3,550 research outputs found
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
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
Molecular biology versus conventional methods – Complementary methodologies to understand occupational exposure to fungi
Contribuição para o controlo da infeção nosocomial de Candida parapsilosis por tipagem com marcadores de DNA microssatélite
Fungal infections constitute a relevant problem in hospitals from all over the world. Their easy
transmission through the air, water, or by direct or indirect contact with contaminated persons,
surfaces or objects lead to an increasing preoccupation with those infections, particularly in
immunocompromised patients.
Regarding the enormous importance of Candida parapsilosis as agent of bloodstream infections
and hospital outbreaks, the presented work aimed to contribute to the development of new
molecular tools able to answer to epidemiological questions related with infections by this species
and their application in favor of patient safetyAs infecções fúngicas constituem um problema hospitalar relevante em todo o mundo. A fácil
transmissão dos fungos pelo ar, água ou pelo contato direto ou indireto com pessoas, superfícies
ou objetos contaminados conduz a uma crescente preocupação com este tipo de infecções,
principalmente em pacientes imunocomprometidos.
Considerando a grande relevância da levedura Candida parapsilosis em Portugal como agente
etiológico de infecções sistémicas e de surtos hospitalares, o trabalho apresentado teve como
objetivo contribuir para o desenvolvimento de novas ferramentas moleculares que permitissem
responder a questões epidemiológicas relacionadas com esta espécie e posterior aplicação desta
ferramenta em prol da segurança do paciente
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
Ventilation influence in occupational exposure to fungi and volatile organic compounds: poultry case
Introduction - In poultry houses, large-scale production has led to increased bird densities within buildings. Such high densities of animals kept within confined spaces are a source of human health problems related to occupational organic dust exposure. This organic dust is composed of both non-viable particles and viable particulate matter (also called bioaerosols). Bioaerosols are comprised by airborne bacteria, fungi, viruses and their by-products, endotoxins and mycotoxins.
Exposure to fungi in broiler houses may vary depending upon the applied ventilation system. Ventilation can be an important resource in order to reduce air contamination in these type of settings. Nevertheless, some concerns regarding costs, sensitivity of the animal species to temperature differences, and also the type of building used define which type of ventilation is used. Aim of the study - A descriptive study was developed in one poultry unit aiming to assess occupational fungal and volatile organic compounds (VOCs) exposure
The burden of serious fungal infections in Portugal
Using published data, we were able to estimate the incidence or prevalence of the above referred fungal infections and ~194 293 (1.8%) people in Portugal suffer from those fungal infections each year
Surveillance of environmental fungi, with focus on Aspergillus, in a Portuguese Central Hospital.
Objectives: Because immunocompromised patients are more prone to acquire nosocomial infections caused by fungi isolated from the environment, e.g. Aspergillus, this study aimed to screen the hospital environment for the presence of fungi and to understand their epidemiology in the different hospital wards analyzed.
Methods: During one-year period, four seasonal samplings, i.e., air and hard surface, were performed. A total of 101 air samples and 99 surface samples were collected from the Hematology, Oncology, and Intensive Care Unit (ICU) wards of a Portuguese Central Hospital. Aspergillus isolates were plated for growth as single colonies on malt extract agar with chloramphenicol to check the colony purity and observe colonial morphology. The universal fungal primers ITS1 and ITS4 were used to amplify DNA from all Aspergillus isolates, amplimers were sequenced, and isolates identified to the species-complex level. Statistical analyses were done using SPSS v15.0 program for Windows.
Results: Aspergillus was the most frequently recovered fungal genus (20.9%), followed by Cladosporium (18.7%), and Penicillium (17.2%). Thirty-five Aspergillus isolates were collected from the wards with hematological patients (bone marrow transplant and hemato-oncology wards), whereas 15 isolates were recovered from ICU. Among Aspergillus isolates from the hospital environment, those belonging to the species-complexes of versicolores (n = 26; 32.5%), nigri (n = 12; 15.0%), flavi (n = 11; 13.7%), and circumdati (n = 6; 7.5%) dominated. Hemato-Oncology was the ward with higher fungal counts, whereas the bone marrow transplant ward, which is protected by HEPA-filtration of the supply air, showed the lowest numbers in all sampling periods. A significant association (p = 0.001) was found between the season and the Aspergillus complexes isolated, with spring and summer having a larger number of different species-complexes detected in the hospital´s air and on the surfaces. Nevertheless, air counts showed that the autumn was the season with the highest proportion of Aspergillus (one third of the total number of fungi detected). This could be due in part to the presence of construction work near these wards.
Conclusion: The knowledge of the epidemiology of environmental fungi in each hospital may allow the establishment of preventive or corrective measures to decrease nosocomial fungal infections
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