Viegas, C.1, 2 / Faria, T1 / Caetano, L.1,3 / Gomes, A.1, 4
Resumo
Os contaminantes fúngicos aerossolizados têm ganho importância devido aos efeitos sobre a saúde causados pela exposição aos respetivos esporos e aos seus metabolitos. Uma adequada avaliação da contaminação fúngica deve combinar a utilização de métodos convencionais e moleculares para assegurar uma adequada caraterização de uma amostra. O objetivo deste trabalho foi analisar dados obtidos provenientes de avaliações anteriores sobre a carga fúngica, com o intuito de propor um protocolo específico para avaliação fúngica em ambientes ocupacionais muito contaminados. Em todos os contextos foram detetadas espécies/estirpes que não foram identificadas pelos métodos convencionais. Além disso, em todos os ambientes ocupacionais foram identificadas espécies fúngicas nas superfícies que não foram identificadas em colheitas de ar. Foi proposto um protocolo que segue o recomendado pelas referências legais Europeias e sugere a utilização de técnicas moleculares simples, de modo a permitir uma avaliação mais criteriosa dos cenários reais em relação à exposição ocupacional a fungos.
Palavras-chave: Avaliação da contaminação fúngica; Protocolo específico; Exposição
ocupacional; Métodos convencionais; Ferramentas moleculares Abstract
Airborne fungal contaminants are increasingly gaining importance in view of health hazards caused by the spores themselves or by microbial metabolites A proper fungal assessment should combine culture based-methods and molecular tools to ensure the best possible characterization of fungal burden in a given sampling source. The aim of this work was to analyze integrated data obtained from previous assessments of fungal burden, in order to propose a specific protocol to ensure a proper fungal burden assessment in highly contaminated occupational environments. All settings presented sampling sites where detection of specific species/strains was possible yet not identified by conventional methods. In addition, all occupational environments assessed presented fungal species in surfaces that were not identified in air samples. Was proposed a protocol that follows the European legal trends, and additionally suggests the use of simple molecular techniques in order to enable a more refined assessment of the real scenarios concerning occupational exposure to fungi.
Keywords: Fungal contamination assessment; Specific protocol; Occupational exposure; Culture based-methods; Molecular tools
1. Theory
An outbreak of research about occupational exposure to bioaerossols is due, among others factors, to (i) an emerging interest in the role of environmental exposure to biological agents (in public and occupational health), (ii) the development of new biotechnologies used in some industrial sectors, and (iii) the access to new molecular tools allowing finer and faster bioaerosols characterization (Oppliger, 2014).
Bioaerosols are defined as airborne particles including fungal spores and hyphae, bacteria, endotoxins, β(1 3)-glucans, mycotoxins or high-molecular-weight allergens, and organic dusts in general that are composed of or derived from biological matter (Oppliger, 2014). Airborne fungal contaminants are increasingly gaining importance in view of health hazards caused by the spores themselves or by microbial metabolites (Fischer and Dott 2003). Each
1 Environment and Health Research Group, Escola Superior de Tecnologia da Saúde de Lisboa, ESTeSL, Instituto
Politécnico de Lisboa, Lisbon, Portugal
2 Centro de Investigação em Saúde Pública, Escola Nacional de Saúde Pública, New University of Lisbon, Lisbon,
Portugal
3 Research Institute for Medicines (iMed.ULisboa), Faculty of Pharmacy, University of Lisbon, Lisbon, Portugal. 4 Institute of Molecular Medicine, Faculty of Medicine of Lisbon, Lisbon, Portugal
bioaerosol sample for fungal assessment is unique as its composition varies in time and space leading not only to high variation between samples from the same workplace, which can be due to external factors, but also to the dynamic evolution of the colonized substrate and fungi species present (Oppliger et al., 2014; Viegas et al., 2015a). In addition, fungi from some typical work sectors are associated with well-known diseases such as Penicillium glabrum complex, which is commonly linked to suberosis in the cork industry (Viegas et al., 2015b). Finally, fungal resistant strains may develop in the environment through the exposure of the fungus to azole fungicides used in agriculture and in material preservation. This type of resistance development consequently places workers at risk of exposure to potential infections by azole-resistant strains in the environment (Mortensen et al., 2010).
