SARS-CoV-2 outbreak investigation in a German meat processing plant.

Accepted Article This article has been accepted for publication and undergone full peer review but has not been through the copyediting, typesetting, pagination and proofreading process, which may lead to differences between this version and the Version of Record. Please cite this article as doi: 10.15252/emmm.202013296 This article is protected by copyright. All rights reserved SARS-CoV-2 outbreak investigation in a German meat processing plant Thomas Günther1§, Manja Czech-Sioli2, Daniela Indenbirken1, Alexis Robitaille1, Peter Tenhaken3, Martin Exner4, Matthias Ottinger5, Nicole Fischer2*, Adam Grundhoff1§*, Melanie M. Brinkmann6,7* 1Heinrich Pette Institute, Leibniz Institute for Experimental Virology, Hamburg, Germany; 2Institute for Medical Microbiology, Virology and Hygiene, University Medical Center Hamburg-Eppendorf, Hamburg, Germany; 3Health Office, Osnabrück, Germany; 4Institute of Hygiene and Public Health, University of Bonn, Bonn, Germany; 5Omikron Systems GmbH, Braunschweig, Germany; 6Viral Immune Modulation Research Group, Helmholtz Centre for Infection Research, Braunschweig, Germany; 7Institute of Genetics, Technische Universität Braunschweig, Braunschweig, Germany §equal contribution * Corresponding authors: Melanie M. Brinkmann, m.brinkm[email protected], phone: +49-531-6181-3069; Nicole Fischer, [email protected], phone: +49-40-741055171; Fax: +49-40-7410-53250; Adam Grundhoff, [email protected]; phone: +49-40-48051-275; Fax: +49-40-48051-277 Character count of the manuscript: 25,613 characters/4,732 words Accepted Article This article is protected by copyright. All rights reserved Abstract We describe a multifactorial investigation of a SARS-CoV-2 outbreak in a large meat processing complex in Germany. Infection event timing, spatial, climate and ventilation conditions in the processing plant, sharing of living quarters and transport, and viral genome sequences were analyzed. Our results suggest that a single index case transmitted SARS-CoV-2 to co-workers over distances of more than 8 meters, within a confined work area in which air is constantly recirculated and cooled. Viral genome sequencing shows that all cases share a set of mutations representing a novel sub-branch in the SARS-CoV-2 C20 clade. We identified the same set of mutations in samples collected in the time period between this initial infection cluster and a subsequent outbreak within the same factory, with the largest number of confirmed SARS-CoV-2 cases in a German meat processing facility reported so far. Our results indicate climate conditions, fresh air exchange rates, and airflow as factors that can promote efficient spread of SARS-CoV-2 via long distances and provide insights into possible requirements for pandemic mitigation strategies in industrial workplace settings. Keywords Aerosol transmission/meat processing plant outbreak/SARS-CoV-2 super spreading event/viral genome sequencing Accepted Article This article is protected by copyright. All rights reserved Introduction The first wave of SARS-CoV-2 infections peaked in Europe from March to mid of May 2020. Implementation of social and physical distancing measures resulted in declining infection numbers in most European countries. Currently, countries seek to implement alternative measures, for example infection management focused on hotspots, contact tracing and sentinel testing. Given this, it is important to immediately follow up on local infection clusters to prevent re-emergence of large-scale community transmission as seen during the first wave of SARS-CoV-2 infections. Transmission of SARS-CoV-2 is thought to mainly occur via respiratory uptake of droplets (van Doremalen, Bushmaker et al., 2020) or aerosols. Aerosols are believed to be particularly important in cases where a single source transmits the virus to a large number of individuals, so-called super spreading events (Dyal, Grant et al., 2020, On Kwok, Hin Chan et al., 2020, Schwierzeck, Konig et al., 2020, Xu, Liu et al., 2020, Yusef, Hayajneh et al., 2020, Zhang, Diao et al., 2020, Zhang, Li et al., 2020). Whereas droplets typically travel no farther than 2 m, aerosols can stay in the air for prolonged periods of time and may deliver infectious viral particles substantially beyond 2m distances, especially in indoor settings with low fresh air exchange rates (Asadi, Wexler et al., 2019, Asadi, Wexler et al., 2020, Liu, Li et al., 2017). Factors such as temperature, humidity and air circulation are thought to significantly influence stability and transport of droplets and aerosols and consequently