Fungal contamination in two Portuguese wastewater treatment plants

The presence of filamentous fungi was detected in wastewater and air collected at wastewater treatment plants (WWTP) from several European countries. The aim of the present study was to assess fungal contamination in two WWTP operating in Lisbon. In addition, particulate matter (PM) contamination data was analyzed. To apply conventional methods, air samples from the two plants were collected through impaction using an air sampler with a velocity air rate of 140 L/min. Surfaces samples were collected by swabbing the surfaces of the same indoor sites. All collected samples were incubated at 27��C for 5 to 7 d. After lab processing and incubation of collected samples, quantitative and qualitative results were obtained with identification of the isolated fungal species. For molecular methods, air samples of 250 L were also collected using the impinger method at 300 L/min airflow rate. Samples were collected into 10 ml sterile phosphate-buffered saline with 0.05% Triton X-100, and the collection liquid was subsequently used for DNA extraction. Molecular identification of Aspergillus fumigatus and Stachybotrys chartarum was achieved by real-time polymerase chain reaction (RT-PCR) using the Rotor-Gene 6000 qPCR Detection System (Corbett). Assessment of PM was also conducted with portable direct-reading equipment (Lighthouse, model 3016 IAQ). Particles concentration measurement was performed at five different sizes: PM0.5, PM1, PM2.5, PM5, and PM10. Sixteen different fungal species were detected in indoor air in a total of 5400 isolates in both plants. Penicillium sp. was the most frequently isolated fungal genus (58.9%), followed by Aspergillus sp. (21.2%) and Acremonium sp. (8.2%), in the total underground area. In a partially underground plant, Penicillium sp. (39.5%) was also the most frequently isolated, also followed by Aspergillus sp. (38.7%) and Acremonium sp. (9.7%). Using RT-PCR, only A. fumigatus was detected in air samples collected, and only from partial underground plant. Stachybotrys chartarum was not detected in any of the samples analyzed. The distribution of particle sizes showed the same tendency in both plants; however, the partially underground plant presented higher levels of contamination, except for PM2.5. Fungal contamination assessment is crucial to evaluating the potential health risks to exposed workers in these settings. In order to achieve an evaluation of potential health risks to exposed workers, it is essential to combine conventional and molecular methods for fungal detection. Protective measures to minimize worker exposure to fungi need to be adopted since wastewater is the predominant internal fungal source in this setting.

Assessment of fungal contamination in waste sorting and incineration: case study in Portugal

Organic waste is a rich substrate for microbial growth, and because of that, workers from waste industry are at higher risk of exposure to bioaerosols. This study aimed to assess fungal contamination in two plants handling solid waste management. Air samples from the two plants were collected through an impaction method. Surface samples were also collected by swabbing surfaces of the same indoor sites. All collected samples were incubated at 27���C for 5 to 7 d. After lab processing and incubation of collected samples, quantitative and qualitative results were obtained with identification of the isolated fungal species. Air samples were also subjected to molecular methods by real-time polymerase chain reaction (RT PCR) using an impinger method to measure DNA of Aspergillus flavus complex and Stachybotrys chartarum. Assessment of particulate matter (PM) was also conducted with portable direct-reading equipment. Particles concentration measurement was performed at five different sizes (PM0.5; PM1; PM2.5; PM5; PM10). With respect to the waste sorting plant, three species more frequently isolated in air and surfaces were A. niger (73.9%; 66.1%), A. fumigatus (16%; 13.8%), and A. flavus (8.7%; 14.2%). In the incineration plant, the most prevalent species detected in air samples were Penicillium sp. (62.9%), A. fumigatus (18%), and A. flavus (6%), while the most frequently isolated in surface samples were Penicillium sp. (57.5%), A. fumigatus (22.3%) and A. niger (12.8%). Stachybotrys chartarum and other toxinogenic strains from A. flavus complex were not detected. The most common PM sizes obtained were the PM10 and PM5 (inhalable fraction). Since waste is the main internal fungal source in the analyzed settings, preventive and protective measures need to be maintained to avoid worker exposure to fungi and their metabolites.

