Portuguese cork industry: filling the knowledge gap regarding occupational exposure to fungi and related health effects.

Introduction: The presence of the Penicillium section Aspergilloides (formerly known as Penicillium glabrum) in the cork industry involves the risk of respiratory diseases such as suberosis. Methods: The aim of this study was to corroborate the predominant fungi present in this occupational environment by performing a mycological analysis of 360 workers' nasal exudates collected by nasal swabs. Additionally, evaluation of respiratory disorders among the cork workers was also performed by spirometry. Results: Penicillium section Aspergilloides was detected by qPCR in 37 out of the 360 nasal swabs collected from workers' samples. From those, 25 remained negative for Penicillium sp. when using culture-based methods. A significant association was found between ventilatory defects and years of work in the cork industry, with those people working for 10 or more years in this industry having an approximately two-fold increased risk of having ventilatory defects compared to those working less time in this setting. Among the workers who detected the presence of Penicillium section Aspergilloides, those with symptoms presented slightly higher average values of CFU. Discussion: Overall, the results obtained in this study show that working in the cork industry may have adverse effects on worker's respiratory health. Nevertheless, more studies are needed (e.g., using serological assays) to clarify the impact of each risk factor (fungi and dust) on disease etiology.

Microbial Contamination in the Coffee Industry: An Occupational Menace besides a Food Safety Concern?

Respiratory abnormalities among workers at coffee roasting and packaging facilities have already been reported; however, little is known about microbiological contamination inside coffee production facilities. This study intends to assess the microbial contamination (fungi and bacteria) in two coffee industries from Brazil with a multi-approach protocol for sampling and for subsequent analyses using four main sources of samples: filtering respiratory protection devices (FRPD) used by workers, settled dust, electrostatic dust cloths (EDC) and coffee beans. The fungal contamination in the assessed industries was also characterized through the molecular detection of toxigenic species and antifungal resistance. Total bacteria contamination presented the highest values in FRPD collected from both industries (7.45 × 104 CFU·m-2; 1.09 × 104 CFU·m-2). Aspergillus genera was widespread in all the environmental samples collected and sections with clinical relevance (Fumigati) and with toxigenic potential (Nigri and Circumdati) were recovered from FRPD. Circumdati section was observed in 4 mg/mL itraconazole. Sections Circumdati (EDC, coffee beans and settled dust) and Nidulantes (EDC, coffee beans and FRPD) were detected by qPCR. Some of the targeted Aspergillus sections that have been identified microscopically were not detected by qPCR and vice-versa. Overall, this study revealed that microbial contamination is a potential occupational risk in the milling stage and should be tackled when assessing exposure and performing risk assessment. In addition, a multi-sampling campaign should be the approach to follow when assessing microbial contamination and FRPD should be included in this campaign. Occupational exposure to mycotoxins should be considered due to high fungal diversity and contamination. A One Health approach should address these issues in order to prevent consumption of coffee crops and beans infected by fungi and, more specifically, to avoid widespread azole resistance.

Bioburden in sleeping environments from Portuguese dwellings.

A wider characterization of indoor air quality during sleep is still lacking in the literature. This study intends to assess bioburden before and after sleeping periods in Portuguese dwellings through active methods (air sampling) coupled with passive methods, such as electrostatic dust cloths (EDC); and investigate associations between before and after sleeping and bioburden. In addition, and driven by the lack of information regarding fungi azole-resistance in Portuguese dwellings, a screening with supplemented media was also performed. The most prevalent genera of airborne bacteria identified in the indoor air of the bedrooms were Micrococcus (41%), Staphylococcus (15%) and Neisseria (9%). The major indoor bacterial species isolated in all ten studied bedrooms were Micrococcus luteus (30%), Staphylococcus aureus (13%) and Micrococcus varians (11%). Our results highlight that our bodies are the source of the majority of the bacteria found in the indoor air of our homes. Regarding air fungal contamination, Chrysosporium spp. presented the highest prevalence both in after the sleeping period (40.8%) and before the sleeping period (28.8%) followed by Penicillium spp. (23.47% morning; 23.6% night) and Chrysonilia spp. (12.4% morning; 20.3% night). Several Aspergillus sections were identified in air and EDC samples. However, none of the fungal species/strains (Aspergillus sections Fumigati, Flavi, Nidulantes and Circumdati) were amplified by qPCR in the analyzed EDC. The correlations observed suggest reduced susceptibility to antifungal drugs of some fungal species found in sleeping environments. Toxigenic fungal species and indicators of harmful fungal contamination were observed in sleeping environments.

Exposure assessment in one central hospital: A multi-approach protocol to achieve an accurate risk characterization.