transmission efficiency (van Doremalen et al., 2020). Meat processing plants have recently emerged as hotspots of SARS-CoV-2 around the world. This is thought to result not only from operational practices (e.g. close proximity of workers in the production line combined with physically demanding work that promotes Accepted Article This article is protected by copyright. All rights reserved heavy breathing), but also from sharing of housing and transportation that may facilitate viral transmission (Dyal et al., 2020). The requirement to operate at low temperature in an environment with low air exchange rates is another factor that may promote spread of the virus among workers. However, direct scientific evidence for the nature of transmission events in a meat processing plant or the role of shared housing and transportation has not been reported yet. Here, we report a transmission cluster in a German meat processing plant in May 2020 and provide data suggesting that environmental conditions promoted viral transmission from a single index case to more than 60% of co-workers within a distance of 8 meters. Viral sequence analyses revealed a previously unreported SARS-CoV-2 genotype that is not only shared by all individuals of the initial cluster, but also by samples collected shortly before a subsequent outbreak in mid-June, which represents the largest outbreak in a meat processing plant seen in Germany thus far. Our findings indicate that a physical distance of 2 meters does not suffice to prevent transmission in environmental conditions such as those studied here; additional measures such as improved ventilation and airflow, installation of filtering devices or use of high-quality face masks are required to reduce the infection risk in these environments. Results We studied an outbreak in the largest meat processing plant in Germany, located in RhedaWiedenbrück, county of Gütersloh, state of North Rhine Westfalia (referred to as MPP-R in the following). MPP-R performs slaughter and fine processing as well as packaging of Accepted Article This article is protected by copyright. All rights reserved beef and pork. A second, independently operated processing plant specialized on sow deboning (MPP-D in the following) is located in Dissen (county of Osnabrück, state of Lower Saxony), approximately 30 km away from MPP-R. Due to occasional SARS-CoV2 positive cases in the German meat industry, several state governments in Germany arranged SARS-CoV-2 PCR-based series testing of the entire staff of meat processing plants in May 2020, including MPP-D and MPP-R. Series of events preceding the outbreak in meat processing plant R (MPP-R) As shown in Fig. 1A, government mandated series testing in MPP-D and MPP-R took place in the week of May 11. Test results were reported on Sunday May 17. 94 out of 279 tested MPP-D employees were found to be SARS-CoV-2 positive, suggesting an ongoing outbreak among MPP-D workers. In MPP-R, only four out of a total of 6,289 employees were found to be positive. None of the four cases in MPP-R was involved in meat processing and the cases were judged to likely be independent. On Tuesday May 19 (Fig. 1B), two MPP-R workers from the early shift (referred to as cases B1 and B2 in the following) reported to the management of having had a brief contact with employees from MPP-D (D1 and D2 in the following) on Sunday May 17, both of whom had received positive test results later that day (Fig. 1A). Cases B1 and B2 reported to have no symptoms. SARS-CoV-2 outbreaks in MPP-R during May and June 2020 B1 and B2 were tested in the company’s test center on May 20 (Fig. 1B). Because the contact with MPP-D workers was not classified as high risk, both continued to work. On Accepted Article This article is protected by copyright. All rights reserved May 21, the early shift did not work due to a holiday. Upon receiving positive test results on May 21, B1 and B2 and five workers with whom they had shared an apartment were quarantined. B1 and B2 were moved to a separate apartment, whereas their flat mates remained in their original quarters. On Monday May 25, all remaining workers from the early shift (n=140) were tested. Test results from May 27 found 18 early shift workers to be positive. All early shift workers were immediately quarantined thereafter. Follow-up Accepted Article This article is protected by copyright. All rights reserved testing performed between May 27 and June 3 identified another 11 positive cases among the already quarantined workers. Following this outbreak in May, riskand evidence-based screening performed by health authorities, general practitioners and the internal