Electrostatic Dust Cloth: A Useful Passive Sampling Method When Assessing Exposure to Fungi Demonstrated in Studies Developed in Portugal (2018–2021)

Electrostatic dust cloths (EDC) have been widely used for microbiologic contamination assessment in different indoor and occupational environments. This paper reviews sixteen studies performed in Portugal between 2018 and 2021 for evaluating the exposure to microbiological agents and focusing on fungi using EDC as a passive sampling method. The findings suggest that EDC can be applied as a screening method for particulate matter-exposure assessment and as a complementary method to characterize microbial exposures in occupational environments. Overall, EDC should be included, side by side with other sampling methods, in sampling campaigns focused on exposure assessments due to the advantages such as the straightforward extraction protocol favoring the employment of different assays, which allows us to assess exposure to a wide range of microbial agents, and presents higher accuracy regarding the fungal diversity.


Exposure Assessment and the Use of Electrostatic Dust Cloths
Current sampling strategies used for microbial exposure assessment may ineffectively describe significant exposures. Even if we apply the state-of-the-art regarding analyses, the information can be biased if our sampling techniques are not properly selected [1]. Thus, it is critical to select the best sampling approach to allow the accurate measurement and identification of the microbiological agents present in the indoor environments to be assessed.
In a recent study performed by Adams et al. (2021) [2] in a school's environment and using electrostatic dust collectors (EDC) it was possible to identify the microorganisms related to inspection-based building moisture damage and then examine the links between those microbial exposures and health effects [2]. Indeed, this sampling method has been widely used for microbiologic contamination assessment in different indoor and occupational environments (Table 1). If the intention is to perform viability studies, the electrostatic cloth used should not be impregnated with any kind of biocide to avoid impairing the viability of the microorganisms viability.

Electrostatic Dust Cloths' Features
As all passive sampling methods, it allows a more integrated time exposure assessment (workshift, days, weeks, or months), since it can collect during different periods of time depending on the activities, work shifts, and expected contamination [2,15,17,18]. In fact, this sampling method can be applied for prolonged periods of time, and, because of that, they allow us to overcome the major drawback of short-term active air sampling, which is highly sensitive to large temporal fluctuations in the airborne microbial load that might be associated to specific events that occur only sporadic in a specific workplace or indoor environment [4]. They have a very low cost (petri dish and an electrostatic cloth) and do not require microbiological training to set up and can be applied by the study subjects themselves in their dwellings [4]; however, in the workplaces, the EDC should be placed by a trained technician to select the proper sampling sites considering the study aim and the most suitable surfaces (preferably elevated surfaces at the height of 1.5-2.5 m) avoiding sampling sites with major airflow disturbances [2,15,17,18]. EDC placed on an elevated surface, besides collecting particles over a known time period, allows capturing airborne dust instead of floor-based particles that may never become sufficiently airborne to contribute to human exposure by inhalation [6]. Previously, a study performed by Madsen and colleagues (2012) [3] reported the need to place the EDC on open surfaces during sampling and that obtained can be frozen at −80 • C with glycerol without disturbing the microorganisms' number considerably [3].