The bioburden in a Hospital building originates not only from patients, visitors and staff, but is also disseminated by several indoor hospital characteristics and outdoor environmental sources. This study intends to assess the exposure to bioburden in one central Hospital with a multi-approach protocol using active and passive sampling methods. The microbial contamination was also characterized through molecular tools for toxigenic species, antifungal resistance and mycotoxins and endotoxins profile. Two cytotoxicity assays (MTT and resazurin) were conducted with two cell lines (Calu-3 and THP-1), and in vitro pro-inflammatory potential was assessed in THP-1 cell line. Out of the 15 sampling locations 33.3% did not comply with Portuguese legislation regarding bacterial contamination, whereas concerning fungal contamination 60% presented I/O > 1. Toxigenic fungal species were observed in 27% of the sampled rooms (4 out of 15) and qPCR analysis successfully amplified DNA from the Aspergillus sections Flavi and Fumigati, although mycotoxins were not detected. Growth of distinct fungal species was observed on Sabouraud dextrose agar with triazole drugs, such as Aspergillus section Versicolores on 1 mg/L VORI. The highest concentrations of endotoxins were found in settled dust samples and ranged from 5.72 to 23.0 EU.mg-1. While a considerable cytotoxic effect (cell viability < 30%) was observed in one HVAC filter sample with Calu-3 cell line, it was not observed with THP-1 cell line. In air samples a medium cytotoxic effect (61-68% cell viability) was observed in 3 out of 15 samples. The cytokine responses produced a more potent average cell response (46.8 ± 12.3 ρg/mL IL-1ß; 90.8 ± 58.5 ρg/mL TNF-α) on passive samples than air samples (25.5 ± 5.2 ρg/mL IL-1ß and of 19.4 ± 5.2 ρg/mL TNF-α). A multi-approach regarding parameters to assess, sampling and analysis methods should be followed to characterize the biorburden in the Hospital indoor environment. This study supports the importance of considering exposure to complex mixtures in indoor environments.

Aspergillus spp. prevalence in Primary Health Care Centres: Assessment by a novel multi-approach sampling protocol.

Exposure 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 analysed PHCC has greatly contributed to risk characterization, highlighting the need to implement corrective measures in order to avoid fungal presence in those settings.

Aspergillus prevalence in air conditioning filters from vehicles: Taxis for patient transportation, forklifts, and personal vehicles.

The frequency and importance of Aspergillus infections is increasing worldwide. This study aimed to assess the occupational exposure of forklifts and taxi drivers to Aspergillus spp. Nineteen filters from air conditioning system of taxis, 17 from forklifts and 37 from personal vehicles were assessed. Filters extract were streaked onto MEA, DG18 and in azole-supplemented media. Real-time quantitative PCR amplification of selected Aspergillus species-complex was also performed. Forklifts filter samples presented higher median values. Aspergillus section Nigri was the most observed in forklifts filters in MEA (28.2%) and in azole-supplemented media. DNA from Aspergillus sections Fumigati and Versicolores was successfully amplified by qPCR. This study enlightens the added value of using filters from the air conditioning system to assess Aspergillus spp. occupational exposure. Aspergillus azole resistance screening should be included in future occupational exposure assessments.

Fungal burden exposure assessment in podiatry clinics from Ireland.

Fungi are amongst the bioaerosols of most importance, as indicated by the growing interest in this field of research. The aim was to characterize the exposure to fungal burden in podiatry clinics using culture-based and molecular methods. METHODS: Airborne fungi were collected using an impaction air sampler and surface samples were also performed. Fourteen air samples were collected for direct detection of fungal DNA from filamentous fungi and dermatophytes. Overall, 63.6 % of the evening samples and 46 % of the morning samples surpassed the threshold values (150 CFU/m3). Molecular detection, by real time PCR, of the target fungal species/strains (Aspergillus and Stachybotrys species) was negative for all samples collected. Trichophyton rubrum was detected by PCR analysis in one DNA sample collected on day six. Results suggest the use of both culture-based and molecular methodologies are desirable for a complete evaluation of fungal burden in this particular health care setting.

Aspergillus spp. prevalence in different Portuguese occupational environments: What is the real scenario in high load settings?