Commonly, fungal burden has been studied by plate cultivation, and essentially all information on their occurrence and prevalence relies on viability studies (Pitkäranta et al., 2008). However, the restrictions of cultivation are known to bias the real scenario regarding characterization of fungal contamination (Hawksworth 1991; Viegas et al., 2014) and this situation is exacerbated when dealing with highly contaminated occupational environments. Different species have distinct growth requirements and, in mixed cultures, fast growth of a given species may lead to its overgrowth inhibiting the growth of other species (Carlile et al., 2001). The main benefits of using molecular methods instead of culture-based methods are the speed, accuracy, and analytical sensitivity of detection and the possibility to detect and identify dead or dormant organisms (Amann et al., 1995; MacNeil et al., 1995). With molecular methods we can identify specific species/strains that are representative of harmful fungal contamination in the setting to be analyzed (Viegas et al., 2016 a, b).
A proper fungal assessment should combine both techniques to ensure the best possible characterization of fungal burden in a given sampling source (Viegas et al., 2015c).
The aim of this work was to analyze integrated data obtained from previous assessments of fungal burden ensured by the Research Group Environment & Health, focusing on highly contaminated occupational environments (poultries, waste water treatment plants, waste treatment plants, slaughterhouses and cork and feed industries) in order to propose a specific protocol to ensure a proper fungal burden assessment in these settings.
2. Methodology
A review of fungal burden data collected between 2010 and 2016 (already published) in six different occupational environments was conducted, focusing on: Poultries (Viegas et al., 2014a), Waste Water Treatment Plants – WWTP (Viegas et al., 2014b), Waste Treatment Plants – WTP (Viegas et al., 2015c) Cork industries (Viegas et al., 2015b), Slaughterhouses (Viegas et al., 2016a) and Feed industries (Viegas et al., 2016b). These settings were selected, among others also assessed, because of their potential to enhance fungal load, due to characteristic environmental variables present in those environments, such as raw material processed or stored (Straumfors et al. 2015), animal density (HSE, 2008), nutrients (Douwes et al. 2003), and dust (Tsapko et al. 2011). The several assessments highlighted the need of a refined protocol to determine occupational exposure to fungi in highly contaminated workplaces
3. Evidence
In all the assessments done we applied in parallel culture-based methods (conventional) and molecular tools to proper assess fungal burden (Table 1).
Table 1 – Fungal burden assessed in different Portuguese occupational settings (2010-2016)
CONVENTIONAL METHODS MOLECULAR METHODS
SETTING Air range CFU/m3 Surface range CFU/m2
Most prevalent species found
(%) Species/strains targeted Species/strains detected
Poultries 320 - 24040 1x10
4
- 6x10-2
Air: Scopulariopsis brevicaulis 40.5 Among Aspergillus spp. : A. flavus complex 74.5
Surfaces: A. versicolor complex 31.4 ; A. flavus complex 16.5; F. solani 15.3. A. fumigatus complex A. flavus complex (toxigenic strains) Stachybotrys chartarum complex A. fumigatus complex A. flavus complex (toxigenic strains) WWTP 20 - 1260 6x10 4 - 2.23x106 Air: Penicillium spp. 58.9 – 38.9 Surfaces: Penicillium spp. 76.8; Cladosporium spp. 36.9 A. fumigatus complex A. flavus complex (toxigenic strains) Stachybotrys chartarum complex A. fumigatus complex WTP 20 - 1280 1x10 4 – 6x105
Air: Waste Sorting Plant (A. niger
complex 73.9;A. fumigatus complex 16.0; A. flavus complex 14.2); Incineration plant
(Penicillium sp. 62.9; A. fumigatus complex 18.0; A. flavus complex 6.0)
Surfaces: Waste Sorting Plant (A.
niger complex 66.1; A. flavus complex 14.2; A. fumigatus complex 13.8); Incineration plant (Penicillium sp. 57.5; A. fumigatus complex 22.3; A. niger complex 12.8) A. fumigatus complex A. flavus complex (toxigenic strains) Stachybotrys chartarum complex A. fumigatus complex
Cork Countless Countless
Air: C. sitophila (countless), A.
fumigatus complex and Penicillium spp.
