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Toxics
  • Editor’s Choice
  • Review
  • Open Access

14 April 2023

One Health Approach to Tackle Microbial Contamination on Poultries—A Systematic Review

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1
CE3C—Center for Ecology, Evolution and Environmental Change, Faculdade de Ciências, Universidade de Lisboa, 1749-016 Lisbon, Portugal
2
H&TRC—Health & Technology Research Center, ESTeSL—Escola Superior de Tecnologia e Saúde, Instituto Politécnico de Lisboa, 1990-096 Lisbon, Portugal
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NOVA National School of Public Health, Public Health Research Centre, Comprehensive Health Research Center, CHRC, NOVA University Lisbon, 1600-560 Lisbon, Portugal
4
CISAS—Center for Research and Development in Agrifood Systems and Sustainability, Instituto Politécnico de Viana do Castelo, 4900-347 Viana do Castelo, Portugal
This article belongs to the Special Issue Risk Assessment of Occupational Exposures for Better Health

Abstract

This study reports the search of available data published regarding microbial occupational exposure assessment in poultries, following the PRISMA methodology. Air collection through filtration was the most frequently used. The most commonly used passive sampling method was material collection such as dust, cages, soils, sediment, and wastewater. Regarding assays applied, the majority of studies comprised culture-based methods, but molecular tools were also frequently used. Screening for antimicrobial susceptibility was performed only for bacteria; cytotoxicity, virological and serological assays were also performed. Most of the selected studies focused on bacteria, although fungi, endotoxins, and β-glucans were also assessed. The only study concerning fungi and mycotoxins reported the carcinogenic mycotoxin AFB1. This study gives a comprehensive overview of microbial contamination in the poultry industry, emphasizing this setting as a potential reservoir of microbial pathogens threatening human, animal, and environmental health. Additionally, this research helps to provide a sampling and analysis protocol proposal to evaluate the microbiological contamination in these facilities. Few articles were found reporting fungal contamination in poultry farms worldwide. In addition, information concerning fungal resistance profile and mycotoxin contamination remain scarce. Overall, a One Health approach should be incorporated in exposure assessments and the knowledge gaps identified in this paper should be addressed in further research.

