Browsing by Author "Verissimo, C."
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- Air contaminants in animal production – poultry casePublication . Viegas, C.; Viegas, S.; Monteiro, A.; Carolino, E.; Sabino, R.; Verissimo, C.A descriptive study was developed in order to assess air contamination caused by fungi and particles in seven poultry units. Twenty seven air samples of 25 litters were collected through impaction method. Air sampling and particle concentration measurement were performed in the pavilions’ interior and also outside premises, since this was the place regarded as reference. Simultaneously, temperature and relative humidity were also registered. Regarding fungal load in the air from the seven poultry farms, the highest value obtained was 24040 CFU/m3 and the lowest was 320 CFU/m3. Twenty eight species/genera of fungi were identified, being Scopulariopsis brevicaulis (39.0%) the most commonly isolated species and Rhizopus sp. (30.0%) the most commonly isolated genus. From the Aspergillus genus, Aspergillus flavus (74.5%) was the most frequently detected species. There was a significant correlation (r=0.487; p=0.014) between temperature and the level of fungal contamination (CFU/m3). Considering contamination caused by particles, in this study, particles with larger dimensions (PM5.0 and PM10) have higher concentrations. There was also a significant correlation between relative humidity and concentration of smaller particles namely, PM0.5 (r=0.438; p=0.025) and PM1.0 (r=0.537; p=0.005). Characterizing typical exposure levels to these contaminants in this specific occupational setting is required to allow a more detailed risk assessment analysis and to set exposure limits to protect workers’ health.
- Comparison of fungal contamination between hospitals and companies food unitsPublication . Viegas, C.; Ramos, C.; Almeida, M.; Sabino, R.; Verissimo, C.; Rosado, L.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.
- Comparison of indoor and outdoor fungi and particles in poultry unitsPublication . Viegas, C.; Viegas, S.; Monteiro, A.; Carolino, E.; Sabino, R.; Verissimo, C.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.
- Dermatophytes’ identification by Matrix-assisted laser desorption ionization-time of flight mass spectrometry. (MALDI-TOF MS) - the experience of a clinical laboratoryPublication . Verissimo, C.; Simões, H.; Sabino, R.; Simões, D.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.
- Frequency and molecular epidemiology of Aspergillus isolated from patients with suspicion of respiratory fungal infectionPublication . Oliveira, M.; Simões, H.; Verissimo, C.; Sabino, R.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.
- Imported African histoplasmosis by Histoplasma capsulatum var. duboisii in an HIV-2 infected patientPublication . Toscano, C.; Batista, J.; Carvalho, R.; Espírito-Santo, C.E.; Marcos, R.; Sabino, R.; Verissimo, C.; Viana, I.; Marques, T.Objectives: African histoplasmosis caused by the fungus Histoplasma capsulatum var. duboisii, is a rare endemic mycosis occuring in western and central regions of sub-Saharian Africa. For unknown reasons, although HIV infection and H. capsulatum var. duboisii coexist in Africa, this coinfection remains rare. In Europe, diagnosed cases of African histoplasmosis are all imported. We describe a case of African histoplasmosis on a Portuguese war veteran co-infected with HIV-2 who fought in Guiné-Bissau in 1963-65 and Angola in 1972. Methods: We report a case of a 76-year-old man, diagnosed with HIV-2 infection in the previous year (under combined antiretroviral therapy) presenting an ulcerated skin lesion on the right tight (image 1), just above the knee. He was diagnosed pulmonary tuberculosis the year before and was finishing one year treatment. The solitary skin lesion begun as a small non-pruriginous eritematous papule, evolving in 6 month to a painless 3-4cm ulcer with raised borders surrounded by a hiperpigmented halo. There were no adenopathies or bone lesions. Respiratory samples and blood cultures were systematically negative for Histoplasma capsulatum. He was treated with IV liposomal Amphotericin-B for one month, followed by oral itraconazol (now on the first month), with