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

Severe Thyroid-associated Ophtalmopathy and Hashimoto´s Thyroiditis in Euthyroid Patient

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Leishmania (Viannia) spp. dissemination and tissue tropism in naturally infected dogs (Canis familiaris).

First evidence is presented for Leishmania (Viannia) spp. dissemination and tissue tropism in the domestic dog. Using PCR and histology, parasites were detected in the conjunctiva, lung, lymph nodes and ovaries of 2 naturally infected Peruvian dogs. The detection of parasites in the blood indicates that parasite dissemination to those organs may have been haematogenous

Assessment of fungal contamination in a Portuguese maternity unit

A descriptive study was developed to monitor air fungal contamination in one Portuguese maternity. Sixty air samples were collected through impaction method. Air sampling was performed in food storage facilities, kitchen, food plating, canteen, pharmacy, sterilization areas, genecology wards, intensive care unit, operating rooms, urgency and also, outside premises, since this was the place regarded as reference. Besides air samples, forty three samples were collected by swabbing the surfaces using a 10 by 10 cm square stencil. Simultaneously, temperature, relative humidity and particles counting (PM10) were registered. Twenty three species of fungi were identified in air, being the two most commonly isolated the genera Penicillium (41,5%) and Cladosporium (28,4%). Regarding yeasts, only Rhodotorula sp. (45,2%), Trichosporon mucoides (51,6%) and Cryptococcus neoformans (3,2%) were found. Thirteen species of fungi were identified in surfaces, being the most frequent the Penicillium genus (91,6%). Concerning yeasts found in surfaces, four species were identified being Rhodotorula sp. (29,1%) the most frequent. There was no coincidence between prevailing genera indoors and outside premises. Moreover, some places presented fungal species different from the ones isolated outside. In the inside environment, Aspergillus species were isolated in air and surfaces. There was no significant relationship (p>0,05) between fungal contamination and the studied environmental variables. Keywords: air, surfaces, fungal contamination, environmental variables, maternity

Light-cone averages in a swiss-cheese universe

We analyze a toy swiss-cheese cosmological model to study the averaging problem. In our model, the cheese is the EdS model and the holes are constructed from a LTB solution. We study the propagation of photons in the swiss-cheese model, and find a phenomenological homogeneous model to describe observables. Following a fitting procedure based on light-cone averages, we find that the the expansion scalar is unaffected by the inhomogeneities. This is because of spherical symmetry. However, the light-cone average of the density as a function of redshift is affected by inhomogeneities. The effect arises because, as the universe evolves, a photon spends more and more time in the (large) voids than in the (thin) high-density structures. The phenomenological homogeneous model describing the light-cone average of the density is similar to the concordance model. Although the sole source in the swiss-cheese model is matter, the phenomenological homogeneous model behaves as if it has a dark-energy component. Finally, we study how the equation of state of the phenomenological model depends on the size of the inhomogeneities, and find that the equation-of-state parameters w_0 and w_a follow a power-law dependence with a scaling exponent equal to unity. That is, the equation of state depends linearly on the distance the photon travels through voids. We conclude that within our toy model, the holes must have a present size of about 250 Mpc to be able to mimic the concordance model.Comment: 20 pages, 14 figures; replaced to fit the version accepted for publication in Phys. Rev.

Pricing and Hedging Asian Basket Options with Quasi-Monte Carlo Simulations

In this article we consider the problem of pricing and hedging high-dimensional Asian basket options by Quasi-Monte Carlo simulation. We assume a Black-Scholes market with time-dependent volatilities and show how to compute the deltas by the aid of the Malliavin Calculus, extending the procedure employed by Montero and Kohatsu-Higa (2003). Efficient path-generation algorithms, such as Linear Transformation and Principal Component Analysis, exhibit a high computational cost in a market with time-dependent volatilities. We present a new and fast Cholesky algorithm for block matrices that makes the Linear Transformation even more convenient. Moreover, we propose a new-path generation technique based on a Kronecker Product Approximation. This construction returns the same accuracy of the Linear Transformation used for the computation of the deltas and the prices in the case of correlated asset returns while requiring a lower computational time. All these techniques can be easily employed for stochastic volatility models based on the mixture of multi-dimensional dynamics introduced by Brigo et al. (2004).Comment: 16 page

Analysis of tomato glycoalkaloids by liquid chromatography coupled with electrospray ionization tandem mass spectrometry.

Steroidal glycoalkaloids (SGAs) extracted from tomato leaves and berries (Lycopersicon esculentum Mill.) were separated and identified using optimized reversed-phase liquid chromatography with electrospray ionization (ESI) and ion trap mass spectrometry (ITMS). The ESI source polarity and chromatographic conditions were evaluated. The ESI spectra contain valuable information, which includes the mass of SGAs, the mass of the aglycones, and several characteristic fragment ions. Cleavage at the interglycosidic bonds proximal to the aglycones is the most prominent process in the ESI process. A protonated molecule, [M+H]+, accompanied by a mixed adduct ion, [M+H+Na]2+, was observed for a-tomatine (i.e., m/z 1034.7 and 528.9) and dehydrotomatine (i.e., m/z 1032.6 and 527.9) in positive ion mode spectra. The structures of these tomato glycoalkaloids were confirmed using tandem mass spectrometry. The identification of a new a-tomatine isomer glycoalkaloid, named filotomatine (MW 1033), which shares a common tetrasaccharide structure (i.e., lycotretraose) with a-tomatine and dehydrotomatine, and soladulcidine as an aglycone, is described for the first time. It occurs in significant amounts in the extracts of wild tomato foliage. Multistage mass spectrometry both of the protonated molecules and of the doubly charged ions was used for detailed structural elucidation of SGAs. Key fragmentations and regularities in fragmentation pathways are described and the fragmentation mechanisms involved are propose