The genus Aspergillus is one of the most prevalent regarding fungi in several highly contaminated occupational environments. The goal of the current study was to assess the prevalence of Aspergillus spp. in different settings, focusing on those where a higher load of fungal contamination is expected according to the European Agency for Safety and Health at Work. A specific protocol to ensure a more accurate assessment of the exposure to Aspergillus spp. is proposed aimed at allowing a detailed risk characterization and management. Two wastewater treatment plants, one wastewater elevation plant, four waste treatment plants, three cork industries, five slaughter houses, four feed industries, one poultry pavilion, and two swineries, all located in the outskirts of Lisbon, were assessed. In total, 125 air samples and 125 surface samples were collected and analysed by culture-based methods. Real-time polymerase chain reaction was performed to detect fungal presence in 100 samples, targeting the Aspergillus sections Circumdati, Flavi, and Fumigati. The highest prevalence of Aspergillus spp. was found in wastewater treatment plants (69.3%; 31.1%), waste treatment plants (34.8%; 73.6%), and poultry feed industry (6.3%; 26.1%), in air and surfaces, respectively. Aspergillus spp. was also prevalent in cork industry (0.9%; 23.4%), slaughter houses (1.6%; 17.7%), and swineries (7.4%; 9.5%), in air and surfaces, respectively. The Aspergillus sections more prevalent in the air and surfaces of all the assessed settings were the Nigri section (47.46%; 44.71%, respectively), followed by Fumigati (22.28%; 27.97%, respectively) and Flavi (10.78%; 11.45%, respectively) sections. Aspergillus section Fumigati was successfully amplified by qPCR in 18 sampling sites where the presence of this fungal species had not been identified by conventional methods. It should be highlighted that the occupational exposure burden is due not only to the Aspergillus load, but also to the toxigenic potential of this genus. Based on our results, a protocol relied in the application of conventional and molecular methods in parallel is herein suggested aimed at allowing a better risk characterization and management.

Fungal contamination in green coffee beans samples: A public health concern.

Studies on the microbiology of coffee cherries and beans have shown that the predominant toxigenic fungal genera (Aspergillus and Penicillium) are natural coffee contaminants. The aim of this study was to investigate the distribution of fungi in Coffea arabica L. (Arabica coffee) and Coffea canephora L. var. robusta (Robusta coffee) green coffee samples obtained from different sources at the pre-roasting stage. Twenty-eight green coffee samples from different countries of origin (Brazil, Timor, Honduras, Angola, Vietnam, Costa Rica, Colombia, Guatemala, Nicaragua, India, and Uganda) were evaluated. The fungal load in the contaminated samples ranged from 0 to 12330 colony forming units (CFU)/g, of which approximately 67% presented contamination levels below 1500 CFU/g, while 11% exhibited intermediate contamination levels between 1500 and 3000 CFU/g. Contamination levels higher than 3000 CFU/g were found in 22% of contaminated coffee samples. Fifteen different fungi were isolated by culture-based methods and Aspergillus species belonging to different sections (complexes). The predominant Aspergillus section detected was Nigri (39%), followed by Aspergillus section Circumdati (29%). Molecular analysis detected the presence of Aspergillus sections Fumigati and Circumdati. The% coffee samples where Aspergillus species were identified by culture-based methods were 96%. Data demonstrated that green coffee beans samples were contaminated with toxigenic fungal species. Since mycotoxins may be resistant to the roasting process, this suggests possible exposure to mycotoxins through consumption of coffee. Further studies need to be conducted to provide information on critical points of coffee processing, such that fungal contamination may be reduced or eliminated and thus exposure to fungi and mycotoxins through coffee handling and consumption be prevented.

Analysis of surfaces for characterization of fungal burden - Does it matter?

OBJECTIVES: Mycological contamination of occupational environments can be a result of fungal spores' dispersion in the air and on surfaces. Therefore, it is very important to assess it in both types of the samples. In the present study we assessed fungal contamination in the air and in the surface samples to show relevance of surfaces sampling in complementing the results obtained in the air samples. MATERIAL AND METHODS: In total, 42 settings were assessed by the analysis of air and surfaces samples. The settings were divided into settings with a high fungal load (7 poultry farms and 7 pig farms, 3 cork industries, 3 waste management plants, 2 wastewater treatment plants and 1 horse stable) and a low fungal load (10 hospital canteens, 8 college canteens and 1 maternity hospital). In addition to culture-based methods, molecular tools were also applied to detect fungal burden in the settings with a higher fungal load. RESULTS: From the 218 sampling sites, 140 (64.2%) presented different species in the examined surfaces when compared with the species identified in the air. A positive association in the high fungal load settings was found between the presence of different species in the air and surfaces. Wastewater treatment plants constituted the setting with the highest number of different species between the air and surface. CONCLUSIONS: We observed that surfaces sampling and application of molecular tools showed the same efficacy of species detection in high fungal load settings, corroborating the fact that surface sampling is crucial for a correct and complete analysis of occupational scenarios.

Occupational exposure to fungi and particles in animal feed industry.