MPP-R test center identified increasing numbers (>110) of positive cases across different parts of the plant in June, suggesting an ongoing and more wide-spread second outbreak event. Indeed, subsequent series testing by health authorities between June 17 and 23 identified more than 1,400 positive cases, constituting the largest outbreak in a German meat processing facility seen thus far (Fig. 1C). Viral genotypes in the May 2020 outbreak The timing of events suggested employees B1 and B2 as the most likely source(s) of the early MPP-R infection cluster. To further substantiate this hypothesis, we performed full viral genome sequencing of the 20 cases tested positive by May 27. In Fig. 2A, we present a heat map showing positions and color-coded frequency values of nucleotide deviations from the Wuhan SARS-CoV-2 reference strain. A total of eight exchanges were found with near 100% frequency across all samples. A search against 56,366 full length sequences available through GISAID identified six of these mutations to be commonly present in the 20C clade of SARS-CoV-2, a branch which accounts for approximately 17% of all SARS-CoV-2 sequences deposited in GISAID at the time of this writing. Interestingly, however, we did not find GISAID entries sharing the two remaining mutations (marked with asterisks in Fig. 2A; see Appendix Table S1 and Figure S3 for further details). Combined, the eight mutations therefore represent a novel Accepted Article This article is protected by copyright. All rights reserved sub-branch within the 20C clade that defines the prototypical viral genome signature of the infection cluster (submitted to GISAID, accession number 476705, strain id NRW-MPP1). Whereas the B1 sequence is an exact prototype representative, we find an additional nucleotide exchange at 100% frequency (C7735T) in B2. The fact that this mutation is absent from the other samples rules out B2 as a possible source of the cluster with near certainty. Another six cases also exhibit a single additional nucleotide exchange that is not shared with any other sample. Taken together, these observations suggest prototype virus transmission by B1 as the common source of infection in the cluster. Given the overall scarcity of non-prototypical nucleotide variants, the presence of additional exchanges most likely resulted from ongoing viral mutagenesis in a subset of newly infected individuals. However, the sequencing data alone cannot rule out the formal possibility that at least some of these variants represent independent infection events. Potential transmission routes in the May 2020 outbreak Given the above, we investigated potential transmission routes between the suspected index case B1 and the other employees within the cluster. The universal point of potential contact among all cases was work in the early shift of the beef processing plant. The shift comprises 147 individuals, most of whom work at fixed positions in a conveyor-belt processing line. The processing line occupies an elongated area approximately 32 meters (m) long and 8.5 m wide (see floor plan in Appendix Fig. S1A). Quarters of beef enter at one end of the line (referred to as proximal in the following) and are processed as they move in longitudinal direction across the room, until cuts are finally packaged near the far Accepted Article This article is protected by copyright. All rights reserved end of the line (referred to as distal in the following). Eight air conditioning units placed near the ceiling in the proximal half of the room constantly cool the air. Fans project the air in a lateral direction, either directly from frontal openings in the unit or via perforated Accepted Article This article is protected by copyright. All rights reserved committee issued a statement of no objection to publish the study under reference number 337/20. SARS-CoV-2 amplicon sequencing and bioinformatic analysis Sample preparation for SARS-CoV-2 amplicon sequencing was performed as described (J., 2020) with modifications (Pfefferle, Guenther et al., 2020). Primer sequences are provided in the Appendix Table S4. Samples were sequenced on an Illumina MiSeq using 500cycle MiSeq v2 reagent kits (Illumina). All samples were sequenced twice (including independent cDNA synthesis and library preparation reactions) to exclude the possibility of variant frequencies resulting from random amplification artifacts. Except for sample B14 (in which one sequencing reaction was excluded due to insufficient quality), reported variant frequencies reflect the average values from independent replicates. Bioinformatic analysis was performed as described (Pfefferle et