a favourable clinical outcome. Results: Histopathology of skin biopsy revealed a superficial ulceration with underlying granulomatous infiltrate with many giant cells, where numerous round mononucleated yeasts measuring 7-8µm were evident and highlighted with PAS and Grocott stain (image 2, left side). Skin biopsy was observed on a wet mount with KOH and revealed numerous round yeasts that were also seen on Gram stain, measuring 7-8µm. Culture of skin biopsy on two Sabouraud dextrose agar (with and without cicloheximide) showed growth of a filamentous fungus compatible with Histoplasma capsulatum (image 2, right side), with large thick-walled spherical macroconidia with finger-like projections (tuberculate conidia) that arise from short conidiophores, and small oval microconidia arising on short stalks from undifferentiated hyphae. Reversion to the yeast fase has not been succeed yet. Identification was further confirmed by sequencing of genomic DNA fragments using the universal fungal primers ITS1 and ITS4. The sequences obtained were compared with sequences deposited in the GenBank and the result was: Histoplasma capsulatum var. duboisii (99% homology). Conclusion: With banalization of business or leisure trips, endemic mycosis are becoming frequently diagnosed in countries outside their natural geographic endemic areas and only a high index of suspicion makes the diagnosis possible. Apart from trips, nowadays in Portugal 4% of the resident population is immigrant, mostly from Brasil (25.5%) but also from Angola and Guiné-Bissau (9.2%), being the former an endemic country of American histoplasmosis and the later of both American and African histoplasmosis. We consider histoplasmosis a probable underdiagnosed disease that should be suspected mainly in immunodeficient HIV positive individuals with a past history of travel or residence in an endemic area.
- Majocchi’s Granuloma by Trichophytum rubrum in a kidney transplant patient - A case reportPublication . Matos Cruz, S.; Silva, L.; Catorze, G.; Sabino, R.; Verissimo, C.; Toscano, C.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.
- Molecular diagnosis of invasive aspergillosis in a child with clinical suspicion of mucormycosisPublication . Sabino, Raquel; Ramos, A.; Simões, H.; Palaré, M.J.; Moreno, R.; Ferrão, A.; Lourenço, F.; Marques, J.G.; Martins, C.; Morais, A.; Verissimo, C.Purpose: Invasive aspergillosis is difficult to manage in immunocompromised patients. We present a clinical case of a 13-year-old boy from Cabo Verde who was transferred to the Hematology Unit of a Central Hospital in Lisbon, Portugal, and diagnosed with aplastic anemia. Weeks after hospital admission, in June 2016, he presented febrile neutropenia with negative blood cultures. In August, the patient showed a necrotic lesion of the left wing of the nose. A CT scan of the sinuses showed densification and thickening of the soft tissues of the nasal pyramid, protruding into the nose. A rapid and accurate diagnosis was required since the patient was going to be subjected to bone marrow transplantation. Methods: A biopsy of the nose tissue was performed in the hospital (August 2016) and sent to the Mycology Reference Laboratory of the Portuguese NIH. Tissue fragments were sliced in small fragments and inoculated in Sabouraud dextrose agar and brain heart infusion. Samples were incubated at 30º and 35ºC. In parallel to culture, DNA was extracted from the tissue using the High Pure PCR Template Preparation Kit (Roche Diagnostics Corp., Indianapolis, IN, USA), according to the manufacturer’s instructions. A panfungal PCR reaction was performed in order to detect any fungal DNA present in the tissue sample. For that purpose, the universal fungal primers ITS1 and ITS2 were used. Positive PCR products are then sequenced by Sanger method. A specific PCR directed to Aspergillus was performed using the AsperGenius® multiplex real-time PCR assay (PathoNostics, Maastricht, The Netherlands), following the manufacturer's instructions. The detection of mutations in the Cyp51A gene for A. fumigatus conferring azole resistance was also performed. Results: A positive panfungal PCR was obtained and PCR products were sent to sequencing. Due to the urgency of the