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- Assessment of azole resistance in clinical settings by passive samplingPublication . Aranha Caetano, Liliana; Almeida, Beatriz; Viegas, CarlaThe exposure to the fungal burden is an increased risk factor for the development of respiratory symptoms and fungal diseases. The emergence worldwide of azole resistance in fungal species is a major concern for public health. Clinical settings must provide a clean and safe environment to protect patients and staff from fungal diseases. The aim of this study was to assess the fungal burden and prevalence of azole resistance in clinical settings in Portugal using passive sampling methods. Ten Primary Health Care Centers were evaluated through passive sampling, including electrostatic dust cloths, heating ventilation, and air conditioning filters, and settled dust. All samples were plated onto Sabouraud dextrose agar media and screened for azole resistance using agar media supplemented with itraconazole, voriconazole and posaconazole through incubation at 27 °C, for 5 days. Fungal species were identified based on macro and micromorphology. Fungal load ranged from 348 to 424628 CFU·m−2 in EDC, 0 to 56500 CFU·m−2 in HVAC filter, and 2 to 514 CFU·g−1 in settled dust. EDC samples presented the highest fungal loads and wider diversity. Azole resistance was observed in all Primary Health Care Centers. Multi-azole resistance (fungal growth in two or more azoles) was observed in 90% units for Penicillium sp., C. sitophila, and Cladosporium sp. None of the samples containing Aspergillus sp. colonies presented resistance to the tested azoles. The knowledge of the fungal burden and prevalence of resistance to azole-based antifungal drugs in clinical environments will allow a better risk characterization regarding fungal burden.
- Aspergillus spp. prevalence in Primary Health Care Centres: assessment by a novel multi-approach sampling protocolPublication . Viegas, Carla; Almeida, Beatriz; Gomes, Anita Quintal; Carolino, Elisabete; Aranha Caetano, LilianaExposure to Aspergillus conidia may cause adverse effects on human health; however, no specific recommendations for routine assessments of Aspergillus in the clinical environment have been suggested so far. This study intended to determine the prevalence of Aspergillus in the clinical environment, focusing on ten Primary Health Care Centres (PHCC) through a novel multi-approach sampling protocol. Air and passive sampling, culture-based methods and a probe-based real-time assay for the detection of four clinically relevant Aspergillus sections were performed. Aspergillus spp. was observed in all PHCC, with highest prevalence on floor surface swabs (n=81) (18% on MEA; 6.94% on DG18). Regarding air samples (n=81), highest Aspergillus counts were found in the waiting room (94% MEA; 18% DG18), where Nigri was the most prevalent Aspergillus section. The use of a multi-approach sampling protocol to assess Aspergillus burden in the analyzed PHCC has greatly contributed to risk characterization, highlighting the need to implement corrective measures in order to avoid fungal presence in those settings.
- Effect of microbiome from waste sorting industry in THP-1 cell homeostasis and inflammatory responsesPublication . Viegas, Carla; Aranha Caetano, Liliana; Cox, Jennie; Korkalainen, Merja; Viegas, Susana; Reponen, TinnaOrganic dust and related microbial exposures are the main inducers of several respiratory symptoms and have been reported in diverse occupational settings. In vitro tests using relevant cell cultures can be useful for characterizing the toxicity of complex mixtures present in the air of occupational environments. In this study, cell viability was determined using the cell proliferation test WST-1 and inflammatory responses by ELISA assays measuring the production of proinflammatory cytokines TNFα and IL-1β. Human macrophages derived from THP-1 monocytic cells were exposed to extracts of 17 filters belonging to the filtration system from forklifts operating in one waste sorting industry located in the Lisbon region and one control filter. Additionally, bacterial and fungal diversity was assessed by RTL Genomics on an Illumina MiSeq with bacterial 16S 515F and 806R sequencing primers and fungal ITS1F and ITS2aR ribosomal DNA primers with 2x300bp chemistry. All filter samples were positive for microbial contamination, for both bacterial (350-1329 operational taxonomic units [OTUs]) and fungal (157-650 OTUs) load. Three filter samples revealed only moderate cytotoxicity in vitro. On average, the reduction in cell viability was between 0-20%, and 35% at the highest. All filter samples caused in vitro proinflammatory effects, regarding the elicited TNFα and IL-1β levels. The highest inflammatory responses were observed in filter samples 1, 4 and 5. A moderate negative correlation was found between bacterial load (OTUs) and inflammatory response for both TNFα (r=-0.61; p=0.02) and IL-1β (r=-0.46; p=0.07). No significant correlation was found between fungal load and inflammatory response in vitro. In light of the results, we should consider that, besides microbiome, others pollutants such as dust, metabolites or particles are probably influencing the increased production of cytokines. These findings corroborate, once more, the importance of considering exposure to complex mixtures in occupational settings. More studies are needed to drive robust conclusions on the effects of exposure to complex mixtures in occupational settings, in order to better estimate health risks for workers.
