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

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

Análise comparativa da quantificação da acção da neve de acordo com o RSA, a norma espanhola NBE e o EC1

A quantificação da acção da neve de acordo com a regulamentação em vigor em Portugal, Espanha e com o estabelecido no Eurocódigo 1 fornece resultados muito distintos. Neste trabalho é realizada uma análise comparativa entre o método de cálculo indicado pelo Regulamento de Segurança e Acções para Estruturas de Edifícios e Pontes, a norma espanhola NBE e o Eurocódigo 1. Foram analisados e comparados os vários parâmetros intervenientes na quantificação da acção da neve

The Influence of Solvent and Extraction Time on Yield and Chemical Selectivity of Cuticular Waxes from Quercus suber Leaves

The cuticular lipid compounds, usually named cuticular waxes, present in the cuticular layering of Quercus suber adult leaves were extracted with solvents of different polarities (n-hexane, dichloromethane and acetone) and analysed by GC–MS. Q. suber leaves have a substantial cuticular wax layer (2.8% of leaf mass and 239 g/cm2), composed predominantly by terpenes (43–63% of all compounds), followed by aliphatic long chain molecules, mainly fatty acids, and by smaller amounts of aliphatic alcohols and n-alkanes. The major identified compound was lupeol (1.2% of leaves in n-hexane extract). The recovery and composition of cuticular lipids depended on the solvent and extraction time. The non-polar or weak polar solvents n-hexane and dichloromethane extracted similar lipid yields (77% and 86% of the total extract, respectively) while acetone solubilised other cellular compounds, namely sugars, with the lipid compounds representing 43% of the total extract. For cuticular lipids extraction, solvents with a low polarity such as n-hexane are the more suitable with an adequate extraction duration, e.g., n-hexane with a minimum extraction of 3 hinfo:eu-repo/semantics/publishedVersio

Experiments on enlarging a lexical ontology

This paper presents two simple experiments performed in order to enlarge the coverage of PULO, a Lexical Ontology, based and aligned with the Princeton WordNet. The first experiment explores the triangulation of the Galician, Catalan and Castillian wordnets, with translation dictionaries from the Apertium project. The second, explores Dicionário-Aberto entries, in order to extract synsets from its definitions. Although similar approaches were already applied for different languages, this document aims at documenting their results for the PULO case

On the Identity of the Adventive Species of Eufriesea Cockerell in the USA: Systematics and Potential Distribution of the Coerulescens Species Group (Hymenoptera, Apidae)

In the summer of 2010, two male specimens of the neotropical orchid bee genus Eufriesea Cockerell were collected in the Guadalupe Mountains of western Texas and southeastern New Mexico, USA. We tentatively identified them as E. coerulescens (Lepeletier de Saint Fargeau) because of the uncertainty surrounding the limits of this taxon and hypothesized that they were members of a persistent bee population, rather than long-distance transient vagrants. The goals of this paper are to clarify the identity of these specimens, assess the species limits of E. coerulescens, and to evaluate suitability of habitats in the USA for this adventive species. Herein, we revise the species in the coerulescens group using morphological features of both sexes and confirm that the specimens of Eufriesea from the USA are E. coerulescens. We recognize the following six species in the coerulescens group: E. coerulescens, E. micheneri Ayala & Engel, E. simillima (Moure & Michener), which is reinstated from synonym with E. coerulescens, and three new species from Mexico (E.barthelli Gonzalez & Griswold, sp. n., E. engeli Gonzalez & Griswold, sp. n., and E. oliveri Gonzalez & Griswold, sp. n.). To facilitate the identification of these taxa, we present a fully illustrated account of the species, comparative diagnoses, descriptions, and an updated key to all Mexican species of Eufriesea. Our analyses using species distribution modelling show an absence of suitable habitat for E. coerulescens in western Texas and southeastern New Mexico, thus favoring the long-distance dispersal hypothesis. The analyses also suggest high suitability of habitats across the Caribbean and some areas in Florida, as well as in other regions in Mexico and Central America. We discuss the implications of these results and compare them with the predicted distribution available for the other two known adventive orchid bee species in the USA

Alternative species for the forest industry as forms of diversify the landscape

Together the forest and industrial activities within the Portuguese forest sector have a great importance in the national economy. The most used wood species in Portugal for industry (wood panel, sawmill, wood crates) are pine and eucalypt, which leads to extreme dependence and competition between the various industries for the same material, and thus unsustainable pressure on these forest resources. This is one of the causes of the decrease of pinewood area in recent years. On the other hand, this dependence leads to extensive areas of forest monocultures and, subsequently, increased risk of the forest fire propagation. This work intends to stimulate the diversification of the wood products used in the national industry of pulp and to provide a pulp with appropriate characteristics for incorporation as fibber for paper reinforcement. At the level of forest producers, the use of this prime-material would increase competitiveness among tree species and revitalization of less favoured rural areas and, turning them into a possible solution for the lack of wood and an incentive to the reforestation of these areas. Wood from species Cupressus sempervirens and Cupressus arizonica, Acacia delbata and Acacia melanoxylon were analysed. Content of extractives and of Klason lignin, fibre length and coarseness were determined. Representative wood samples from Pinus pinaster grown in Portugal and from Pinus sylvestris grown in Finland were used as reference. The wood from Cupressus sempervirens showed lower Klason lignin and a fibre quality that appears to be more adequate to pulp and paper. Acacia species, with their relatively short, flexible and collapsible fibres, have potential to produce papers with good relationships light scattering/tensile strength and smoothness/tensile strength, at low energy consumption in refining. The studied acacia species showed slightly better performance in pulping than the Eucalyptus globulus sample used as a comparison