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

Majocchi’s Granuloma by Trichophytum rubrum in a kidney transplant patient - A case report

Introduction: Trichophytum rubrum is a filamentous fungus, with worldwide distribution, that usually causes superficial infections of skin and nails, namely tinea pedis, tinea corporis, tinea cruris and onychomycosis. Rarely, severe dermatophytosis can occur, presenting as deep dermatophytosis, Majocchi’s Granuloma or extensive dermatophytosis. Objectives and Methods: Case report of Majocchi’s Granuloma in a kidney transplant patient. Results: A case of a 55-year-old woman who underwent a kidney transplant 7 months before, under immunosuppressive therapy with tacrolimus and mycophenolate mofetil. She attended a Dermatology consultation to clarify skin lesions that appeared 6 months earlier. The skin exam revealed hard and painful plaque lesions on both legs, with an ulcer on the left leg lesion, violaceous papular lesions on the dorsum of the left foot and toes and a hard consistency nodule on the left leg. Some of the toe nails presented dystrophy or onycholysis. The patient denied any previous trauma or contact with plants or soil. Biopsies of lesions of the left leg and foot dorsum where sent for histology and mycological culture and toe nails for mycological culture. The histological examinations showed, in the reticular dermis and reaching the hypodermis, suppurative granulomas with multinucleated giant cells and areas of necrosis. PAS (Periodic Acid- Schiff) and GMS (Grocott’s Methenamine Silver) staining revealed multiple spores and septate hypha within the granulomas but not in the stratum corneum. No remnants of hair follicles where found. Culture of skin biopsies were positive for Tricophytum rubrum but nails´ culture was negative. Identification was further confirmed by sequencing of ITS region of ribosomal DNA (GenBank accession number MK967277). Oral Itraconazole 100mg bid and topic Sertoconazole where initiated. The patient was observed one month after and reported general malaise, tiredness, exertional dyspnea, whitish stools and increased abdominal volume. The physician chose to discontinue itraconazole and initiate oral terbinafine 250mg id. After two months on oral terbinafine, there was regression of the legs´ and left foot lesions with ulcer healing and disappearance of the left leg nodule. Conclusion: Diagnosis of deeper dermatophytosis is difficult, in part because there is no specific clinical presentation and, in many cases, it is even polymorphic. However, especially in patients with immunodeficiency, this hypothesis should be weighed. Confirmation is achieved by finding hyphae compatible with dermatophytes in the dermis and a positive culture for a dermatophyte. Treatment should include systemic antifungal agents, to which topical medication may be associated. Multiple therapeutic regimens have been proposed, but randomized trials or large case series are lacking. Antifungal therapy should be continued until the lesions are completely resolved. Surgical treatment has been reported as an option for highly localized lesions.info:eu-repo/semantics/publishedVersio

Comparison of fungal contamination between hospitals and companies food units

A descriptive study was developed to compare air and surfaces fungal contamination in ten hospitals’ food units and two food units from companies. Fifty air samples of 250 litres through impaction method were collected from hospitals’ food units and 41 swab samples from surfaces were also collected, using a 10 by 10 cm square stencil. Regarding the two companies, ten air samples and eight surface samples were collected. Air and surface samples were collected in food storage facilities, kitchen, food plating and canteen. Outdoor air was also collected since this is the place regarded as a reference. Simultaneously, temperature, relative humidity and meal numbers were registered. Concerning air from hospitals’ food units, 32 fungal species were identified, being the two most commonly isolated genera Penicillium sp

O NOME NAS CANTIGAS SATÍRICAS: ESTUDO DE (POSSÍVEIS) PERSONAGENS MOUROS À LUZ DA INTERPRETATIO NOMINIS

O interesse desta Tese centra-se na análise de cantigas de escárnio e maldizer galego-portuguesas que tenham como satirizados personagens mouros com nomes. A partir da interpretatio nominis, das questões que envolvem o gênero satírico trovadoresco peninsular (sobretudo a hequivocatio) e da noção de cantigas como jogo de palavras (jugar de palabras) para entretenimento cortesão, o processo de investigação dessa modalidade de sátira medieval nos possibilita inquirir a respeito da motivação nominal, sendo tal recurso, o da utilização de nomes onomasticamente ativos como um dos signos que sustentam o jogo risível, um componente retórico valioso para a poética escarninha. Diante dessa constatação, é possível que o nome satírico do personagem mouro naquelas cantigas possa ampliar o entendimento e as significações muito mais que um nome de registro o pudesse fazer. Acolhemos especialmente, para a argumentação deste trabalho, os estudos de Carlos Paulo Martínez Pereiro, a respeito da interpretatio nominis; os de Manuel Rodrigues Lapa, Graça Videira Lopes, Giuseppe Tavani e Giulia Lanciani, acerca do gênero cantiga de escárnio e maldizer; os de Ana Echeverría Arsuaga, Maria Filomena Lopes de Barros, Juan Eslava Galán, Eugenio López- Aydillo, Marta Madero, Abdelwahab Bouhdiba, Camilo Álvarez de Morales, John Boswell, James A. Brundage, Francisco Nodar Manso e Xosé Bieito Arias Freixedo, a propósito dos mouros; os de Jesús Montoya Martínez, sobre a dimensão lúdica das cantigas trovadorescas peninsulares, além das edições críticas das cantigas, sobretudo, de Manuel Rodrigues Lapa e Graça Videira Lopes