BACKGROUND: Very few studies regarding fungal and particulate matter (PM) exposure in feed industry have been reported, although such contaminants are likely to be a significant contributing factor to several symptoms reported among workers. The purpose of this study has been to characterize fungal and dust exposure in one Portuguese feed industry. MATERIAL AND METHODS: Air and surface samples were collected and subject to further macro- and microscopic observations. In addition we collected other air samples in order to perform real-time quantitative polymerase chain reaction (PCR) amplification of genes from Aspergillus fumigatus and Aspergillus flavus complexes as well as Stachybotrys chartarum. Additionally, two exposure metrics were considered - particle mass concentration (PMC), measured in 5 different sizes (PM0.5, PM1, PM2.5, PM5, PM10), and particle number concentration (PNC) based on results given in 6 different sizes in terms of diameter (0.3 µm, 0.5 µm, 1 µm, 2.5 µm, 5 µm and 10 µm). RESULTS: Species from the Aspergillus fumigatus complex were the most abundant in air (46.6%) and in surfaces, Penicillium genus was the most frequently found (32%). The only DNA was detected from A. fumigatus complex. The most prevalent in dust samples were smaller particles which may reach deep into the respiratory system and trigger not only local effects but also the systemic ones. CONCLUSIONS: Future research work must be developed aiming at assessing the real health effects of these co-exposures. Med Pr 2016;67(2):143-154.

Assessment of exposure to the Penicillium glabrum complex in cork industry using complementing methods.

Cork oak is the second most dominant forest species in Portugal and makes this country the world leader in cork export. Occupational exposure to Chrysonilia sitophila and the Penicillium glabrum complex in cork industry is common, and the latter fungus is associated with suberosis. However, as conventional methods seem to underestimate its presence in occupational environments, the aim of our study was to see whether information obtained by polymerase chain reaction (PCR), a molecular-based method, can complement conventional findings and give a better insight into occupational exposure of cork industry workers. We assessed fungal contamination with the P. glabrum complex in three cork manufacturing plants in the outskirts of Lisbon using both conventional and molecular methods. Conventional culturing failed to detect the fungus at six sampling sites in which PCR did detect it. This confirms our assumption that the use of complementing methods can provide information for a more accurate assessment of occupational exposure to the P. glabrum complex in cork industry.

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 µ 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 29×10(4) to 109×10(4) CFU m(-2) before cleaning and from 11×10(4) to 89×10(4) 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.

Non-coding RNAs: multi-tasking molecules in the cell.

In the last years it has become increasingly clear that the mammalian transcriptome is highly complex and includes a large number of small non-coding RNAs (sncRNAs) and long noncoding RNAs (lncRNAs). Here we review the biogenesis pathways of the three classes of sncRNAs, namely short interfering RNAs (siRNAs), microRNAs (miRNAs) and PIWI-interacting RNAs (piRNAs). These ncRNAs have been extensively studied and are involved in pathways leading to specific gene silencing and the protection of genomes against virus and transposons, for example. Also, lncRNAs have emerged as pivotal molecules for the transcriptional and post-transcriptional regulation of gene expression which is supported by their tissue-specific expression patterns, subcellular distribution, and developmental regulation. Therefore, we also focus our attention on their role in differentiation and development. SncRNAs and lncRNAs play critical roles in defining DNA methylation patterns, as well as chromatin remodeling thus having a substantial effect in epigenetics. The identification of some overlaps in their biogenesis pathways and functional roles raises the hypothesis that these molecules play concerted functions in vivo, creating complex regulatory networks where cooperation with regulatory proteins is necessary. We also highlighted the implications of biogenesis and gene expression deregulation of sncRNAs and lncRNAs in human diseases like cancer.

RNA interference knockdown of hU2AF35 impairs cell cycle progression and modulates alternative splicing of Cdc25 transcripts.

U2AF is a heterodimeric splicing factor composed of a large (U2AF65) and a small (U2AF35) subunit. In humans, alternative splicing generates two U2AF35 variants, U2AF35a and U2AF35b. Here, we used RNA interference to specifically ablate the expression of each isoform in HeLa cells. Our results show that knockdown of the major U2AF35a isoform reduced cell viability and impaired mitotic progression, leading to accumulation of cells in prometaphase. Microarray analysis revealed that knockdown of U2AF35a affected the expression level of approximately 500 mRNAs, from which >90% were underrepresented relative to the control. Among mRNAs underrepresented in U2AF35a-depleted cells we identified an essential cell cycle gene, Cdc27, for which there was an increase in the ratio between unspliced and spliced RNA and a significant reduction in protein level. Furthermore, we show that depletion of either U2AF35a or U2AF35b altered the ratios of alternatively spliced isoforms of Cdc25B and Cdc25C transcripts. Taken together our results demonstrate that U2AF35a is essential for HeLa cell division and suggest a novel role for both U2AF35 protein isoforms as regulators of alternative splicing of a specific subset of genes.