Evaluation of disinfection efficacy of vaporized hydrogen peroxide on foreign animal viruses

Summary

In this paper, the evaluation of the disinfection efficacy of equipment and surface of objects (potentially contaminated by foreign animal viruses) using vaporized hydrogen peroxide in the transfer chamber was performed. Different viral agents were studied in the experiment, including several representative families of the virus family ( Orthomyxoviridae Orthomyxoviridae , Reoviridae Reoviridae , Flaviviridae Flaviviridae , Paramyxoviridae Paramyxoviridae) infected from poultry and mammals. Herpesviridae herpesviridae, Picornaviridae picornavirus family, Caliciviridae Calicivirus and Rhabdoviridae Rhabdoviridae , with particular emphasis on the study of exotic animal viruses in Canada. The effect of vaporized hydrogen peroxide on the fumigation of different laboratory equipment is also the purpose of this experimental study. The virus suspension in the cell culture medium, egg white or blood was dried into a glassware and stainless steel vessel, and then exposed to vaporized hydrogen peroxide for 30 minutes, and the virus survival rate was evaluated. In all test virus under all conditions, the sterilization process the avian virus (avian influenza and Newcastle disease virus) titers decreased to 0ELD 50, the mammalian viruses (African swine fever African swine plague, bluetongue bluetongue, hog cholera hog cholera, pseudorabies phobia rabies, swine vesicular disease swine vesicular disease, vesicular exanthema of swine vesicular disease transfection, vesicular stomatitis viruses vesicular stomatitis virus) reduced to less than 1 TCID 50. The laboratory equipment was simultaneously exposed to hydrogen peroxide gas without any damage. For Biosafety Laboratories III, which treats foreign animal viral agents, vaporized hydrogen peroxide can be used as a safe and effective means of disinfection to eliminate potential viral contaminants.

Materials and Methods

Equipment: A hydrogen peroxide generator connected to a 18-cubic-foot stainless steel transfer chamber through a 1.5-inch polyethylene conduit. Two circulating fans are built into the transfer compartment to ensure an even distribution of vaporized hydrogen peroxide. The chambers are pressure monitored at each stage of sterilization, and temperature monitoring probes are placed at three different locations within the chamber for temperature monitoring. The transfer chamber is pressurized to 2.0 in. water column pressure and the attenuation rate is calculated to exceed 0.08 in./h by 18 hours of attenuation.

Cycle parameters: The biological sterilization system operates according to the program settings. With a 30% aqueous solution of hydrogen peroxide as a reagent, the injection rate of hydrogen peroxide was 2 g/min at a gas flow rate of 10 cfm, the injection time was 30 minutes, and it was maintained at a steady state of about 1.73 mg/l or 1211 ppm. Next, in the state where the air flow rate was 10 cfm, the time of the ventilation phase was 3.5 hours. In all cycles, the cabin maintains a negative pressure equivalent to 2.0 to 3.0 inches of water.

Biological indicator: The cells were placed in the transfer chamber using Bacillus stearothermophilus. When the cycle was over, the transfer chamber was opened and the indicator was collected, placed in tryptone soy broth medium, and cultured at 56 ° C for 7 days. The turbidity of the medium is a sign of bacterial growth. If the sterilization process kills 3.4*10 5 spores, the sterilization process is considered successful.

At the same time, eight different viruses from eight different virus families were used (Table 1). All viruses operate in a standard way. The virus is inoculated in one of three media: i. minimal essential medium with 5% fetal bovine serum (standard growth medium required for intracellular propagation of the virus); ii. allantoic fluid (viruses propagated in eggs) ); iii. Pig blood (a virus that is propagated in animal blood). After inactivation by vaporized hydrogen peroxide, the virus can be evaluated under two conditions: suspending the virus in a liquid or drying on a solid support.