1. Introduction

The One Health approach incorporates human, animal, and plant health, as well as the health of their shared environment, for supporting a multidisciplinary and holistic approach that integrates monitoring, planning, and evaluation to optimize co-benefits and public health outcomes [,]. In addition, the One Health approach supports global health by fostering coordination, collaboration, and communication among different sectors at the human–animal–environment interface to address common health threats such as antimicrobial resistance (AMR), food safety, zoonotic diseases, and several others [,].
The industrialization of the poultry sector poses a considerable negative impact on air, soil, and water. The increase in waste management problems can be considered as one of the major drivers fostering harmful effects on environmental health []. Indeed, pathogens can be disseminated by unrecognized pathways, for example, on airborne dust and animal wastes utilized in agriculture and, consequently, water and soil quality may be impacted [].
Poultry production intensification needs increases in livestock numbers and densities, the use of particular feed to raise conversion ratios, and shorter production cycles [,]. Consequently, such changes may potentially alter transmission patterns and the evolutionary conditions of dominant pathogens, leading to emergence of zoonotic diseases [,]. The environment of animal husbandry, such as humidity level, number of animals, ventilation type, and hygiene measures may influence microbial development []. In fact, intensive animal production is also considered as one of the causes for biodiversity loss and potentially for upcoming pandemics [,].
Agricultural expansion and intensification bring wildlife, livestock, and people into closer contact, allowing animal microbes to spill over into people and causing infections, sometimes outbreaks, and less frequently epidemics and pandemics [,]. Production intensification of livestock raises concerns about the feasibility of the One Health model for animal production regarding the protection of the health of animals, workers, and consumers []. Thus, intensive poultry farming not only poses a significant risk to workers [,] but can also act as a potential public health menace [,].
Human and animal well-being is also in the scope of a One Health approach. Animal diseases threaten human health, food safety, and security, driven by the transmission of zoonotic diseases or by the loss of animal productivity. Adequate hygiene management is therefore critical to avoiding the negative human health and economic repercussions of foodborne diseases [].
We shouldn´t consider the close linkage and interdependencies of human and animal health without considering maintenance of stable ecosystem services that can be threatened by livestock rearing methods and/or excessive exploitative human activities []. In 2013, the European Union (EU) through the Directive 2003/99/EC aimed to improve the system for monitoring and collection of information on zoonoses, antimicrobial resistance, and foodborne outbreaks (EU, 2013). In 2017, the European Centre for Disease Prevention and Control (ECDC), European Food Safety Authority (EFSA), and European Medicines Agency (EMA) jointly established a list of harmonized outcome indicators to assist EU Member States’ assessment of the progress in reducing the use of antimicrobials and AMR in both humans and food-producing animals (ECDC, EFSA, EMA; 2017). More recently, in 2021, a list of harmonized outcome indicators was presented per country in the scope of the One Health approach (ECDC, EFSA, EMA; 2021). Additionally, since 2011, EFSA has reported zoonoses, zoonotic agents, and foodborne outbreaks and, in 2019, the annual EU Summary Reports were renamed the “EU One Health Zoonoses Summary Report” which is co-authored by EFSA and ECDC. In the scope of food safety, the poultry industry remains a public health issue, since foodborne pathogens can be in contact at all phases of the producing chain. Thus, identifying the sources and routes of transmission of pathogens is required in order to reduce their occurrence. Some regulatory guidelines have been designed to answer these concerns, namely the Regulation (EC) No 2073/2005, concerning microbiological contaminants for food safety criteria. However, the food industry is known to be very committed to food safety assurance, but less concerned with the safety of workers, and biological risk assessment is usually neglected by occupational health professionals due to the lack of systematized information about the biological agents involved []. In fact, the microorganisms’ occupational exposure is being neglected in a wide range of industrial sectors (besides poultry production), being less recognized and not so well described in comparison with other occupational agents [].
Concerning the occupational health legal framework, Portuguese employers are obliged by regulation to assess and prevent exposure to occupational risks [] and specifically to biological agents. The Directive 2000/54/EC of the European Parliament and the Council of September 18 sets the rules regarding risk assessment if exposure to biological agents cannot be avoided []. However, it is not common to include zoonosis and AMR as a source of risk in studies on occupational risk assessment of animal-related occupations. In 2020, the European Agency for Safety and Health at Work (EU-OSHA) published a review about biological agents and prevention of work-related diseases, and animal farming was considered a high-risk occupation [].
Due to the lack of studies regarding poultry farms this study aimed to perform a systematic review to provide a broad overview of the state of the art in the developed subject, describing the microbiological contamination reported in previous studies developed in poultry farms and indicating which parameters and methods were applied to perform the microbial contamination assessment in this setting in different scopes (occupational/food safety/animal health).

2. Materials and Methods

2.1. Registration

The Preferred Reporting Items for Systematic Reviews and Meta-Analyses (PRISMA) checklist [] was completed (Supplementary Material—Table S1).

2.2. Search Strategy, Inclusion and Exclusion Criteria

This study reports the search of available data published in the period of 1 January 2000 to 20 January 2023. The search terms aimed to identify studies on microbial occupational exposure assessments, selecting studies on sawmills that included the terms “exposure” AND “microorganisms” AND (“poultry” OR “broilers”), with English as the chosen language. The databases chosen were PubMed (https://pubmed.ncbi.nlm.nih.gov/ (accessed on 20 January 2023)), Scopus (https://www.scopus.com/ (accessed on 20 January 2023)), and Web of Science (WoS) (www.webofscience.com (accessed on 20 January 2023) following the PRISMA methodology. This search strategy identified 258 papers in all databases. Articles that did not fulfill the inclusion criteria were not subjected to additional review (but some of them were used for Introduction and Discussion sections) (Table 1).
Table 1. Inclusion and exclusion criteria for the articles selected.

2.3. Study Selection and Data Extraction

The selection of the articles was performed through the Rayyan intelligent systematic review application, which is a free web-tool that greatly speeds up the process of screening and selecting papers for academics working on systematic reviews, in three rounds.
The first round was conducted by one investigator (BG) and comprised the screening of all titles to eliminate papers that were duplicated or unrelated to the subject.
Rayyan was then used to analyze the papers that were chosen. The second round was a screening of all abstracts carried out by two investigators (BG and RC). The full texts of all potentially relevant studies were evaluated in the third round, taking into account the inclusion and exclusion criteria. Potential divergences in the selection of the studies were analyzed and resolved by four investigators (BG, MD, RC, and PP). Data extraction was then conducted by BG. It was also checked over by MD and CV. The following details were manually extracted: (1) databases, (2) title, (3) country, (4) environment assessed, (5) objective, (6) microorganisms and metabolites, (7) analyzed matrices, (8) sampling methods, (9) analytical methods, (10) main findings, (11) references.