al., 2020), (see Appendix Table S5 for details on the amplicon-Seq statistics), with the following modifications: Input thresholds were set to at least 10 variant supporting reads with a minimum base quality of 30 (-C10 - q30). Only high confidence variants present in > 20% of reads in at least one individual sample were included and annotated using ANNOVAR 16. Minor frequency variants resulting in frame shift, stopgain or startloss were excluded. Comparison of viral genotypes with GISAID database entries We performed a blast search of the prototypical NRW-MPP-1 genotype identified in this study against all 56,366 sequences deposited in GISAID as of July 6, 2020. None of the entries contained the combination of the two nucleotide variants C6406T and G18972Athat Accepted Article This article is protected by copyright. All rights reserved are shared across all samples investigated in our study. As shown in Appendix Table S6, a very limited number (23 out of 56,366 sequences) contain one of the two mutations. Two samples from the US (collected on the same date as B1 and B2) also carry the variant C6406T, but additionally exhibit another 7 and 8 mutations. These samples clearly belong to a different sub-branch of clade 20C defined by a previously introduced mutation at position 27964. 21 samples from the UK also contain one of the two mutations but belong to the separate clade 20B. The occurrence of these variants in different clade identifies them as homoplasies and suggests that these isolates are not closely related to the NRWMPP-1 genotype. Statistical analysis P-values in Fig. 3B and Appendix Table S2 indicate the cumulative probability of infection rates among workers with fixed stations in the indicated distance ranges being equal or higher than observed, under the null hypothesis that the probability of any given individual being positive is independent of spatial location and reflects overall positive rates among workers with fixed stations around the index case (20 out of 78 = 25.6%; see Appendix Table S2). Similarly, for each shared unit in Fig. 3C and Appendix Table S3 we calculated p-values for infection frequencies being equal or higher than observed among all individuals who share one or more unit (22 out of 65 = 33.8%; please note that due to data protection regulations we cannot reveal which worker IDs belong to a shared unit). Cumulative probability mass values were calculated using the BINOM.DIST.RANGE function from Microsoft Excel for Microsoft 365 MSO (v16.0.12827.20328) with the following input values: probability p: average infection rate among workers with fixed Accepted Article This article is protected by copyright. All rights reserved stations (0.256) or workers sharing one or more unit (0.338), minimum number of successes s: observed number of positive workers in distance range or shared unit, trials t and maximum number of successes s2: : total number of workers in distance range or shared unit. Description of housing conditions, work area conditions, and working conditions Housing conditions: Many of the workers share apartments and those usually commute together to their workplace in vans organized by the company. The company provided us with anonymized information about the housing situation of the workers regarding information on shared apartments, bedrooms and carpools. The largest housing unit encompassed seven workers for the initial outbreak in May in the beef processing plant (see Appendix Table S3; note that due to data protection regulations we cannot reveal which workers belong to individual shared units). In addition, we collected information about the work area and the working conditions during our on-site visit on June 2 2020. During that visit, we visited the beef processing plant during operating hours accompanied by technical staff from MPP-R. Work area conditions: The plant comprises separate areas in which slaughtering and meat processing is performed. While slaughtering takes place at ambient temperatures with higher air exchange rates, beef and pork processing are performed in rooms cooled to approximately 10°C with a high proportion of recirculated cooled air. The beef processing plant has a size of 2,800 m2 and is 6.1 m high (Appendix Figure S1A-B). The entire room and the production line are cleaned and disinfected daily according to food hygiene regulations in Germany. On the day of the on-site visit, the temperature in area 1 and 2 in Accepted Article This article is protected by copyright. All rights reserved the beef processing plant ranged between 9.5-10.7°C and between 5.4-8.7°C in area 3. Relative humidity was measured to be 