Fungal contamination assessment in Portuguese elderly care centers

Individuals spend 80-90% of their day indoors and elderly subjects are likely to spend even a greater amount of time indoors. Thus, indoor air pollutants such as bioaerosols may exert a significant impact on this age group. The aim of this study was to characterize fungal contamination within Portuguese elderly care centers. Fungi were measured using conventional as well as molecular methods in bedrooms, living rooms, canteens, storage areas, and outdoors. Bioaerosols were evaluated before and after the microenvironments' occupancy in order to understand the role played by occupancy in fungal contamination. Fungal load results varied from 32 colony-forming units CFU m(-3) in bedrooms to 228 CFU m(-3) in storage areas. Penicillium sp. was the most frequently isolated (38.1%), followed by Aspergillus sp. (16.3%) and Chrysonilia sp. (4.2%). With respect to Aspergillus genus, three different fungal species in indoor air were detected, with A. candidus (62.5%) the most prevalent. On surfaces, 40 different fungal species were isolated and the most frequent was Penicillium sp. (22.2%), followed by Aspergillus sp. (17.3%). Real-time polymerase chain reaction did not detect the presence of A. fumigatus complex. Species from Penicillium and Aspergillus genera were the most abundant in air and surfaces. The species A. fumigatus was present in 12.5% of all indoor microenvironments assessed. The living room was the indoor microenvironment with lowest fungal concentration and the storage area was highest.

Potential pathogenic fungi assessment through molecular biology in cork industry

Portugal has been the world leader in the cork sector in terms of exports, employing ten thousands of workers. In this working activity, the permanent contact with cork may lead to the exposure to fungi, raising concerns as potential occupational hazards in cork industry. The application of molecular tools is crucial in this setting, since fungal species with faster growth rates may hide other species with clinical relevance, such as species belonging to P. glabrum and A. fumigatus complexes. A study was developed aiming at assessing fungal contamination due to Aspergillus fumigatus complex and Penicillium glabrum complex by molecular methods in three cork industries in the outskirt of Lisbon city.

Avalia����o da exposi����o a fungos e part��culas em explora����es av��colas e suin��colas: estudo

