While a possible association between respiratory symptoms, such as rhinitis, and the presence of fungi in the indoor environment has been documented by several authors, other studies have observed no significant relationship. The development of standardised sampling, detection and diagnostic tests will be essential to understand the proper role of fungi in the indoor atmosphere and their impact on public health.

Estimation of the dissociation rate of unlabelled ligand-receptor complexes by a 'two-step' competition binding approach.

BACKGROUND AND PURPOSE: Because the in vivo effectiveness of ligands may also be determined by the rate by which they dissociate from their target receptors, drug candidates are being increasingly screened for this kinetic property. The dissociation rate of unlabelled ligand-receptor complexes can be estimated indirectly from their ability to slow the association of subsequently added radioligand molecules. EXPERIMENTAL APPROACH: We used the 'two-step competition' binding approach consisting of pre-incubating the receptor preparation with a wide range of ligand concentrations, washing off free ligand molecules, adding radioligand and monitoring its receptor binding after a fixed time. Based on the rationale that binding of both ligands is mutually exclusive and that they bind according to the law of mass action to a single class of sites, the unlabelled ligand's dissociation rate can be estimated from the upward shift that the competition curve experiences after washing. KEY RESULTS: The relevance of the 'two-step competition' approach was explored by computer simulations and by comparing the dissociation behaviour of unlabelled D(2) dopamine and CB(1) cannabinoid receptor antagonists in this and alternative approaches. Besides providing satisfactory estimations of dissociation rates, the method also detects the ability of the unlabelled ligand molecules to be released from 'sinks' such as the cell membrane. CONCLUSIONS AND IMPLICATIONS: As the 'two-step competition' requires rapid intermediate washing steps and needs radioligand binding to be measured at only one time point, this approach is particularly suited for binding studies on intact plated cells. LINKED ARTICLES: This article is part of a themed section on Analytical Receptor Pharmacology in Drug Discovery. To view the other articles in this section visit http://dx.doi.org/10.1111/bph.2010.161.issue-6.

Ligands, their receptors and ... plasma membranes.

Ligand-receptor interactions are customarily described by equations that apply to solutes. Yet, most receptors are present in cell membranes so that sufficiently lipophilic ligands could reach the receptor by a two-dimensional approach within the membrane. As summarized in this review, this may affect the ligand-receptor interaction in many ways. Biophysicians calculated that, compared to a three-dimensional approach from the liquid phase, such approach could alter the time the ligands need to find a receptor. Biochemists found that ligand incorporation in lipid bilayers modifies their conformation. This, along with the depth at which the ligands reside in the bilayer, will affect the probability of successful receptor interaction. Novel mechanisms were also introduced, including "exosite" binding and ligand translocation between the receptor's alpha-helical transmembrane domains. Pharmacologists focused attention at ligand concentrations in membrane, their adsorption and release rates and the effects thereof on ligand potency and residence time at the receptor.

Molecular mechanisms for the persistent bronchodilatory effect of the beta 2-adrenoceptor agonist salmeterol.

BACKGROUND: Beta(2)-adrenoceptor agonists are effective bronchodilators. In vitro studies demonstrated long-lasting airway smooth muscle relaxation by salmeterol after washout, the quick disappearance of this effect in presence of antagonists and its recovery after antagonist removal. Current explanations invoke salmeterol accumulation in the membrane ('diffusion microkinetic' model) or the existence of salmeterol-binding 'exosites'. An alternative model based on 'rebinding' of a dissociated ligand to the receptor molecules also produces an apparent decrease in the ligand's dissociation rate in the absence of competing ligands. PURPOSE AND APPROACH: Computer-assisted simulations were performed to follow the receptor-occupation by a salmeterol-like ligand and a competing ligand as a function of time. The aptness of the models to describe the above in vitro findings was evaluated. KEY RESULTS: The 'diffusion microkinetic' model is sufficient to explain a long-lasting beta(2)-adrenoceptor stimulation and reassertion as long as the membrane harbors a high concentration of the agonist. At lower concentration, 'rebinding' and, in second place, 'exosite' binding are likely to become operational. CONCLUSIONS AND IMPLICATIONS: The 'rebinding' and 'exosite' binding mechanisms take place at a sub-cellular/molecular scale. Pending their demonstration by experiments on appropriate, simple models such as intact cells or membranes thereof, these mechanisms remain hypothetical in the case of salmeterol. Airway smooth muscle contraction could also be governed by additional mechanisms that are particular to this macroscopic approach.