key: cord-1028196-sd8bqjpt authors: Hu, Qiaoyun; Ma, Pei; Hong, Junyi; Wang, Yulong; Huang, Dong; Tan, Yadi; Yu, Zhengjun title: Thermal Fogging with Disinfectant Didecyl Dimethyl Ammonium Bromide Effectively Kills A Coronavirus, An Influenza Virus and Two Indicator Bacteria in Subzero Cold-Chain Environment date: 2021-03-26 journal: bioRxiv DOI: 10.1101/2021.03.25.436894 sha: 75e3a487ba3a0ed42dabae282530d715170f2e56 doc_id: 1028196 cord_uid: sd8bqjpt Background The origin of several local COVID-19 outbreaks in China in 2020 were confirmed to be frozen food or packages contaminated with SARS-CoV-2, revealing the lack of effective disinfection measures in the frozen food chain. Objective To evaluate the disinfection efficacy at −20°C of a recently marketed thermal fogging disinfection product, with disinfectant-antifreeze combination being didecyl dimethyl ammonium bromide (DDAB) - propylene glycol (PPG). Method Carriers with porcine epidemic diarrhea virus (PEDV) YN1, a coronavirus, and a swine influenza virus H1N1 (SIV-H1N1), and three indicator bacteria, E. coli, S. aureus, and B. subtilis endospores, were respectively placed in a −20°C freezer warehouse with or without DDAB-PPG fogging and activities of the microorganisms were tested. Results At −20°C, DDAB-PPG fogging, which fully settles in 3.5−4.5 hours, fully inactivated PEDV of 10−3.5 TCID50/0.1ml and SIV-H1N1 of 26 hemagglutination titer within 15-30 min, and inactivated S. aureus and E. coli vegetative cells (106/ml) within 15 or 60 min, respectively, but had little effect on B. subtilis spores. The bactericidal effect lasted at least 3 hours for bacteria on carrier plates and for 6 hours for airborne bacteria. Conclusions A practical subzero temperature disinfection technology was confirmed its efficacy in killing enveloped viruses and vegetative bacteria. It would help to meet the urgent public health need of environmental disinfection in frozen food logistics against pandemic and other potential pathogens and to enhance national and international biosecurity. Highlights First report of efficacy of disinfectant didecyl dimethyl ammonium bromide (DDAB) at subzero temperatures The customized thermal fogging machine makes fine droplets of DDAB-propylene glycol (PPG) fog which can suspend for ~4 hours at −20°C DDAB-PPG thermal fogging at −20°C effectively inactivated a coronavirus and an influenza virus DDAB-PPG thermal fogging at −20°C for effectively killed S. aureus and E. coli DDAB-PPG thermal fogging at −20°C inactivated airborne microorganism for up to 6 h Facing the situation, China issued a number of technical guidances on food cold-chain disinfection 9 , 74 including Technical guidance for prevention and control of COVID-19 in cold food production chain and 75 Technical guidance for disinfection in cold food production chain to prevent and control COVID-19" Guidance [2020]-245 (Oct 16, 2020) 10 and "Work plan for preventive and comprehensive disinfection of imported cold-77 chain food" Guidance [2020]-255 (Nov 8, 2020) 11 . A key focus of these guidelines is preventive disinfection 78 of food product packaging and interior surfaces of shipping containers, particularly in the frozen food 79 import chain from port to market, instead of food content within packaging. These guidelines require 80 disinfection be carried out at critical steps and efficacy of disinfection checked, and call for "sum up good 5/18 experience and practice in SARS-CoV-2 detection and disinfection" instead of giving specifics on how to 82 disinfect, particularly for the frozen chain. Thus it can be said that the disinfection practices in the frozen 83 food chain are still being explored and optimized. Many disinfection products have been used since the 84 COVID-19 pandemic, but data are needed to support their efficacy and continued use in the frozen food 85 cold-chain. The concentration of DDAB-PPG fog used in the study are listed in Table 1 , which included two 115 concentrations,~0.4 g/m 3 and~1.0 g/m 3 , corresponding to~8 or~20 min of