Material Compatibility Testing: To ensure that vaporized hydrogen peroxide was successfully defined as a non-destructive biocide, several different types of facilities were placed in the experiment for exposure fumigation and then evaluated for damage. These include telephones, cameras, unused film, X-ray film, photographed Polaroid photos, computer hard drives (running five programs), laptops (running five programs), electric drills, watches, electronic timers, electric Aspirator.

result

Incubation of the collected biological indicator after the end of the cycle showed that all spores were inactivated and that no spore growth was observed in the medium, demonstrating that the sterilization cycle was successful.

Table 2 shows the effect of the sterilization process on the potency of different viruses. In addition to the pig cholera virus suspended in the blood, the sterilization process reduces the virus titer of the avian influenza virus from 8.5 or 9.6 half of the embryo lethal dose (ELD 50 ) to 0 ELD 50 , and the mammalian virus from 6.5 to 9.0 half of the tissue. The amount of cultured infection (TCID 50 ) was reduced to less than 1 TCID 50 . This observation will not change regardless of whether the virus is in liquid or dry conditions. For swine herpes virus, a single heating can reduce several log units of extractable virus. For cell culture of herpes virus, swine cholera virus, and vesicular stomatitis virus, single drying can result in extractable virus levels below 1 TCID 50 . Only the avian influenza virus in the blood, the pig cholera virus, and the Newtown chicken plague virus, pseudorabies virus, and vesicular stomatitis virus in the allantoic fluid remain more than one log unit of potency after drying and heating in the transfer compartment ( Fumigation without vaporization of hydrogen peroxide) (Table 2). In most cases, there is no significant difference between the amount of virus extracted from the glass article and the amount of virus extracted from the tempered product. Whether it is a virus in cell culture fluid or allantoic fluid, the rate of killing is almost the same after fumigation by vaporized hydrogen peroxide, as shown in the vesicular stomatitis virus shown in Table 2.

discuss

The results of this study show that high concentrations of vaporized hydrogen peroxide can be safely stored in confined spaces. The hydrogen peroxide concentration during the sterilization process was shown to reduce the viral titer of avian viruses to 0 ELD 50 and the viral titer of mammalian viruses to 1 TCID 50 . Previous studies have shown that B. stearothermophilus is the most resistant, so vaporized hydrogen peroxide can kill biological indicators, so it should be completely effective against microorganisms such as viruses that are less resistant. This is consistent with the findings of the scholar Klapes, who found that pathogenic bacteria, yeast, fungal spores, viruses and bacterial spores can be rapidly inactivated (3). The only exception is when the virus finds different results in the blood. Although 5% of burdock serum or egg white is also rich in protein, the possible explanation is that blood is a protein-rich substance that protects the virus from the oxidizing properties of the gas to some extent. It is also possible that the cells naturally contain peroxidase and catalase, and these endogenous enzymes of red blood cells can neutralize certain hydrogen peroxide or all hydrogen peroxide, making the sterilization process ineffective. Biosafety laboratories III or IV in various parts of the world have traditionally used formaldehyde to treat foreign viruses. This study shows that vaporized hydrogen peroxide can replace formaldehyde well. Vaporized hydrogen peroxide is very effective in killing viruses during fumigation without destroying any laboratory equipment, making it the best disinfection option for Biosafety Laboratories III or IV.

references:

[1] Block, SS 1991. Peroxygen compounds, p. 167–181. In SS Block (ed.), Disinfection, sterilization and preservation, 4th ed. Lea and Febiger, Philadelphia, Pa.

[2] Johnson, JW, JF Arnold, SF Nail, and E. Renzi. 1992. Vaporized hydrogen peroxide sterilization of freeze dryers. J. Parenter. Sci. Technol. 46:215–225.

[3] Klapes, NA 1990. New applications of chemicals germicides: hydrogen peroxide, abstr. 20, p. 14–15. In Program and abstracts of the ASM International Symposium on Chemical Germicides. American Society for Microbiology,
Washington, DC

Etc.

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