Studies Performed in Portugal
This paper reviews several studies performed in Portugal between 2018 and 2021 for assessing the exposure to microbiological agents using EDC as a passive sampling method ( Table 6). The conducted studies were exploratory, since they were the first efforts to assess exposure to microorganisms in different Portuguese indoor environments. In fact, different indoor environments were assessed: veterinary clinic, bakeries, dwellings, health care facilities (including ambulances for patients 'transportation), and firefighters' headquarters (FFH).
Fifteen studies were included in the table, two of them being dedicated specifically to the use of EDC, aiming to investigate the adequacy of this device for characterizing the distribution patterns and exposure concentrations of particulate matter and microbial contaminants [10,15]. In both of them, the suggested procedures reported previously [3] were followed.
EDC was applied only with one other sampling method in the studies performed in dwellings to avoid disturbance in the occupant's routine. In fact, from the four studies performed in dwellings, two filters of 47 mm diameter quartz fiber were also used to collect particulate matter [17,23]. In the remaining two, in one, PM2.5 and PM2.5-10 were measured with a medium volume sampler [16], and in the other, active sampling by impaction method was also performed [20].
EDC was one among the several sampling methods applied in the other studies reported in Table 6, presenting, in most of the studies, a higher number of passive methods than active sampling methods employed. In fact, besides being used in parallel with devices that allow air sampling, other different passive sampling methods were employed, such as surface swabs and settled dust and different environmental matrices (e.g., filters from HVAC, mops, cleaning cloths, uniform ranks, and identification badges) were collected depending on the indoor environment/setting being assessed [11][12][13][14].
The sampling period used for the EDC varied between studies. Fifteen days of sampling was followed in the two studies dedicated to bakeries, while in the other occupational environments, 30 days were applied. This difference was due to the expected microbial contamination in the assessed indoor environments. In the assessed dwellings, some constraints were faced due to the occupant's availability when the sampling period ended, and although 30 days of sampling was to be followed, an extended period of sampling was performed in some cases. This difficulty was overcome by applying a different formula, (CFU.m-2.day =(1 x/(3.14*EDC area))/days of sampling) (1) where the sampling days were considered, to obtain fungal densities [21]. This different approach for the fungal density's quantification was the one followed after the first study performed in dwellings [16]. The protocol used was common to all studies where the EDC was employed in the dedicated sampling campaigns and following the procedures previously published [3] and as follows: Each EDC cloth was washed with 20 mL 0.9% NaCl with 0.05% Tween80™ (Merck S.A, Lisbon, Portugal) by orbital shaking (250 rpm, 60 min, at room temperature), and 150 µL of the wash suspension was inoculated on to two different culture media: 2% malt extract agar (MEA) with 0.05 g/L chloramphenicol media and dichloran glycerol (DG18) agar-based media. After incubation of the plates with the selected media, bacteria and/or fungal densities were determined.
In all studies, the fungal contamination was characterized focusing on Aspergillus genera due to clinical and toxicological relevance from the Aspergillus sections [12,21], EDC provided information concerning fungal azole resistance in all the performed studies presented in Table 6, unveiling more data regarding this public and occupational health threat. Additionally, it was also possible to focus on Aspergillus sections indoors [12,21], as well as other fungal species, providing a more complete fungal characterization with a wider number of different fungal species being identified than the other active and passive sampling methods [13,14,17,18,20,31]. Although the bakeries setting presented the highest fungal contamination due to the role as contamination sources of the raw materials [10,14], the setting presenting the highest Aspergillus sp. contamination was the FFH due to the observed buildings damage and leakages [21]. Mycotoxin's detection [18,22] and cytotoxicity assessment using different cell lines [13,18,23] were also assays employed in enlarged studies dedicated to microbiologic agents.
Almost all the studies used culture-based methods and qPCR for the detection of Aspergillus sections. The exception, where only culture-based methods were applied, was in the study performed in 12 bakeries [10], the study performed in different settings where molecular identification was achieved [31], and a study concerning Aspergillus section Fumigati where the isolates were recovered by culture-based methods for further analyses [23]. In the studies where culture-based methods and qPCR detection were applied side by side, complementary results were obtained with higher detection of Aspergillus sections, mainly section Fumigati, by molecular tools. The sampling protocol in veterinary clinics should comprise active and passive sampling methods. Culture-dependent and independent methods should be used to achieve a more complete characterization of the microbial contamination. [11] Twelve bakeries To analyze the adequacy of EDC for identifying the distribution patterns and exposure concentrations of particulate matter and microbial contaminants in bakeries.
Passive sampling method (EDC N = 33) and Particle counts and size distribution (0.3 µm, 0.5 µm, 1 µm, 2.5 µm, 5 µm, and 10 µm) measurement Culture-based methods (bacteria and fungi) Higher EDC mass was significantly correlated with higher fungal load on DG18 and with particle size distribution in different dimensions Penicillium sp. (42.56%) was the most frequent fungi.
EDC was useful for identifying critical workplaces regarding exposure to particulate matter and microbial contamination. Results obtained suggest that EDC can be applied as a screening method in exploratory studies concerning particulate matter-exposure assessment and to quantify exposures in specific occupational environments. [10] Thirteen bakeries To assess workersé xposure to fungi and mycotoxins in Portuguese bakeries.
Active methods (Air impaction and impingement each N = 53) and passive (surface swabs N = 58, EDC N = 36 and settled dust N = 11) methods

Culture-based methods (fungi) and qPCR (Aspergillus sections)
A. section Fumigati presented 50% of prevalence on DG18. it was possible to detect section Fumigati in 7.4% on EDC samples A wide number of sampling methods (active and passive) and different assays (culture-based and molecular methods) should be employed to obtain a refined risk characterization regarding fungi and mycotoxins. Culture-based methods (bacteria and fungi) and qPCR (Aspergillus sections) In MEA A. section Fumigati was observed in smaller counts (0.01%) and it was identified in the cleaning supply room.
The EDC was useful for unveiling the microbial contamination on the assessed PHCC. [15] One Central Hospital from Oporto To assess the exposure to bioburden in one central hospital with a multi-approach protocol using active and passive sampling methods.
Mycotoxins and endotoxins profile were also assessed. Two cytotoxicity assays were conducted with two cell lines and in vitro pro-inflammatory potential was assessed Fumigati section was observed in all the samples where culture-independent tools were applied including EDC (100%, 15 samples out of 15).
A multi-approach concerning sampling and analysis methods should be applied in the hospital environment