O projeto ���Avalia����o da Exposi����o a Fungos e Part��culas em Explora����es Av��colas e Suin��colas��� contemplou um elevado n��mero de colheitas ambientais e biol��gicas e respectivo processamento laboratorial, sendo apenas poss��vel a sua concretiza����o gra��as ao financiamento disponibilizado pela Autoridade para as Condi����es de Trabalho. Foi realizado um estudo transversal para avaliar a contamina����o causada por fungos e part��culas em 7 explora����es av��colas e 7 explora����es suin��colas. No que concerne �� monitoriza����o biol��gica, foram medidos os par��metros espirom��tricos, utilizando o espir��metro MK8 Microlab, avaliada a exist��ncia de sintomas cl��nicos associados com a asma e outras doen��as al��rgicas, atrav��s de question��rio adaptado European Community Respiratory Health Survey e, ainda, avaliada a sensibiliza����o aos agentes f��ngicos (IgE). Foram ainda adicionados dois objetivos ao estudo, designadamente: aferir a exist��ncia de tr��s esp��cies/estirpes potencialmente patog��nicas/toxinog��nicas com recurso �� biologia molecular e avaliar a exposi����o dos trabalhadores �� micotoxina aflatoxina B1 por recurso a indicador biol��gico de exposi����o. Foram colhidas 27 amostras de ar de 25 litros nas explora����es av��colas e 56 de 50 litros nas explora����es suin��colas atrav��s do m��todo de impacto. As colheitas de ar e a medi����o da concentra����o das part��culas foram realizadas no interior e no exterior dos pavilh��es, sendo este ��ltimo considerado como local de refer��ncia. Simultaneamente, a temperatura e a humidade relativa tamb��m foram registadas. As colheitas das superf��cies foram realizadas atrav��s da t��cnica de zaragatoa, tendo sido utilizado um quadrado de metal inoxid��vel de 10 cm de lado, de acordo com a International Standard ISO 18593 ��� 2004. As zaragatoas obtidas (20 das explora����es av��colas e 48 das explora����es suin��colas) foram inoculadas em malte de extract agar (2%) com cloranfenicol (0,05 g/L). Al��m das colheitas de ar e de superf��cies, foram tamb��m obtidas colheitas da cama das explora����es av��colas (7 novas e 14 usadas) e da cobertura do pavimento das explora����es suin��colas (3 novas e 4 usadas) e embaladas em sacos esterilizados. Cada amostra foi dilu��da e inoculada em placas contendo malte extract agar. Todas as amostras foram incubadas a 27,5��C durante 5 a 7 dias e obtidos resultados quantitativos (UFC/m3; UFC/m2; UFC/g) e qualitativos com a identifica����o das esp��cies f��ngicas. Para a aplica����o dos m��todos de biologia molecular foram realizadas colheitas de ar de 300 litros utilizando o m��todo de impinger com a velocidade de recolha de 300 L/min. A identifica����o molecular de tr��s esp��cies potencialmente patog��nicas e/ou toxinog��nicas (Aspergillus flavus, Aspergillus fumigatus e Stachybotrys chartarum) foram obtidas por PCR em tempo real (PCR TR) utilizando o Rotor-Gene 6000 qPCR Detection System. As medi����es de part��culas foram realizadas por recurso a equipamento de leitura direta (modelo Lighthouse, 2016 IAQ). Este recurso permitiu medir a concentra����o (mg/m3) de part��culas em 5 dimens��es distintas (PM 0.5; PM 1.0; PM 2.5; PM 5.0; PM10). Nas explora����es av��colas, 28 esp��cies/g��neros de fungos foram isolados no ar, tendo Aspergillus versicolor sido a esp��cie mais frequente (20.9%), seguida por Scopulariopsis brevicaulis (17.0%) e Penicillium sp. (14.1%). Entre o g��nero Aspergillus, Aspergillus flavus apresentou o maior n��mero de esporos (>2000 UFC/m3). Em rela����o ��s superf��cies, A. versicolor foi detetada em maior n��mero (>3 �� 10���2 UFC/m2). Na cama nova, Penicillium foi o g��nero mais frequente (59,9%), seguido por Alternaria (17,8%), Cladosporium (7,1%) e Aspergillus (5,7%). Na cama usada, Penicillium sp. foi o mais frequente (42,3%), seguido por Scopulariopsis sp. (38,3%), Trichosporon sp. (8,8%) e Aspergillus sp. (5,5%). Em rela����o �� contamina����o por part��culas, as part��culas com maior dimens��o foram detectadas em maiores concentra����es, designadamente as PM5.0 (part��culas com a dimens��o de 5.0 bm ou menos) e PM10 (part��culas com a dimens��o de 10 bm ou menos). Neste setting a preval��ncia da altera����o ventilat��ria obstrutiva foi superior nos indiv��duos com maior tempo de exposi����o (31,7%) independentemente de serem fumadores (17,1%) ou n��o fumadores (14,6%). Relativamente �� avalia����o do IgE espec��fico, foi apenas realizado em trabalhadores das explora����es av��colas (14 mulheres e 33 homens), n��o tendo sido encontrada associa����o positiva (p<0.05%) entre a contamina����o f��ngica e a sensibiliza����o a antig��nios f��ngicos. No caso das explora����es suin��colas, Aspergillus versicolor foi a esp��cie mais frequente (20,9%), seguida por Scopulariopsis brevicaulis (17,0%) e Penicillium sp. (14,1%). No g��nero Aspergillus, A. versicolor apresentou o maior isolamento no ar (>2000 UFC/m3) e a maior preval��ncia (41,9%), seguida por A. flavus e A. fumigatus (8,1%). Em rela����o ��s superf��cies analisadas, A. versicolor foi detetada em maior n��mero (>3 ��10���2 UFC/m2). No caso da cobertura do pavimento das explora����es suin��colas, o g��nero Thicoderma foi o mais frequente na cobertura nova (28,0%) seguida por A. versicolor e Acremonium sp. (14,0%). O g��nero Mucor foi o mais frequente na cobertura usada (25,1%), seguido por Trichoderma sp. (18,3%) e Acremonium sp. (11,2%). Relativamente ��s part��culas, foram evidenciados tamb��m valores mais elevados na dimens��o PM5 e, predominantes nas PM10. Neste contexto, apenas 4 participantes (22,2%) apresentaram uma altera����o ventilat��ria obstrutiva. Destes, as obstru����es mais graves encontraram-se nos que tamb��m apresentavam maior tempo de exposi����o. A preval��ncia de asma na amostra de trabalhadores em estudo, pertencentes aos 2 contextos em estudo, foi de 8,75%, tendo-se verificado tamb��m uma preval��ncia elevada de sintomatologia respirat��ria em profissionais n��o asm��ticos. Em rela����o �� utiliza����o complementar dos m��todos convencionais e moleculares, �� recomend��vel que a avalia����o da contamina����o f��ngica nestes settings, e, consequentemente, a exposi����o profissional a fungos, seja suportada pelas duas metodologias e, ainda, que ocorre exposi����o ocupacional �� micotoxina aflatoxina B1 em ambos os contextos profissionais. Face aos resultados obtidos, �� importante salientar que os settings alvo de estudo carecem de uma interven����o integrada em Sa��de Ocupacional no ��mbito da vigil��ncia ambiental e da vigil��ncia da sa��de, com o objetivo de diminuir a exposi����o aos dois factores de risco estudados (fungos e part��culas).

