A Comparative Study of Methods To Validate Formaldehyde Decontamination of Biological Safety Cabinets

Methods of validation of formaldehyde decontamination of biological safety cabinets were compared. Decontamination of metal strips inoculated with Mycobacterium bovis, poliovirus, or Bacillus spp. spores was compared with the results obtained with three biological indicators. Conditions for successful decontamination, particularly relative humidity, were defined. The Attest 1291 biological indicator was the only biological indicator which was an aid in the detection of gross decontamination failure.     

Liquid-phase study of ozone inactivation of Venezuelan equine encephalomyelitis virus.

Ozone, in a liquid-phase application, was evaluated as a residue-free viral inactivant that may be suitable for use in an arboviral research laboratory. Commonly used sterilizing agents may leave trace residues, be flammable or explosive, and require lengthy periods for gases or residues to dissipate after decontamination of equipment such as biological safety cabinets. Complete liquid-phase inactivation of Venezuelan equine encephalomyelitis virus was attained at 0.025 mg of ozone per liter within 45 min of exposure. The inactivation of 10(6.5) median cell culture infective doses (CCID50 of Venezuelan equine encephalomyelitis virus per milliliter represented a reduction of 99.99997% of the viral particles from the control levels of 10(7.25-7.5) CCID50/ml. A dose-response relationship was demonstrated. Analysis by polynomial regression of the logarithmic values for both ozone concentrations and percent reduction of viral titers had a highly significant r2 of 0.8 (F = 63.6; df = 1, 16). These results, together with those of Akey (J. Econ. Entomol. 75:387-392, 1982) on the use of ozone to kill a winged arboviral vector, indicate that ozone is a promising candidate as a sterilizing agent in some applications for biological safety cabinets and other equipment used in vector studies with arboviruses.     

Inactivation of Semliki Forest Virus in aerosols.

Purified Semliki forest virus in aerosols is inactivated rapidly at 40% and above 70% relative humidity. At all humidities tested the decay of virus infectivity runs parallel with the decrease in hemagglutination activity, whereas the biological integrity of the virus ribonucleic acid is preserved. Also, free infectious ribonucleic acid is stable after spraying at all relative humidities. Evidence is presented for the hypothesis that above 20% relative humidity, virus inactivation in aersols is mainly due to surface-dependent factors, damaging the virus coat.     

Physicochemical stability and inactivation of human and simian rotaviruses

The effects of various physical and chemical treatments on the stability of a human serotype 1 rotavirus and simian agent 11 (SA11) were compared by using a fluorescence focus assay. The infectivity of both strains was retained after storage at room temperature for 14 days, 4 degree C for 22 days, and -20 degree C for 32 days; lyophilization; and treatment at pH 3 to 11. Both viruses were inactivated at pH 12, as was the human virus at pH 2, although this pH resulted in only partial inactivation of SA11. The human virus also appeared to be more sensitive than SA11 to the action of ether and chloroform. The infectivity of both viruses was lost after UV irradiation for 15 min and after treatment with 8% formaldehyde for 5 min, 70% (vol/vol) ethanol for 30 min, and 2% lysol, 2% phenol, and 1% H2O2 for 1 h each.     

Physicochemical stability and inactivation of human and simian rotaviruses.

The effects of various physical and chemical treatments on the stability of a human serotype 1 rotavirus and simian agent 11 (SA11) were compared by using a fluorescence focus assay. The infectivity of both strains was retained after storage at room temperature for 14 days, 4 degree C for 22 days, and -20 degree C for 32 days; lyophilization; and treatment at pH 3 to 11. Both viruses were inactivated at pH 12, as was the human virus at pH 2, although this pH resulted in only partial inactivation of SA11. The human virus also appeared to be more sensitive than SA11 to the action of ether and chloroform. The infectivity of both viruses was lost after UV irradiation for 15 min and after treatment with 8% formaldehyde for 5 min, 70% (vol/vol) ethanol for 30 min, and 2% lysol, 2% phenol, and 1% H2O2 for 1 h each.     

Effects of temperature, relative humidity, absolute humidity, and evaporation potential on survival of airborne Gumboro vaccine virus

Survival of airborne virus influences the extent of disease transmission via air. How environmental factors affect viral survival is not fully understood. We investigated the survival of a vaccine strain of Gumboro virus which was aerosolized at three temperatures (10°C, 20°C, and 30°C) and two relative humidities (RHs) (40% and 70%). The response of viral survival to four metrics (temperature, RH, absolute humidity [AH], and evaporation potential [EP]) was examined. The results show a biphasic viral survival at 10°C and 20°C, i.e., a rapid initial inactivation in a short period (2.3 min) during and after aerosolization, followed by a slow secondary inactivation during a 20-min period after aerosolization. The initial decays of aerosolized virus at 10°C (1.68 to 3.03 ln % min(-1)) and 20°C (3.05 to 3.62 ln % min(-1)) were significantly lower than those at 30°C (5.67 to 5.96 ln % min(-1)). The secondary decays at 10°C (0.03 to 0.09 ln % min(-1)) tended to be higher than those at 20°C (-0.01 to 0.01 ln % min(-1)). The initial viral survival responded to temperature and RH and potentially to EP; the secondary viral survival responded to temperature and potentially to RH. In both phases, survival of the virus was not significantly affected by AH. These findings suggest that long-distance transmission of airborne virus is more likely to occur at 20°C than at 10°C or 30°C and that current Gumboro vaccination by wet aerosolization in poultry industry is not very effective due to the fast initial decay.     