Surfaces: C. sitophila (countless),
A. fumigatus complex, Penicillium spp. and Trichoderma spp. A. fumigatus complex P. glabrum complex A. fumigatus complex P. glabrum complex Slaughte r houses 10 - 970 0 - 90000
Air: Poultry Slaughterhouse
(Scopulariospis candida 59.5); Swine and Bovine Slaughterhouse (Cladosporium 47.5); Large Animal Slaughterhouse (Penicillium sp. 80.8)
Surfaces: Poultry Slaughterhouse
(Mucor sp. 100); Swine and Bovine Slaughterhouse (no fungi
isolated); Large Animal Slaughterhouse (Scopulariopsis brumptii 40.0) A. fumigatus complex A. flavus complex (toxigenic strains) A. ochraceus complex Stachybotrys chartarum complex A. fumigatus complex Feed 0 -144 0 - 18x104
Air: A. fumigatus complex 46.6;
Penicillium spp. 26.7. Surfaces: Penicillium spp. 32.0; A. flavus complex 12.5. A. fumigatus complex A. flavus complex (toxigenic strains) A. ochraceus complex Stachybotrys chartarum complex
All settings presented sampling sites where detection of specific species/strains was possible yet not identified by conventional methods. In addition, all occupational environments assessed presented fungal species in surfaces that were not identified in air samples.
Based on the previous experience from field and bench work, and also due to the already mentioned constraints from using only culture-based methods, the protocol to ensure a proper fungal burden a ssessment should comprise the following stages:
First. Apply a low cost exposure assessment, so that the intervention in the assessed settings can be prioritized for the implementation of safety measures based on comparison with already
established limit values (EN 14042:2003). Culture-based (conventional) methods should be used to obtain fungal load information (air and surfaces) regarding the most critical scenario previously considered;
Second. Compare the results obtained with relevant guidelines or legal requirements/limits suggested by scientific and/or technical organizations. The results of the quantitative analysis should be based on the limits set by respective countries, due to the lack of pan-European standards (Gutarowska et al., 2015). However, since in Portugal there are no guidelines to be used as terms of reference, we suggest the application of the guideline proposed by the World Health Organization (WHO) (maximum value of 150 CFU.m–3) (WHO, 2009), since it is the most strict limit for occupational assessment purposes.
Third. Select the most suitable indicators of harmful fungal contamination for each setting and apply conventional-culture methods together with molecular tools to determine fungal load. For clinical relevant fungal species, namely for the Fumigati section, isolates should be grown in medium containing antifungal drugs in order to detect resistant strains, which should be further analyzed to determine the susceptibility to the drug by measuring the Minimum Inhibitory Concentration (MIC) by reference microdilution methods (Arendrup et al., 2012; Hope et al., 2013). This methodology will ensure an indebted characterization of fungal burden in each setting while enabling the identification of further measures regarding assessment of fungal metabolites, thus, allowing a more adequate health surveillance of workers (Viegas et al., 2016 a, b).
The protocol herein proposed follows the European legal trends, and additionally suggests the use of simple molecular techniques, such as quantitative polymerase chain reaction, in order to enable a more refined assessment of the real scenarios concerning occupational exposure to fungi. In a near future, although not possible to currently apply ‘in the field’, we envisage to characterize mycobiota and detect their species composition using next-generation DNA sequencing technologies in order to assess occupational risks (Oplliger et al., 2014; Hoisington et al., 2014).
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