Fungal burden in waste industry: an occupational risk to be solved

High loads of fungi have been reported in different types of waste management plants. This study intends to assess fungal contamination in one waste-sorting plant before and after cleaning procedures in order to analyze their effectiveness. Air samples of 50 L were collected through an impaction method, while surface samples, taken at the same time, were collected by the swabbing method and subject to further macro- and microscopic observations. In addition, we collected air samples of 250 L using the impinger Coriolis �� air sampler (Bertin Technologies) at 300 L/min airflow rate in order to perform real-time quantitative PCR (qPCR) amplification of genes from specific fungal species, namely Aspergillus fumigatus and Aspergillus flavus complexes, as well as Stachybotrys chartarum species. Fungal quantification in the air ranged from 180 to 5,280 CFU m���3 before cleaning and from 220 to 2,460 CFU m���3 after cleaning procedures. Surfaces presented results that ranged from 29��������104 to 109��������104 CFU m���2 before cleaning and from 11��������104 to 89��������104 CFU m���2 after cleaning. Statistically significant differences regarding fungal load were not detected between before and after cleaning procedures. Toxigenic strains from A. flavus complex and S. chartarum were not detected by qPCR. Conversely, the A. fumigatus species was successfully detected by qPCR and interestingly it was amplified in two samples where no detection by conventional methods was observed. Overall, these results reveal the inefficacy of the cleaning procedures and that it is important to determine fungal burden in order to carry out risk assessment.