2.4. Quality Assessment

The assessment of the risk of bias was performed by two investigators (BG and CV). Within each study, we evaluated the risk of bias across three parameters divided into key criteria (environment assessed, microorganisms and metabolites, sampling methods, analytical methods).
Each parameter’s risk of bias was rated as “low,” “medium,” “high,” or “not applicable.” The studies for which all the key criteria and most of the other criteria were characterized as “high” were excluded.

3. Results

The workflow diagram for selecting studies is illustrated in Figure 1. Initially, 259 studies were found in the database search, from which 197 abstracts were examined and 97 complete texts were assessed for eligibility. After considering the inclusion and exclusion criteria, a total of 39 studies were disregarded, mostly because they were related to biocontrol efficacy, clinical trials, or biological samples. A total of 58 studies related to microbial exposure in poultry facilities were selected.
Figure 1. PRISMA-based selection of articles.

Characteristics of the Selected Studies

Table 2 describes the main characteristics of the selected studies. Of the reviewed studies (n = 58), 34 were conducted in Europe (9 in Germany [,,,,,,,,], 8 in Poland [,,,,,,,], 4 in Portugal [,,,], 3 in Italy [,,], 1 in the Netherlands [], Spain [], Austria [], Lithuania [], France [], Denmark [], 1 in Denmark and Switzerland [], and 2 in several European countries (Belgium, Bulgaria, Denmark, France, Germany, Italy, the Netherlands, Poland, and Spain [] and Denmark, Switzerland, and Spain []). Thirteen studies were performed in Asia, namely, six in China [,,,,,], four in Korea [,,,], one in Lebanon [], one in India [], and one in Iran []. Eight studies were carried out in America, including six in the United States of America [,,,,,] and two in Canada [,]. In Africa, three studies were conducted in Egypt [,,] while, in Oceania, two studies were performed in Australia [,].
Table 2. Characteristics of and Data Obtained in the Selected Studies.
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The majority of studies (31 out of 58, 53%) were performed on poultry farms [,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,]; followed by animal farms (19 out of 58%, 33%), namely, six on poultry and swine farms [,,,,,], two on poultry and dairy farms [,], two on poultry farms, sheep sheds, and horse stables [,], one on cattle, chicken, swine, and sheep [], one on poultry, swine, and cattle [], one on cow barns, swine, sheep sheds, poultry houses, horse stables, and buildings for storage of hay [], one on poultry, pork, and beef farms [], one on a duck hatchery [], one on a turkey hatchery [], one on swine, chicken, and cattle farms [], one on poultry farms and live poultry markets [], and one on poultry processing plants []. Additionally, three studies (5%) covered several workplaces (one on a cement plant, composting plant, poultry farm, and cultivated area [], one on a poultry farm, flourmill, and the textile and food industry [], and one on a poultry farm, swinery, feed preparing and storing house, grain mill, wooden panel-producing factory, and organic waste and recycling facilities [].
Most of the studies focus on occupational health (33 out of 58, 57%) [,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,], followed by food safety (6 out of 58, 10%) [,,,,,] and human health (5 out of 58, 9%) [,,,,]. Some studies encompass occupational and animal health (4 out of 58, 7%) [,,,], while some studies focus only on animal health (4 out of 58, 7%) [,,,]. Few studies focused on human and animal health (2 out of 58, 3%) []. One study focused on environmental health and food safety [], one on human and environmental health [], and one on environmental health [].
Regarding microbial contamination, most of the selected studies focused on bacteria (31 out 58, 53%) [,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,], while some considered bacteria and fungi (8 out 59, 14%) [,,,,,,,]. Eight studies (14%) considered microorganisms and metabolites, six studies (10%) focused on bacteria, fungi, and endotoxins [,,,,,], one focused on bacteria, fungi, endotoxins, and β-glucans [], and one focused on bacteria and endotoxins []. In addition, six studies (10%) focused only on fungi [,,,,,], three studies (5%) on endotoxins [,,], one study on fungi and mycotoxins [], and one study on viruses [].