case, a real time PCR directed to Aspergillus was also performed directly from the DNA extracted from the biopsy. A positive signal was obtained for Aspergillus fumigatus and no mutations in Cyp51A gene were detected. Sequencing results revealed 100% homology with A. fumigatus sensu stricto. Results were immediately reported to the physician and posaconazole was administered with regression of the lesion. After 30 days, the cultures of the tissue remained negative. In September 2016, nodular lesions appeared in the patients’ lower limbs and voriconazole therapy was then initiated. A complete regression was observed. Multiresistant Klebsiella pneumoniae and Enterobacter asburiae were further isolated from a perianal ulcer and in October 2016 the patient revealed positive bloodcultures of multiresistant Klebsiella pneumoniae. Facing difficulties in the infection control, a multidisciplinary team decided to perform allogeneic bone marrow transplantation, with complete hematological and infections recovery. Conclusion: An early diagnosis and prompt initiation of appropriate antifungal therapy are imperative and essential for a favorable clinical outcome. In this case, the necrotic lesions of the nose and patient’s risk factors conducted to the clinical suspicion of mucormycosis. The molecular approach performed led to the rapid identification of Aspergillus fumigatus and therefore to the adequate antifungal therapy of the patient.
- Molecular Epidemiology of Aspergillus collected from Cystic Fibrosis PatientsPublication . Sabino, R.; Ferreira, J.A.G.; Moss, R.B.; Valente, J.; Verissimo, C.; Carolino, E.; Clemons, K.V.; Everson, C.; Banaei, N.; Penner, J.; Stevens, D.A.Background: Aspergillus respiratory infection is a common complication in cystic fibrosis (CF) and is associated with loss of pulmonary function and allergic disease. Methods: Fifty-three Aspergillus isolates recovered fromCF patients were identified to species by Internal Transcribed Spacer Region (ITS), β-tubulin, and calmodulin sequencing. Results: Three species complexes (Terrei, Nigri, and Fumigati) were found. Identification to species level gave a single Aspergillus terreus sensu stricto, one Aspergillus niger sensu stricto and 51 Aspergillus fumigatus sensu stricto isolates. No cryptic species were found. Conclusions: To our knowledge, this is the first prospective study of Aspergillus species in CF using molecular methods. The paucity of non-A. fumigatus and of cryptic species of A. fumigatus suggests a special association of A. fumigatus sensu stricto with CF airways, indicating it likely displays unique characteristics making it suitable for chronic residence in that milieu. These findings could refine an epidemiologic and therapeutic approach geared to this pathogen
- Occupational exposure to aflatoxin b1 in swine production and possible contamination sourcesPublication . Viegas, S.; Veiga, L.; Figueiredo, P.; Almeida, A.; Carolino, E.; Sabino, R.; Verissimo, C.Although the adverse health consequences of ingestion of food contaminated with aflatoxin B1 (AFB1) are known, relatively few studies are available on the adverse effects of exposure in occupational settings. Taking this into consideration, our study was developed aiming to elucidate the possible effects of occupational exposure to AFB1 in Portuguese swine production facilities using a specific biomarker to assess exposure to AFB1. In total, 28 workers participated in this study, providing blood samples, and a control group (n = 30) was composed of subjects without any type of agricultural activity. Fungal contamination was also studied by conventional methods through air, surfaces, and new and used floor coverage. Twenty-one workers (75%) showed detectable levels of AFB1 with values ranging from <1 ng/ml to 8.94 ng/ml and with a mean value of 1.91 ± 1.68 ng/ml. In the control group, the AFB1 values were all below 1 ng/ml. Twelve different Aspergillus species were identified. Aspergillus versicolor presented the highest airborne spore counts (3210 CFU/m3) and was also detected in higher values in surfaces (>300 CFU/cm2). Data indicate that exposure to AFB1 occurs in swine barns, and this site serves as a contamination source in an occupational setting.