fogging. A photo of the 116 fogging machine in operation is shown in Figure 1 . The PEDV virus stock was prepared to be 10 -3.5 TCID 50 /0.1ml. Carrier polystyrene plates were prepared by 126 dropping 60 μl virus stock in each clean plate and let air dry, which were placed either on the floor or on a 127 shelf in the freezer warehouse and exposed to disinfection thermal fogging for 15-60 min as indicated. Some plates served as freeze-only controls. The plates were taken out, rinsed with 1.2 ml DMEM 129 (containing 2% penicillin and streptomycin), and the eluents were collected and stored at -80C freezer for 130 TCID 50 assay later, which is done according to previous described method. with 0.85% saline, counted, and then diluted to 10 6 cfu/ml saline. Then 100 μl each was spread on a 60 mm 149 diameter empty PS plate, let air fry in biosafety hood and then covered. Carrier plates were placed in a - 20°C freezer warehouse either on the floor (2 sites) or on a shelf (1.2 m height, 2 sites) at 2-3 plates each 151 site, and exposed to disinfection fog for 15-60 min as indicated. The disinfection efficacy of DDAB fog for PEDV and the 3 bacteria were assessed by calculating the log 10 TCID 50 /0.1ml or cfu and comparing fog exposed samples to the starting value or freezer only controls. Reduction of >=3log10 is considered qualified. For SIV H1N1, the infectivity is measured by HA titer which is The long hours of suspension gives the disinfectant fog ample time to act on all surfaces exposed and 175 arrive hard-to-reach spots. In addition, based on the settling speed (5 meter high/~3.5 h at RT) the fog 176 droplet size can be estimated by Stokes law, which should give the droplet size around 4~6 μm 14 . In A, the carrier plates were exposed for 1 h (n=6); in B, the sterile plates were exposed for 15 min (n=6). The advantages of the technology presented in this study are summarized below. First, thermal fogging technology disperses the disinfectants throughout the air and into hard-to-reach 249 spots to achieve dead-zone free disinfection of exposed surfaces of all angles, and it is likely to be able to 250 eliminate all airborne viruses as the fine fog droplets permeate the whole space for hours. Second, the product uses familiar chemicals with excellent safety profile. The disinfectant chosen, DDAB, is officially approved and recommended in recent disinfection guidelines 254 for SARS-CoV-2 and had many years of use in numerous products. The fogging fluid, PPG, which also 255 function as antifreeze, is often used to make artificial smoke and mists for fire safety training, theatrical 256 performances and rock concerts, and has been recognized as safe 17 . Third, minimal human labor is required for using the machine-reagent product set. The reagents are ready to use and no dilution with water is required which reduces workload and chances 260 for errors. Once set and powered on, the machine will run and stop automatically. As seen in Figure 1 Cold-chain food contamination as the possible origin of COVID-19 resurgence in Beijing Cold-chain transportation in the frozen food industry may have caused a recurrence of COVID-19 cases in destination: Successful isolation of SARS-CoV-2 virus from the imported frozen cod package surface COVID-19 Among Workers in Meat and Poultry Processing Facilities -19 States KUCB (2020) Positive COVID-19 results grow to 92 aboard factory trawler Seeding outbreaks of COVID-19 by contaminated fresh and frozen food, bioRxiv (2020) Persistence of SARS-CoV-2 in the environment and COVID-19 transmission risk from environmental matrices and surfaces Key characteristics of foods with an elevated risk for viral enteropathogen contamination COVID-19 transmission in cold chain: A safe and green new-generation cold chain is demanded Controlling COVID-19 Transmission due to Contaminated Imported Frozen Food and Food Packaging The Joint Prevention and Control Mechanism of the State Council of the People's Republic of China. 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