The most frequent sampling method was air sampling (36 out of 58, 62%) [,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,]. Considering active sampling, air collection through a filtration method was frequent (24 out of 58, 41%) [,,,,,,,,,,,,,,,,,,,,,,,]. The impaction method was also recurrent (12 out of 58, 21%) [,,,,,,,,,,,], followed by the impingement method (4 out of 58, 7%) [,,,]. Additionally, one study used the two-stage bioaerosol cyclone [].
Regarding passive sampling, material collection was the most frequent methodology applied (13 out of 58, 22%) [,,,,,,,,,,,,], namely of dust (3 out of 58, 5%) [,,], litter samples (3 out of 59, 5%) [,,], bird carcasses (3 out of 58, 5%) [,,], and animal fecal samples (2 out of 59, 3%) [,]. Manure and soil samples [], eggshell [] and specimens, feces, cages, soils, sediment, wastewater, and surface swabs from chopping boards [] were collected. Some studies performed surface swabs (11 out of 58, 19%) [,,,,,,,,,,] of bird cloacas (5 out of 58, 8%) [,,,,], bird cloacas and human sera [], human sera [], the skin of workers [], nose and throat of workers [], fecal droppings [], and the floor []. Furthermore, three studies used both active and passive sampling methods [,,] and two studies performed personal air sampling [,].
Considering analytical procedures for microbial characterization, the majority of studies used culture-based methods (41 out of 58, 71%) [,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,]. Fluorescence quantification was also recurrent (5 out of 58, 9%) [,,,,]. In addition, screening for antimicrobial susceptibility was carried out only for bacteria (13 out of 58 studies, 22%) [,,,,,,,,,,,,]. In addition, two studies performed a cytotoxicity assay [,] and one study used both virological and serological assays [].
Molecular tools were frequently applied (29 out of 58 studies, 50%) [,,,,,,,,,,,,,,,,,,,,,,,,,,,,]. Several studies performed PCR (9 out of 58, 16%) [,,,,,,,,], while some resorted to RT-PCR [,]. Antibiotic resistance gene sequencing was carried out by four studies (7%). While some studies rely on metagenomics analysis (2 out of 58, 3%) [,], others rely on whole-genome sequencing [], while one study used restriction fragment length polymorphism [] and other pyrosequencing analyses []. Several studies performed gene sequencing (20 out of 58, 33%), with the 16s rRNA gene being the most frequently sequenced (11 out of 58, 19%) [,,,,,,,,,,], followed by vanA PCR (2 out of 58, 3%) [,]. Additionally, one study performed 16s rRNA, tetL, tetW, and E. coli gene sequencing [], and one study sequenced tetracycline resistance genes []. Gas chromatographic and spectrophotometric methods were also used by some studies (5 out of 58, 9%) [,,,,].
Regarding metabolite characterization, Limulus amebocyte assay was frequently used for endotoxin assessment (11 out of 58, 19%), while one study used ELISA assay for mycotoxin assessment []. On the other hand, chemical analysis of litter samples was performed by one study []. Furthermore, quantitative kinetic Glucatell assay was used for β-glucan assessment []. Additionally, some studies performed questionnaires [,] and spirometric measures from workers were performed [,] .
According to the microbial assessment, several studies evidence the presence of bacteria (26 out of 58, 45%) [,,,,,,,,,,,,,,,,,,,,,,,,,] in facilities associated with poultry production. Fungal exposure was also evidenced by 14 studies (24%) [,,,,,,,,,,,,,]. Furthermore, microorganisms´ metabolites such as endotoxins were detected by eight studies (14%) [,,,,,,,], while endotoxins and β-glucans were evidenced by one study []. The only study that performed fungal and mycotoxin assessment found the carcinogenic mycotoxin AFB1 []. Overall, occupational exposure to microorganisms is a frequent concern reported by the selected studies (15 out of 58, 26%) [,,,,,,,,,,,,,,]. Indeed, the risk of exposure to potential pathogenic bioaerosols originating in poultry facilities is emphasized in some studies (5 out of 58, 9%) [,,,,].