- Bioburden in health care centers: is the compliance with Portuguese legislation enough to prevent and control infection?Publication . Viegas, Carla; Almeida, Beatriz; Monteiro, Ana; Aranha Caetano, Liliana; Carolino, Elisabete; Gomes, Anita Quintal; Twarużek, Magdalena; Kosicki, Robert; Marchand, Geneviéve; Viegas, SusanaThis study intends to assess the bioburden in ten Portuguese health care centers (PHCC) with a multi-approach protocol using active (air sampling by impaction and impinger) and passive (surface swabs and air-conditioning filters) sampling methods, and compare the results with compliance levels of IAQ Portuguese legislation for the assessment of bioburden. The fungal burden in the PHCC was also characterized through molecular detection of toxigenic species, antifungal resistance and mycotoxins profile. Concerning legal compliance criteria for bacteriota the first criteria [indoor] + 350 CFU. m−3 < [outdoor] was not compiled in all PHCC analyzed. Regarding fungal load, 60% did not comply with the quantitative guideline (I/O < 1). Growth of four fungal genera (Chrysosporium, Cladosporium, Mucor, and Penicillium) was observed in HVAC filter samples collected from 6 out of 10 assessed PHCC when using azole-supplemented SDA media. Overall, Aspergillus/Penicillium/Paecilomyces levels were above the detection limits, except in one PHCC. The presence of mycotoxins was found both in air and HVAC filter samples. From the air samples analyzed, nine were contaminated (ng/ml) with 1–5 different mycotoxins within the same sample, whereas in HVAC filters, four samples contained 1 or 2 mycotoxins in the same filter. Overall, it was possible to conclude that Portuguese legislation is not enough to ensure IAQ in health care settings. The multi-approach sampling protocol used in this study allowed to unveil a more real scenario regarding exposure to bioburden. Detection of mycotoxins reinforces the relevance of studying mycotoxins in the clinical environment.
- EXPOsE: avaliação da exposição a micobiota resistente a antifúngicosPublication . Caetano, Liliana Aranha; Viegas, CarlaMicrobiota in clinical environments: poor hospital indoor air quality (IAQ) may lead to hospital-acquired infections, sick hospital syndrome, and various occupational hazards; microbiological IAQ monitoring and control in hospitals is currently a necessary and integral part of prevention strategies against hospital-acquired infections; implementation of sampling and analysis methods should be adapted to the hospital environment; bacterial, viral and fungal infections are frequently acquired via inhalation, among them pulmonary aspergillosis and pneumocystosis still represent high disease burden; a large number of fungal species can cause severe infections, especially among immunocompromised individuals; most important fungi related with fungal exposure: Cladosporium, Alternaria, Stachybotris, Penicillium, Aspergillus.
- Assessment of azole resistance in clinical settings by passive samplingPublication . Caetano, Liliana Aranha; Almeida, Beatriz; Viegas, CarlaIntroduction - Clinical settings (hospitals, primary health care centers – PHCC), with high occupation rates, present microbiologic agents in their environment. Exposure to mycobiota in indoor environments is related to several adverse human health effects, such as respiratory symptoms. Azole resistance in fungal species and consequent failure of antifungal therapy is a major concern for public health. Portuguese legislation recommends active air sampling for bioburden assessment indoors. Passive sampling can be used in complement to determine bioburden levels from longer periods. Aim of the study - To assess the fungal burden and prevalence of azole resistance in clinical settings in Portugal using passive sampling methods.
- Characterization of occupational exposure to fungal burden in Portuguese bakeriesPublication . Viegas, Carla; Faria, Tiago; Aranha Caetano, Liliana; Carolino, Elisabete; Quintal-Gomes, Anita; Twarużek, Magdalena; Kosicki, Robert; Viegas, SusanaSeveral studies reported adverse respiratory health effects in workers exposed to ambient contaminants in bakeries. The aim of this study was to examine worker exposure to fungi and mycotoxins in Portuguese bakeries in order to develop new policies in occupational health. Environmental samples such as air, surfaces, settled dust and electrostatic dust collector (EDC) were collected in 13 bakeries for fungal and mycotoxins assessment. Air samples obtained by impaction were performed applying malt extract agar (MEA) supplemented with chloramphenicol (0.05%) and dichloran glycerol (DG18) agar-based media. Air samples collected through impinger method were determined as well for fungal detection by molecular tools of Aspergillus sections and mycotoxins. The highest median value for fungal load was 1053 CFU·m-3 and 65.3% (32 out of 49) of the sampling sites displayed higher fungal load than limits imposed by the World Health Organization. Aspergillus genera was found in air, surface swabs and EDC. Molecular tools were effective in measuring Aspergillus section Fumigati in 22.4% on air, 27.8% on surface swabs and in 7.4% in EDC and Aspergillus section Versicolores in one air sample. All settled dust samples showed contamination with six to eight mycotoxins in each sample. The mycotoxins detected were deoxynivalenol-3-glucoside, deoxynivalenol, zearalenone, 15-acetyldeoxynivalenol, monoacetoxyscirpenol, diacetoxyscirpenol, fumonisin B1, fumonisin B2, griseofulvin, HT2, ochratoxin A, ochratoxin B and mycophenolic acid. Industrial hygienists and exposure assessors should rely on different sampling methods (active and passive) and different assays (culture based and molecular methods) to obtain an accurate risk characterization regarding fungal burden (fungi and mycotoxins). Additionally, the awareness for the raw material as a potential mycotoxins indoor contamination source is important.