Fungal infections in neutropenic patients: a 8-year prospective study

In this paper we report a eight-year prospective study designed to further characterize incidence, epidemiology, specific syndromes, treatment and prognosis associated with fungal infections in neutropenic patients. During the study period 30 fungal infections were diagnosed in 30 patients among 313 episodes of fever and neutropenia (10%). There were 15 cases of candidiasis, 5 pulmonary aspergillosis, 3 sinusitis by Aspergillus fumigatus, 5 infections by Fusarium sp., one infection by Trichosporon sp., and one infection due to Rhodotorula rubra. Blood cultures were positive in 18 cases (60%). The predisposing factors for fungal infection in multivariate analysis were the presence of central venous catheter (p<0.001), longer duration of profound (<100/mm��) neutropenia (p<0.001), the use of corticosteroids (p<0.001), gram-positive bacteremia (p=0.002) and younger age (p=0.03). In multivariate analysis only recovery of the neutropenia (p<0.001) was associated with good prognosis whereas the diagnosis of infection by Fusarium sp. (p=0.006) was strongly associated with a poor outcome. The death rate was 43%. There was no statistically significant difference in the death rate between patients who did receive (52%) or did not receive (50%) antifungal treatment. Identifying patients at risk, specific syndromes and prognostic factors may help to reduce the high mortality associated with disseminated fungal infections in neutropenic patients.

Chronic pulmonary histoplasmosis in Brazil: report of two cases with cavitation diagnosed by transthoracic needle biopsy

Two cases of Chronic Pulmonary Histoplasmosis are reported and other eleven cases, collected from Brazilian literature, are commented. After being clinically cured, one of our patients presented an Aspergillus fungus ball inside a cavitation in the wall of which H. capsulatum was disclosed. Comments are also done on the diagnosis of the mycosis

Fungus ball in HIV-infected patients

Aspergillus is a phagocyte opportunistic fungus that causes aspergillosis, an unusual disease in patients with AIDS. Six cases of fungal ball in patients with AIDS are reported here. In this group, all patients had hemoptysis and tuberculosis as the underlying lung disease. The diagnosis of pulmonary fungus ball was based on the clinical and radiographic feature, combined with serological and mycological evidence of Aspergillus fumigatus.

Microbiota f��ngica dos condicionadores de ar nas unidades de terapia intensiva de Teresina, PI

Com intuito de identificar a microbiota f��ngica em condicionadores de ar nas unidades de terapia intensiva de hospitais p��blicos e particulares de Teresina-PI, coletou-se material s��lido de dez UTIs, isolando 33 esp��cies pertencentes ��s Moniliaceae e Dematiaceae, sendo primeira refer��ncia para o Piau��. Registrou-se elevada freq����ncia de Aspergillus niger Van Tieghem (60%); Aspergillus fumigatus Fres (50%); Trichoderma koningii Oudem (50%), Aspergillus flavus Link: Fr (40%). A validade da limpeza dos condicionadores de ar ultrapassou em todas as UTIs, a quantidade de unidades formadoras de col��nia estava al��m do permitido pela Portaria 176/00 do Minist��rio da Sa��de. �� importante que os profissionais estejam munidos de equipamento de prote����o individual, al��m de adotar medidas de controle de infec����o hospitalar, sensibilizar para a exist��ncia de infec����es f��ngicas, melhorar ventila����o de ar, possibilitando arejamento do ambiente e limpar periodicamente os condicionadores de ar, conscientizando os profissionais de sa��de da import��ncia destes fungos no ambiente hospitalar.