4. Discussion

Industrialization has led to increased animal density in enclosed production buildings, resulting in high concentrations of viable and non-viable bacteria and fungi, as well as metabolites in bioaerosols []. The poultry industry has been found to pose a significant global health risk due to microbiological contamination []. Farm facilities housing multiple animals promote complex mixtures of microorganisms in bioaerosols, including dust-containing feathers, skin fragments, feces, feed particles, microorganisms, and chemicals []. Long shifts in manufacturing plants have become common, resulting in workers inhaling complex bioaerosols, which can pose several health hazards in agricultural environments []. This situation has prompted increased studies on occupational health. Bioaerosols from farms can also pose health risks to nearby residents [,], highlighting the importance of research on human health, environmental impact, and the One Health approach to address these concerns. Broilers and laying hens are susceptible to bacterial and viral infections of the upper respiratory tract, as indicated by several studies [,,,]. The transmission of pathogens can occur through inhalation, close contact with infected animals, feces, litter, or contaminated objects, and inadequate biosecurity controls can result in significant economic losses []. As international trade expands, food safety concerns regarding the rapid spread of foodborne pathogens through the global food chain are increasing [].
Moreover, environmental health concerns arise from the utilization of animal by-products, such as poultry manure and litter, in agriculture. Repeated use of these by-products as manure can lead to the accumulation of contaminants in agricultural soils, potentially increasing their bioavailability and toxicity in the environment []. Air sampling has been widely used to characterize occupational exposure to fungi, but it is important to consider the appropriate sampling period and the influence of variables such as ventilation and building features. Passive sampling methods, such as settled dust assessment, have been shown to be more reliable for collecting contamination over a longer period of time. Broiler manure and animal bedding have been identified as the primary sources of indoor air microbial contamination in the poultry industry [,,,,,,,].
It is recommended to use a multiapproach sampling protocol for a more comprehensive understanding of microbial contamination. While culture-based methods have been primarily used for microbial characterization, culture-independent methods such as cloning approaches and quantitative real-time PCR have shown to be suitable for various bioaerosol measurements. Molecular tools, such as whole-genome sequencing, could provide more information on the biodiversity of microorganisms in these environments. Overall, these findings highlight the importance of considering various sampling methods and assays in the assessment of indoor microbial contamination in the poultry industry [,]. Studies on bioaerosols in poultry production are limited and identifying all organisms, both viable and non-culturable, is important for characterizing bioaerosols in these facilities []. Inhalation exposure to non-viable microorganism components such as endotoxins and mycotoxins may cause health hazards, so evaluating non-viable components may be useful for assessing pulmonary disease risk. Microbial assessment of poultry farms shows the presence of numerous microbes, including zoonotic pathogens, which can act as transport agents of airborne diseases [,]. Despite the growing threat of fungal infections to human health, there are fewer studies conducted on fungi (and also viruses) compared to bacteria, and this lack of attention and resources makes it challenging to determine the precise burden of fungal infections and to encourage policy and programmatic action [].
Due to the extensive use of antibiotics in the livestock industry, these facilities are significant sources of antibiotic resistance genes (ARGs). Therefore, multidrug-resistant bacterial pathogens may be transmitted through the inhalation of bioaerosols []. This explains the frequent screening for bacterial antimicrobial susceptibility by several studies [,,,,,,,,,,,].
Several potentially pathogenic bacteria have been identified [,,,]. The potential dispersal pattern and distance of airborne bacteria and ARGs from these animal sources remain unknown []. However, it is important to note that clinically significant multidrug-resistant bacteria Staphylococcus sp. [,], E. coli [], Campylobacter jejuni [], among others, belonging to the WHO priority pathogens list of antibiotic-resistant bacteria (2017), were isolated from poultry farms.
Recently, the World Health Organization (WHO) published the first fungal priority pathogens list [], listing 19 groups of human fungal pathogens associated with a high risk of mortality or morbidity. This formal recognition by the WHO highlights an important group of infections, which has been perennially neglected in terms of the awareness and research funding needed [].
Regarding fungal assessment, despite the low number of studies (14 out of 58, 24%), several fungi comprising the critical priority group of the WHO list (2022), namely, A. fumigatus, were frequent in indoor air samples [,,], along with Candida albicans []. Regarding the high-priority group, Fusarium sp. [,,], the order Mucorales [,], and Candida tropicalis [] were also some of the ones reported.
Concerning microbial components, endotoxin, a major component of the outer membrane of Gram-negative bacteria, poses a serious health risk []. Endotoxins found in airborne organic dust have been linked to respiratory disease in both humans and animals [].
Regarding mycotoxins, some of the literature already evidenced occupational exposure in animal production facilities []. In fact, fungal species recognized as mycotoxin producers were reported in some of the selected studies [,,,]. Even though only one of the selected studies performed mycotoxin assessment, the obtained results are enough to hypothesize that workers in these settings may be at a higher risk of Aspergillus mycotoxicosis. Indeed, elevated concentrations of A. flavus and A. versicolor were recovered through environmental sampling. Additionally, through human biomonitoring, analysis of mycotoxins and/or their metabolites in blood and urine evidence detectable levels of the carcinogenic mycotoxin AFB1 [].
Briefly, to mitigate and decrease such pollutants it is crucial to establish international standards for what constitutes good microbiological indicators from environmental samples, which could be used to guide risk reduction decisions and create effective incentives for people to follow such guidance, which have already been suggested [].
Globally, temperature rises due to climate change have various impacts on ecosystems, human health, animal health, and food production, which also affect AMR [].
The emergence of resistant fungal strains in occupational exposure scenarios has already been demonstrated [,]. Indeed, temperature increases may influence the susceptibility of pathogens (bacteria, fungi, and parasites) in chicken environments []. Thus, as in the case of bacteria, antifungal resistance should be addressed in further research [,]. Additionally, it is crucial to investigate the effects of heat stress on poultry production to formulate various effective mitigation strategies to reduce significant production losses [].
The prevalent airborne microorganisms in animal production buildings are not well characterized in terms of quantity, composition, and risk group. Identification and quantification would be useful for determining the causative agents and performing risk assessments [].
The poultry industry must be sustainable, and it needs to produce more with less, while benefiting all []. The sector must improve human, animal, and environmental health and welfare. Implementing a comprehensive and coordinated One Health approach that incorporates exposure assessment can help tackle threats to health and ecosystems [], ensuring priority areas for action in order to mitigate microbial exposure, promoting a safe environment for workers and animals in poultry facilities, along with less environmental impact.
Overall, these findings highlight the need for improved biosecurity measures and environmental management practices to ensure animal health, food safety, and environmental sustainability in the poultry industry.

