Dermatophytes’ identification by Matrix-assisted laser desorption ionization-time of flight mass spectrometry. (MALDI-TOF MS) - the experience of a clinical laboratory

Objectives: Dermatophytes are a challenging group of fungi that infect the keratinized tissues. The taxonomy of these fungi has changed recently with the reclassification of some species and description of new ones. However, many clinical laboratories still base the identification of dermatophytes on their phenotype. Since dermatophytes are very pleomorphic, macro and micromorphology are often insufficient to reach a correct classification and may lead to misidentifications. The identification based on MALDI-TOF relies on the protein profile of the microorganism. Thus, this study aims to summarize our current laboratorial experience of dermatophyte identification using MALDI-TOF MS. Methods: From january to april 2018, 95 dermatophytes isolates, collected from human keratinized samples and also from quality control programs were characterized by phenotypic analysis, and by VITEK MS V3.2 bioMerieux. Before identification procedure, isolates were inoculated on Sabouraud Dextrose agar plates and incubated at 27°C during 5 to 10 days. Species were identified taking into account clinical features, as well as cultural, microscopic and physiological characteristics. Prior to MALDI-TOF MS analysis, the samples were pre-treated according to the manufacturer’s protocol for filamentous fungi. Molecular identification by sequencing of the internal transcribed spacer 1 (ITS1) was performed in 34 of those isolates Results: Through phenotypic analysis eight different species were identified (54 Trichophyton rubrum; 4 T.soudanense; 22 T.interdigitale; 1 T.mentagrophytes; 3 T.tonsurans; 7 Microsporum canis; 3 M.audouinii; 1 Microsporum spp.- (non canis or audouinii). MALDI-TOF analysis showed an identification agreement in 80 cases (84,2%) with a confidence level of 99,9%. Eight isolates showed divergent identification results: three T.rubrum were identified as T.violaceum, three T.soudanense were identified as T.rubrum, one T.mentagrophytes was identified as T.interdigitale and one T.tonsurans was identified as T.rubrum. In four cases MALDI-TOF analysis did not get a profile. The ITS sequencing analysis of discrepant results corroborated the MALDI-TOF identification in five of them. On the other hand, T.soudanense was only identified by phenotypic analysis since MALDI-TOF and ITS sequencing result was T.rubrum. MALDITOF identification of T.violaceum was not confirmed by ITS sequencing that identified T. rubrum instead, in accordance with the phenotypic identification. Conclusion: Correct identification of dermatophytes to species level requires sequencing of the ITS, LSU, and/or betatubulin regions. The implementation of this methodology in a clinical laboratory is expensive and time consuming. MALDI-TOF identification is a good option for dermatophytes’ identification performed in laboratory routine, since costs of consumables as well as time of sample preparation are lower than for PCR analysis and doesn’t require long training period as phenotypic identification does. In this study, however, both methods failed to identify some species variants like Trichophyton soudanense or T. violaceum. The combined use of both MALDI-TOF and phenotypic methods seems to be the better approach for dermatophytes’ identification since some species show significant phenotypic and clinical differences.info:eu-repo/semantics/publishedVersio

Evaporation waves in superheated dodecane

We have observed propagating adiabatic evaporation waves in superheated liquid dodecane, C_(12)H_(26). Experiments were performed with a rapid decompression apparatus at initial temperatures of 180–300°C. Saturated dodecane in a tube was suddenly depressurized by rupturing a diaphragm. Motion pictures and still photographic images, and pressure and temperature data were obtained during the evaporation event that followed depressurization. Usually, a front or wave of evaporation started at the liquid free surface and propagated into the undisturbed regions of the metastable liquid. The evaporation wave front moved with a steady mean velocity but the front itself was unstable and fluctuating in character. At low superheats, no waves were observed until a threshold superheat was exceeded. At moderate superheats, subsonic downstream states were observed. At higher superheats, the downstream flow was choked, corresponding to a Chapman–Jouguet condition. At the most extreme superheat tested, a vapour content of over 90% was estimated from the measured data, indicating a nearly complete evaporation wave. Our results are interpreted by modelling the evaporation wave as a discontinuity, or jump, between a superheated liquid state and a two-phase liquid–vapour downstream state. Reasonable agreement is found between the model and observations; however, there is a fundamental indeterminacy that prevents the prediction of the observed wave speeds

Frequency and molecular epidemiology of Aspergillus isolated from patients with suspicion of respiratory fungal infection