Ensaios de micologia: contribui����o dos cogumelos patog��nicos em Minas Gerais

O autor diz que, de outubro de 1941 a dezembro de 1943, insulou numerosos fungos patog��nicos de les��es as mais variadas. Obteve culturas puras de 5 casos de Rinocladiose, 3 de Mol��stia de Lutz, apurou um Actinomyces minutissimus, 2 de Malassezia furfur e 2 Actinomyces, ainda em estudos, etc. Uma das observa����es de Esporotricose merece refer��ncia especial. Tratava-se de um indiv��duo, morador em Governador Valadares, que apresentava uma les��o gomosa, nodular e verruc��ide, fistulosa, localizada na regi��o umbilical, provocada pela dentada de um peixe, de esp��cie ignorada, que atacou o paciente quando ��ste se banhava, completamente despido, nas ��guas do Rio Doce. O ponto da dentada sangrou ligeiramente e pouco depois come��avam a aparecer os n��dulos, que se sucederam progressivamente sem perturbar o estado geral do paciente. As sementeiras do pus de uma dessas gomas deram culturas puras do Rhinocladium Beurmanni, ��nica esp��cie que o autor at�� hoje insulou, em 55 casos de Esporotricose em Minas Gerais. O autor j�� havia registrado a mol��stia pela dentada de ratos, pelos ferimentos de espinhos de roseiras, peda��os de madeira, facas de cozinha, marteladas etc. Pensa, por��m, que a mol��stia produzida pela dentada de peixe �� a primeira vez que se faz refer��ncia na literatura. Referiu-se depois o autor aos casos de Otomicoses que lhe chegaram ��s m��os em 1942, no servi��o do Prof. ILDEU DUARTE. D�� o resumo das observa����es e concluiu que se tratava de les��es produzidas pelo Aspergillus fumigatus de FRESENIUS, 1841, de ac��rdo com as les��es histopatol��gicas e as culturas que obteve.

Observa����es sobre aspergilose de passaros

Os autores descrevem a observa����o de dois casos de aspergilose observados em "gaturamo" (Tanagra violacea), com localiza����o nos sacos a��reos. De ambos os casos foi isolado o Aspergillus fumigatus que se mostrou patogenico para cobaios, coelhos e principalmente para pombos. A morte desses animais ocorria ap��s um periodo variavel, dependendo da via de inocula����o, sendo que em todos os casos foram obtidas retroculturas.

Donor-derived aspergillosis from use of a solid organ recipient as a multiorgan donor.

The growing need for organs and the scarcity of donors has resulted in an increased use of extended criteria donors. We report a case where a recipient of a cardiac graft was used as an organ donor. Death of the recipient occurred 9 days after transplantation and was attributed to presumed cerebral hemorrhage, which post mortem was diagnosed as invasive aspergillosis of the brain. One recipient of a kidney transplant lost the graft due to infection with Aspergillus fumigatus, whereas prompt initiation of therapy successfully prevented disseminated aspergillosis in the other recipients. Despite the pressure to extend the use of organs by lowering the acceptance criteria, organs should only be accepted if the cause of death of the donors is unequivocally explained.

Surgery and granulocyte transfusions for life-threatening infections in chronic granulomatous disease.

We report two patients with chronic granulomatous disease (CGD) and life-threatening infections: a 10 10/12-year-old boy had Aspergillus fumigatus spondylitis with destruction of the 11th vertebral body and paravertebral abscess formation, and an 8 5/12-year-old boy had multiple Staphylococcus aureus hepatic abscesses with subphrenic abscess formation. Both patients failed to respond to intense antimicrobial therapy but showed a remarkable recovery following surgical drainage combined with granulocyte transfusions. These results suggest that antimicrobial therapy and surgical drainage followed by granulocyte transfusions may be the ideal mode of treatment for severe infections in patients with CGD.

Ost��og��n��se atypique apr��s s��questrectomies multiples pour fracture ouverte infect��e du f��mur: un cas [Atypical osteogenesis following multiple sequestrectomies for infected open fracture of the femur: a case]

The authors present the case of an open fracture of femur Cauchoix type II with an infection due to Escherichia coli, Clostridium perfringens, Enterococcus and Aspergillus fumigatus. After several sequestrectomies and five hyperbaric sessions, apyrexia was attained at the end of the third month, the femur having been stabilised with an external fixator. The 15 cm gap due to loss of bone substance, filled at each dressing with an antiseptic iodine based ointment, closed itself finishing as continuous bone five months after the accident, the granulation tissue having been recovered by thin skin grafts. A repeated fracture occurring forty-eight hours after the removal of the Hoffmann frame was treated by fitting an Ilizarov fixator arriving at consolidation in seven months. The authors examine different possibilities of accelerating osteogenesis and highlight the potential role of iodine ointment as inductive to osteogenesis stemming from a periosteal layer seemingly held in place.