5. Conclusions

This review allowed us to identify microbiological contamination reported in the poultry industry, sampling methods and assays already employed to assess occupational exposure to microbial contamination within different scopes (occupational health, food safety, and animal health), and knowledge gaps to be tackled in future studies.
Poultry workers are exposed to several microbial contaminants in their workplace. Exposure to bacteria and fungi has been assessed and reported, as well as bacterial metabolites (namely endotoxins), β-glucans, and mycotoxins. Occupational exposure to microorganisms is a frequent concern, and the risk of exposure to potential pathogenic and resistant bioaerosols originating in poultry facilities is emphasized. Future research should aim to identify the main sources of contamination in this setting.
A One Health approach is a vital framework and the use of effective risk assessment tools and strategies can help prevent occupational exposure and protect the health of workers, consumers, and animals.

Supplementary Materials

The following supporting information can be downloaded at: https://www.mdpi.com/article/10.3390/toxics11040374/s1, Table S1: PRISMA checklist.

Author Contributions

Conceptualization, B.G. and C.V.; methodology, B.G. and C.V.; following analysis, B.G., R.C., P.P. and M.D.; investigation, B.G. and C.V.; resources, B.G. and C.V.; writing—original draft preparation B.G., M.D., C.V., M.V.P. and J.S.; writing—review and editing, B.G. and C.V.; supervision, C.V.; funding acquisition, B.G. All authors have read and agreed to the published version of the manuscript.

Funding

This work was supported by national funds through FCT/MCTES/FSE/UE, UI/BD/153746/2022, and CE3C unit UIDB/00329/2020 within the scope of a PhD grant. H&TRC authors gratefully acknowledge the FCT/MCTES national support through UIDB/05608/2020 and UIDP/05608/2020.

Institutional Review Board Statement

Not applicable.

Data Availability Statement

No applicable.

Conflicts of Interest

The authors declare no conflict of interest.

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