34% right below the cooling fans in area 1 and 68% in the remaining part of area 1, and between 67-71% in areas 2 and 3 (Appendix Figure S1A). Cooling fans are cooling recirculated air without filters (C1-8). C3-8 are connected to a perforated hose directed towards area 3 whereas C1 and C2 lack a hose. C1 and C2 turn on only when temperatures rise above 10°C. Cooled air is expelled through the hall up to approximately 12 meters. Cooling fans 3-8 are operating permanently and are expelling cooled air through attached perforated hoses. The air exchange rate for the entire beef processing plant is <1. This means that it takes more than one hour to have the air replaced by fresh air. Specification of the cooling fans is as follows: Manufacturer: Guenther AG & Co. KG, Fuerstenfeldbruck, Germany, Model: S-GGHF 50Hz, Type 050.1E/17-AS, capacity 18,6 kW, airflow 6440 m3/h, air throw 37 m, dimensions: Length 1363 mm, Height 747 mm, Depth 713 mm. Working conditions: The workers in the beef processing plant that are working on the platform (proximal side) and the connected processing line (starting in area 1 and ending in the middle of area 2) are trained for specific cuts and therefore have fixed workplaces (Appendix Figure S1). Hence, workers could be traced in detail during their working hours. While 5-6 workers handle the beef quarters entering the plant on the platform and prepare them for cutting, the quarters are then translocated onto three conveyor-belt processing lines where 24-25 workers separate the meat from the bones. Next, finer cuts are performed (shearing) by 26-27 workers. Towards the distal part of the plant as well as in area 3, the beef is packed into vacuum packaging (Appendix Figure S1B). While the production line Accepted Article This article is protected by copyright. All rights reserved workers have fixed workplaces, the supervisory staff has flexible workplaces and commutes within the beef processing plant. Shifts in the beef processing plant change once per day. The staff for early and late shifts are provided by two independent sub-contractors and hence no staff is exchanged between the shifts. A shift has two 30 min breaks and one break of one hour. During breaks, the workers from a shift visit the canteen. Workers do not have fixed seats in the canteen. Supervisory staff does not spend the break times together with the production line workers. The supervisors do not share housing or transport facilities with the production line workers. Measures implemented by MPP-R during SARS-CoV-2 pandemic With the onset of the SARS-CoV-2 pandemic, additional preventive measures for the production staff were imposed by MPP-R. The company adhered to the recommendations of the relevant occupational Health and safety guidelines (BGN “Ergänzung der Gefährdungsbeurteilung im Sinne des SARS-CoV-Arbeitsschutzstandards Branche Fleischwirtschaft”). Additional measures were developed and implemented by the company. Hygiene regulations like hand hygiene and one-way traffic in hallways were reinforced, and an internal multi-lingual information campaign was enrolled to raise awareness for prevention and self-detection of early COVID-19 symptoms. A body temperature thermo scanner was set up to check all employees’ body temperature entering the building. Workers have been made aware of the company’s SARS-CoV-2 test center and were motivated to report any events where they see themselves being at risk. Specific workplace assessments were performed to decipher possibilities to extend distances Accepted Article This article is protected by copyright. All rights reserved between workers. Simple one-layer face masks were made compulsory. Regulations were in place to prohibit rotation between working places for the workers. Measures in the canteen were imposed to reduce physical contact and to enforce that workers would spend their break times exclusively with workers from their own shift. Since the outbreak of the pandemic, the company managed to prevent intra-company infection chains until the event described in this paper. The implementation of the measures was audited on May 15 and May 29 by unannounced inspections of the Occupational Health and Safety Experts of the competent authority and on May 20 by the Occupational Health and Safety experts of the “Berufsgenossenschaft Nahrungsmittel und Gastgewerbe”. The company had set up their own test center for PCR-based SARS-CoV-2 testing in early May 2020. In the SARS-CoV2 test center trained staff takes oropharyngeal swap samples from workers and other staff. The samples were analysed by RT-qPCR in an accredited laboratory (Labor Kneißler GmbH & Co.KG, Burglengenfeld, Germany). Staff was tested based on self-reported symptoms, possible contacts to other infected persons, returning to work after more than 96 h absence from work, or based on risk-assessment of possible workplace contacts. Data availability Virus sequence data have been deposited at European Nucleotide Archive, ENA, https://www.ebi.ac.uk/ena/; Accession number: PRJEB40387. Virus sequences are also available at GISAID, www.gisaid.org, see Appendix Table S1 for the individual accession link information. Accepted Article This article is protected by copyright. All rights reserved Acknowledgments We thank Gereon Schulze Althoff, Dirk Moormann, Cathrin Becker, Uwe Golling, Holger Scholze and Dennis Junkmann from Tönnies Lebensmittel GmbH & Co. KG, RhedaWiedenbrück, Germany, for sharing information necessary for the outbreak investigation and the possibility to visit the site of the outbreak. We thank Johannes Knobloch and Martin Aepfelbacher, Institute for Medical Microbiology, Virology and Hygiene, UKE, and Thomas Schulz, Institute of Virology, Hannover Medical School, for helpful discussions on the manuscript. We also greatly appreciate the support of and valuable discussions with Anna Kebschull, Landrätin Osnabrück, Germany. We thank the county’s health authorities involved in the outbreak management for their support. This work is supported by the Helmholtz Association, Germany (W2/W3-090). MMB is supported by the SMART BIOTECS alliance between the Technische Universität Braunschweig and the Leibniz Universität Hannover, an initiative supported by the Ministry of Science and Culture (MWK) of Lower Saxony, Germany. AG is supported by the Federal Ministry of Health, Germany (HPI-COVID-19). Author Contributions TG, AG, MO, NF, and MMB designed the study. TG, AG, ME, MO, NF, and MMB performed literature search; AG, MO, NF, and MMB wrote the manuscript; TG, MCS, DI, AG, ME, MO, NF, PT, and MMB collected the data. TG, AR, and AG performed bioinformatic data analysis; TG, AG, MO, NF, and MMB generated the figures and tables, PT reviewed the manuscript. Accepted Article This article is protected by copyright. All rights reserved Conflict of Interest The authors declare that they have no conflict of interest. For More Information GISAID Initiative; https://www.gisaid.org ENA Browser - European Nucleotide Archive; https://www.ebi.ac.uk/ena Accepted Article This article is protected by copyright. All rights reserved The Paper Explained Problem Originating from an index case that had self-reported previous contact with potentially SARS-CoV-2 infected persons, we describe the first cluster of SARS-CoV-2 transmission in a meat processing plant in a confined working area. This infection cluster preceded a large SARS-CoV-2 outbreak in the same meat processing plant shortly thereafter involving 1,413 SARS-CoV-2 positive tested employees. Results By performing a multifactorial analysis of housing and commuting parameters along with spatial and climate conditions in the work area and viral genome sequencing, this study provides evidence that transmission occurred likely via airborne transmission of SARSCoV-2 over long distances in a confined area of the meat processing plant. Physical work and relatively low fresh air exchange rates together with continuous recirculation of cooled air may have favored the transmission of SARS-CoV-2 among employees. Impact Our study implicates that common operational conditions in industrial meat processing plants promote the risk of SARS-CoV-2 super spreading events. Additional measures such as improved ventilation, optimized airflow management, installation of filtering devices or the use of high-quality face masks are required to reduce the infection risk in these environments. References Accepted Article This article is protected by copyright. All rights reserved Asadi S, Wexler AS, Cappa CD, Barreda S, Bouvier NM, Ristenpart WD (2019) Aerosol emission and superemission during human speech increase with voice loudness. Sci Rep 9: 2348 Asadi S, Wexler AS, Cappa CD, Barreda S, Bouvier NM, Ristenpart WD (2020) Effect of voicing and articulation manner on aerosol particle emission during human speech. 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J Hosp Infect Pfefferle S, Guenther T, Kobbe R, Czech-Sioli M, Noerz D, Sauter R, Oh J, Kluge S, Oestereich L, Peldschus D, Indenbirken D, Huang J, Grundhoff A, Aepfelbacher M, Knobloch J, Luetgehetmann M, Fischer N (2020) Low and high infection dose Accepted Article This article is protected by copyright. All rights reserved Accepted Article This article is protected by copyright. All rights reserved