Mycotoxins assessment and cytotoxicity profile was also performed
Aspergillus section Fumigati was detected in 7 EDC samples (7 out of 12; 58.33%).
The use of the two sampling methods-swabs and EDC-allowed us to obtain a more complete characterization of the microbial contamination. Culture-dependent and independent methods used side by side allow to perform an accurate characterization of the A. section Fumigati contamination. [18] Thirty-three dwellings and four schools To assess microbial contamination in the indoor microenvironments more frequented by children PM2.5 and PM2.5-10 was sampled with a medium volume sampler. EDC was placed in the living room (N = 33) and in the children's bedroom (N = 31), and in schools (N = 4) Culture-based methods (fungi and bacteria and azole resistance screening) and qPCR (Aspergillus sections) The fungal species most frequently found in bedrooms was Penicillium sp. (91.79%), while, in living rooms, it was found Rhizopus sp. (37.95%) was the most prevalent. Aspergillus sections with toxigenic potential were observed in bedrooms and living rooms and were able to grow on VOR.
Future studies, applying EDC sampling method coupled with PM assessment, should be performed to allow for a long-term integrated sample of organic dust.
in EDC ranged from 0 to 405.3 CFU/m 2 /day on MEA, while in DG18 Aspergillus species were not observed.
Bacterial increased during the sleeping period. Toxigenic fungal species and indicators of harmful fungal contamination, belonging to Aspergillus genera were identified indoors, as well as reduced susceptibility to antifungal drugs of some fungal species. [20] Thirty dwellings To assess the deposition rates of total settleable dust and microbial contamination in the indoor air of dwellings onto quartz fiber filters andEDC, respectively 47 mm diameter quartz fiber filters were exposed to collect particulate matter and EDC (N = 30) were used for microbial contamination characterization Culture-based methods (fungi and bacteria and azole resistance screening) and qPCR (Aspergillus sections) Fungal contamination ranged from 1.97 to 35.4 CFU m −2 day −1 in MEA, and from undetectable to 48.8 CFU m −2 day −1 in DG18. Penicillium sp. was the most found in MEA (36.2%) and Cladosporium spp. in DG18 (39.2%).
Settleable dust and fungal contamination were increased in dwellings with pets; Indicators of harmful fungal contamination were present indoors; Aspergillus section Candidi was identified in azole supplemented media (VOR and POS); Specific housing typologies and characteristics influenced the microbial contamination. [19]

Exposure Assessment: The Role and Advantages of the EDC
This study only focuses on studies performed in Portuguese indoor environments; however, several others using the sampling approach were mentioned, reporting also the features of the EDC as a sampling method. In addition, a previous review on the use of EDC was performed before this sampling method started to be used as a sampling resource in sampling campaigns [32]. EDC allows a wide array of assays to be employed afterward, featuring an enriched exposure assessment. This sampling method allows overcoming some of the limitations when facing the need to perform an accurate exposure assessment dedicated to fungi in the scope of indoor air quality or occupational health. Indeed, besides the features already mentioned, this sampling method has little impact on occupants/workers daily routine, being also suitable for enlarged studies dealing with several EDC and respective extracts.
Since during EDC extraction, the recovery of microbial contamination can be partially lost (as in all sampling methods) [3], the recommendation is to use more than one sampling method in exposure assessments [33], being the EDC a suitable sampling method to complement active sampling methods, as well as to be used in parallel with more specific passive methods, that can be adjusted to the indoor environment under study (for instance filters from forklifters´HVAC, identification badges from ambulances crew, . . . ).
Although the trend is to apply more refined molecular tools to obtain the fungal diversity from the indoor environment to be assessed [2], since the viable part constitutes only a reduced part of the total composition [34] in exposure assessments culture-based methods are still needed to be applied to allow: (a) guidelines and legal framework comparison [12,21]; (b) to draw conclusions regarding the inflammatory potential variation, since the inflammatory and/or cytotoxic potential can affect the fungal viability [35,36] and; (c) to recover isolates for azole resistance screening, sequencing and mutations detection [31,37].
The findings also suggest that EDC can be applied as a screening method for particulate matter-exposure assessment and as a complementary method to quantify fungal contamination exposures in indoor environments [10,13].

Conclusions
Overall, this study allowed us to conclude that EDC should be included in sampling protocols dedicated to performing exposure assessment due to several advantages raised: (a) straightforward extraction protocol obtaining a liquid sample favoring the employment of different assays; (b) possible to be used to assess exposure to a wide range of microbial agents (fungi and metabolites); (c) allows a higher accuracy regarding the fungal diversity when compared with other sampling methods (active and passive); (d) low cost and little disturbance in the workers/occupants´daily routines that can influence the assessment being performed.  Data Availability Statement: Data sharing is not applicable to this article as no datasets were generated or analyzed during the current study.