Objective: The aim of this study was to determine the frequency of Aspergillus detected in respiratory samples from a cohort of patients with suspicion of fungal infection of the respiratory tract as well as to determine the susceptibility to azoles of the isolates from the Fumigati section. Methods: A retrospective study was performed involving samples obtained from 16 hospitals covering different districts of continental Portugal and Azores islands. One hundred and eighty-seven respiratory samples (101 bronchoalveolar lavage fluids, 52 bronchial lavages, 27 bronchial secretions, 6 expectorations and 1 bronchial aspirate) were collected between November 2011 and December 2017 from a cohort of 146 patients with suspicion of respiratory fungal infection (ages ranging from 20 to 87 years old). Demographic and clinical data were recorded. Detection of Aspergillus was done by culture, immunoenzimatic assay and/or molecular techniques. Aspergillus molecular identification to species level was performed by sequencing of the calmodulin and β-tubulin genes. To detect possible resistance to azoles, isolates belonging to section Fumigati were inoculated into Sabouraud dextrose agar media supplemented with 1 µg/ml or 4 µg/ml of voriconazole, 4 µg/ml of itraconazole and 0.5 µg/ml of posaconazole and their growth was observed and recorded after 7 days of incubation at 27ºC. Doubtful results were confirmed when possible by E-test and by real-time multiplex PCR for the detection of mutations in the Cyp51A gene. Results: Fifty-seven (39.0%) of the studied patients were positive for Aspergillus. From the cases with a positive culture (n=58) the species were identified by sequencing and belonged to six different sections. The most frequently isolated was the section Nigri (42.1%) followed by the Fumigati (33.3%) and Flavi sections (8.6%). Regarding the species, the most frequent was A. niger sensu stricto (33.9%) followed by A. fumigatus sensu stricto (32.1%). Nine cryptic species were also identified which frequency was 21.4%. In order to study the frequency of azole resistance in Fumigati isolates collected from the samples of this cohort as well from other biological products, 52 isolates - Aspergillus fumigatus sensu stricto (n=45), A. lentulus (n=4), A. udagawae (n=2) and A. pseudofelis (n=1) – were tested. The tested A. fumigatus sensu stricto isolates did not show resistance to azoles. An A. udagawae strain revealed low susceptibility to voriconazole (MIC was not determined due to loss of strain viability). An A. pseudofelis strain also showed decreased susceptibility to voriconazole (MIC =1 μg/ml) as well as to and itraconazole (MIC = 2 μg/ml). Conclusion: In this study, the genus Aspergillus was frequently isolated in the respiratory samples tested and a high number of cryptic species was detected. Although resistance to azoles was not a problem identified in the tested isolates, determination of the in vitro susceptibility profile and molecular identification of the Aspergillus species is essential to improve the diagnosis and management of aspergillosis since several cryptic species have intrinsic resistance to antifungal drugs.info:eu-repo/semantics/publishedVersio

Impulsive Heating of Solar Flare Ribbons Above 10 MK

The chromospheric response to the input of flare energy is marked by extended extreme ultraviolet (EUV) ribbons and hard X-ray (HXR) footpoints. These are usually explained as the result of heating and bremsstrahlung emission from accelerated electrons colliding in the dense chromospheric plasma. We present evidence of impulsive heating of flare ribbons above 10 MK in a two-ribbon flare. We analyse the impulsive phase of SOL2013-11-09T06:38, a C2.6 class event using data from Atmospheric Imaging Assembly (AIA) on board of Solar Dynamics Observatory (SDO) and the Reuven Ramaty High Energy Solar Spectroscopic Imager (RHESSI) to derive the temperature, emission measure and differential emission measure of the flaring regions and investigate the evolution of the plasma in the flaring ribbons. The ribbons were visible at all SDO/AIA EUV/UV wavelengths, in particular, at 94 and 131 \AA\ filters, sensitive to temperatures of 8 MK and 12 MK. Time evolution of the emission measure of the plasma above 10 MK at the ribbons has a peak near the HXR peak time. The presence of hot plasma in the lower atmosphere is further confirmed by RHESSI imaging spectroscopy analysis, which shows resolved sources at 11-13 MK associated with at least one ribbon. We found that collisional beam heating can only marginally explain the necessary power to heat the 10 MK plasma at the ribbons.Comment: 21 pages, 15 figure

Integração de sistemas de energia renovável a instalações residenciais

Os sistemas de microgeração domésticos que produzem energia a partir de fontes renováveis necessitam de legislação especifica e de ferramentas para identificar os potenciais e a viabilidade de implantação de soluções tecnológicas adaptadas ao ambiente urbano. Este artigo analisa exemplos de implementação de sistemas eólicos, fotovoltaicos e híbridos em uma edificação, debatendo questões económicas e energéticas

Influence of wood basic density on acacia melanoxylon kraft process

Wood density is a complex physical property related to the anatomical structure and the chemical composition of wood and one of the most important wood quality factors. Sampling was based on a total of 20 trees from four sites in Portugal that were harvested at a sawmill diameter class of 40 cm and wood discs taken at different height levels from the base to the top of the tree. The mean basic density of the Acacia melanoxylon trees measured at 5% height level (near breast height level) was 516 kg/m3 with a 34 kg/m3 standard deviation. The within-tree axial variation of density was of small magnitude, but showing higher values at the stem base and top. Site had no influence on the basic density of the wood. Overall the between tree variation of density was small possibly linked to the narrow genetic diversity of this introduced exotic species. In the Kraft process we can observe variability between stands and an increase of the pulp yield, and fiber width and length with the higher level in the tree. The wood basic density is well correlated with the ISO brightness and well correlated in inverse order with the pulp Yield and fiber width and length. The kappa number don’t present a great variation with the wood basic density