O Nome da Sátira Medieval: Estudo de Três Personagens Dionisinos à Luz da Interpretatio Nominis

Análise dos ciclos satíricos do rei-trovador Dom Dinis, destinados a Melion Garcia e a João Bolo, além da cantiga destinada a João Simeão, à luz da interpretatio nominis. A investigação desses textos assim como de seu contra-texto, resultante do jogo de palabras cubertas nos possibilita inquirir a respeito da motivação nominal nesse fazer poético, tendo em vista que os nomes são um dos muitos signos que estruturam e sustentam o jogo risível das cantigas escarninhas. Esses nomes-burlas são, ao mesmo tempo, pista de identidade e denúncia das falhas dos visados. Com isso, texto propriamente dito e a persona satirizada, numa relação nome/texto, (a)firmam-se como um objeto único, funcionando o personagem seja este representado por uma alcunha, um sobrenome, um apelido ou até mesmo por um anagrama como uma chave de leitura. Palavras-chave: Trovadorismo galego-português; Cantigas de escárnio e maldizer; Dom Dinis (Cantigas satíricas); Interpretatio nominis (Estudo onomástic

Air fungal contamination in ten hospitals’ food units from Lisbon

A descriptive study was developed to monitor air fungal contamination in ten food units from hospitals. Fifty air samples of 250 litres were collected through impaction method. Samples were collected in food storage facilities, kitchen, food plating, canteen and also, outside premises, since this is the place regarded as reference. Simultaneously, environmental parameters were also monitored, including temperature and relative humidity through the equipment Babouc, LSI Sistems and according to the International Standard ISO 7726

Comparison of indoor and outdoor fungi and particles in poultry units

A descriptive study was developed in order to compare indoor and outdoor air contamination caused by fungi and particles in seven poultry units. Twenty eight air samples of 25 litters were collected through the impaction method on malt extract agar. Air sampling and particles concentration measurement were done in the interior and also outside premises of the poultries’ pavilions. Regarding the fungal load in the air, indoor concentration of mold was higher than outside air in six poultry units. Twenty eight species / genera of fungi were identified indoor, being Scopulariopsis brevicaulis (40.5%) the most commonly isolated species and Rhizopus sp. (30.0%) the most commonly isolated genus. Concerning outdoor, eighteen species/genera of fungi were isolated, being Scopulariopsis brevicaulis (62.6%) also the most isolated. All the poultry farms analyzed presented indoor fungi different from the ones identified outdoors. Regarding particles’ contamination, PM2.5, PM5.0 and PM10 had a statistically significant difference (Mann-Whitney U test) between the inside and outside of the pavilions, with the inside more contaminated (p=.006; p=.005; p=.005, respectively). The analyzed poultry units are potential reservoirs of substantial amounts of fungi and particles and could therefore free them in the atmospheric air. The developed study showed that indoor air was more contaminated than outdoors, and this can result in emission of potentially pathogenic fungi and particles via aerosols from poultry units to the environment, which may post a considerable risk to public health and contribute to environmental pollution

Preparation of the conducting nanocomposites using molded inorganic matrix: fibrous cerium(IV) hydrogenphosphate as a self-supported pyrrole polymerization agent

New supported polypyrrole nanocomposites have been prepared and studied in this work. Self-supported sheets of fibrous cerium(IV) hydrogenphosphate (CePf) were used as the inorganic matrix, since the fibrous morphology presented by this material makes molding possible, giving a desired shape to the final polypyrrole/ CePf conducting nanocomposites. The influence of pyrrole concentration on the nanocomposite preparation was studied, as well as the influence of CePf on the polypyrrole characteristics. XRD, Raman, SEM and conductivity measurement data show the conducting polypyrrole coating CePf fibers, yielding self-supported sheets of organic-inorganic conducting nanocomposites.1361378138