key: cord-0799464-rou248d1 authors: Pena, Pedro; Morais, Joana; Gomes, Anita Quintal; Viegas, Carla title: Sampling methods and assays applied in SARS-CoV-2 exposure assessment date: 2021-02-17 journal: Sci Total Environ DOI: 10.1016/j.scitotenv.2021.145903 sha: e9cb7ecef5b6553ce36cd302b2a0dc2644868ac5 doc_id: 799464 cord_uid: rou248d1 The SARS-CoV-2 exposure assessment is critical to implement control measures and guarantee safety of patients and workers from different occupational environments. The aim of this review article was to identify methodologies applied for SARS-CoV-2 sampling and analyses in environmental samples in different occupational and indoor environments. This study reports the search of available data published between May 29th, 2020 and November 1st, 2020. The search strategy used allowed the identification of 48 papers that comply with selected inclusion and exclusion criteria. The most described indoor environment consisted of health care facilities. From all the analyzed studies, 34 sampled surfaces, 27 sampled air (impactors and impingers being the most used), and 9 sampled water. All studies were based on molecular detection by qPCR of viral RNA extracted from collected samples. SARS-CoV-2 was detected in 44 out of the 48 studies. The results suggest that the sampling approach should include both active and passive sampling methods in order to overcome each method limitations. Concerning the assays used, although most studies were based on qPCR detection, the fact that the digital PCR technique allows SARS-CoV-2 detection at lower concentrations, indicates that this should be the chosen method for future detection studies. The diagram describes the different phases of the selection of papers and the papers that were obtained in final phase (Figure 1 ). The most described indoor environment were health care facilities (35 out of 48), followed by different environmental matrices: 9 wastewater treatment plants, rivers and household, 1 cruise, 1 household environment and 2 industrial occupational environments (Table 2) . From all the analyzed studies, 34 sampled surfaces, 27 sampled air and 9 sampled water ( Table 2) . Concerning sampling methods, all surface sampling (34) was collected with swabs and all water samples (9 mentioned above) were collected in to sterile containers. Regarding air Records identified through database searching (n=292) Additional records identified through other sources (n=12) Records after duplicates removed (n=304) Records screened (n=304) Full-text articles assessed for eligibility (n=79) Full-text articles excluded for not meeting the inclusion criteria (n=31) Studies included in qualitative synthesis (n=48) (Table 2) . In all the studies, molecular tool kits were used for RNA extraction which was then subject to detection by PCR methods (Table 2) . SARS-CoV-2 was detected in 44 out of the 48 articles (Table 2) . Considering the environmental matrixes analyzed, SARS-CoV-2 was detected in all the 9 articles that sampled water; in 19 of 27 articles that sampled air and in 31 of the 34 articles that sampled surfaces (Table 2 ). Some discussion has been raised among industrial hygienists concerning the sampling and analyses methods for SARS-CoV-2 exposure assessment. Studies focusing on virus exposure assessment have been critically limited. This is mainly due to the difficulties in collecting and analysing airborne viruses. Among the active sampling methods, several sampling devices can be used to assess the airborne virus, being the most common the impactors and impingers, as well as filters and electrostatic precipitators (Verreault et al., 2008) (Table 2) . Besides active sampling methods (air sampling), also passive methods, such as swabs, can be used. In fact, this was the sampling method mostly used in the selected papers corroborating its importance in the assessment of bioburden exposure (comprising fungi and bacteria) in health care facilities (Viegas et al., 2019) and in other indoor environments (Viegas et al., 2020) . Concerning active sampling methods, it should be stressed that longer active sampling volumes ranging from 60 L (Zhang et al., 2020) to 54720 L (Setti et al., 2020) resulting in either negative or positive detection of SARS-CoV-2 independently of the sampling method used. From these studies the standard consensual condition is the use of an airflow rate of 200 L/min and the minimal of 1 m 3 of air during each sample collection when using Coriolis μ (impinger method device) for SARS-CoV-2 assessment (Bertin Instruments, 2020). However, the sampling duration can affect the integrity of the virus structure and decrease their infectivity, being these drawbacks more emphasized on filters samples (Verreault et al., 2008) . In fact, every virus and strains have a unique response to environmental factors, increasing the difficulty to select the optimal sampling device and conditions (Verreault et al., 2008) . Concerning the assays used to detect SARS-CoV-2, these were mostly based on onestep reverse transcriptase quantitative PCR detection (RT-qPCR), which is much faster than traditional PCR methods (Carter et al., 2020; Chan et al., 2020) . The samples were extracted with different extraction kits/reagents depending on the matrix, with some of them, namely water samples, being subject to concentration prior to analysis. One to three sets of probes for different SARS-CoV-2 viral genome regions were usually used in each assay, with positive results reporting to the amplification of all the regions subject to analysis in each particular study. The CT or cycle threshold that was considered a cut-off, above which samples were considered negative, varied within the studies, ranging from CT 38 to CT 43. A few studies (e.g. Liu et al., 2020 and Gonzalez et al., 2020) have used the recently developed digital PCR technique, which has higher sensitivity and accuracy when compared to standard RT-qPCR, allowing the detection of viral nucleic acid present at low concentrations. With this method, quantification is achieved without the need of PCR cycle threshold values or standard curves. Instead, a PCR sample is portioned into droplets, with each droplet containing the target sequence being detected by fluorescent and considered positive, allowing absolute quantitation of target sequence. As the abundance of viral particles in the environment is usually low, future studies should consider this approach do detect SARS-CoV-2 nucleic acid in environmental samples. include active (with an air volume of, at least 1m 3 , per sample) and passive sampling methods to overcome each method limitations. Concerning the assays used to detect SARS-CoV-2, these were mostly based on onestep reverse transcriptase quantitative PCR detection (RT-qPCR), but an increase in digital PCR technique is expected, since it allows SARS-CoV-2 detection at lower concentration ranges.. This work was supported by Instituto Politécnico de Lisboa, Lisbon, Portugal for funding the Projects "Occupational exposure of ambulance drivers to bioburden" (IPL/2020/BIO-AmbuDrivers_ESTeSL) and "PL Zero Moment: Ensuring the academic activities during pandemic crises". H&TRC authors gratefully acknowledge the FCT/MCTES national support through the UIDB/05608/2020 and UIDP/05608/2020. None. I have full control of all primary data and permission is given to the journal to review the data if requested. Wei, L., Lin, J., Duan, X., Huang, W., Lu, X., Zhou, J., Zong, Z., 2020. Asymptomatic COVID-19 The authors certify that they have NO affiliations with or involvement in any organization or entity with any financial interest (such as honoraria; educational grants; participation in speakers' bureaus; membership, employment, consultancies, stock ownership, or other equity interest; and expert testimony or patent-licensing arrangements), or non-financial interest (such as personal or professional relationships, affiliations, knowledge or beliefs) in the subject matter or materials discussed in this manuscript. This statement is signed by the correspondent author on behalf of all authors. 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