Inactivation of adenovirus and simian virus 40 tumorigenicity in hamsters by vaccine processing methods

The effectiveness of the adenovirus vaccine inactivation process in destroying the tumorigenic potential for hamsters of adenoviruses, simian virus 40 (SV-40), and adenovirus-SV-40 hybrids was studied. Baby hamsters injected with untreated virus and with samples subjected to the complete inactivation process and to portions of the process were observed for tumor development for periods in excess of 300 days. Over 20,000 hamsters were injected. From 1 to 7 hr of exposure to formaldehyde at a concentration of 0.031 m at 37 C was sufficient to destroy the tumorigenicity observed in the nontreated preparations. Since the inactivation process included 48 hr of exposure at 37 C to 0.031 m formaldehyde plus treatment with ultraviolet (UV) and with beta-propiolactone (BPL), it was concluded that the process has a large margin of safety. Adenovirus isolates free from tumorigenic potential are difficult, if not impossible, to obtain. Therefore, a proven inactivation process appears to provide the best assurance for obtaining adenovirus vaccines free from such potential. Data presented suggest that the tumorigenic property of the viruses studied might be independent of the infectivity of the preparation. The tumorigenic property was found to be highly susceptible to formaldehyde, but less sensitive to BPL or UV treatment. In contrast, treatment with UV or BPL decreased viral infectivity more readily than tumorigenicity. The three-stage inactivation process (formaldehyde, UV, and BPL) inactivated both tumorigenicity and infectivity.     

Hydrogen peroxide vapour (HPV) inactivation of adenovirus

Aims: Adenovirus contamination can be problematic in various settings including life science laboratories and during pharmaceutical manufacturing processes. Stringent and effective decontamination procedures are necessary to minimize the risk of personnel exposure or product cross‐contamination in these settings. Hydrogen peroxide vapour (HPV) is sporicidal, tuberculocidal and fungicidal with proven efficacy against some viruses. We investigate the efficacy of HPV for the inactivation of a recombinant adenovirus. Methods and Results: In this study, the survival of a dried recombinant adenovirus (Ad5GFP) was tested before and after HPV exposure to determine the efficacy of HPV at inactivating adenovirus. A > 8‐log TCID₅₀ reduction resulted from 45‐min exposure to HPV in a microbiological safety cabinet. Conclusions: HPV is effective for the inactivation of a recombinant adenovirus. Significance and impact of the study: The results suggest that HPV may be useful for adenovirus decontamination in life science laboratories or in manufacturing facilities.     

The inactivation of foot and mouth disease, Aujeszky's disease and classical swine fever viruses in pig slurry

The aim of the study was to investigate the decontamination of pig slurry containing exotic viruses of pigs, foot AND mouth disease virus (FMDV), Aujeszky's disease virus (ADV) AND classical swine fever virus (CSFV). Laboratory-scale decontamination experiments showed that FMDV, ADV and CSFV were heat inactivated in slurry within 3 min at 67 degrees C, 3 min at 62 degrees C and 3 min at 60 degrees C and in Glasgow Eagles medium within 5 min at 67 degrees C, 4 min at 65 degrees C and 2 min at 65 degrees C, respectively. At pilot scale, FMDV was heat inactivated at 66 degrees C in water and 61 degrees C in slurry, ADV at 61 degrees C in water or slurry and CSFV at 62 degrees C in water and 50 degrees C in slurry. Treatment of pig slurry for the inactivation of exotic viruses may be achieved through the use of a thermal pilot plant operating in continuous mode. The work demonstrates the suitability of thermal treatment in ensuring the safety of pig slurry following a disease outbreak.     

Dynamics of airborne influenza A viruses indoors and dependence on humidity

There is mounting evidence that the aerosol transmission route plays a significant role in the spread of influenza in temperate regions and that the efficiency of this route depends on humidity. Nevertheless, the precise mechanisms by which humidity might influence transmissibility via the aerosol route have not been elucidated. We hypothesize that airborne concentrations of infectious influenza A viruses (IAVs) vary with humidity through its influence on virus inactivation rate and respiratory droplet size. To gain insight into the mechanisms by which humidity might influence aerosol transmission, we modeled the size distribution and dynamics of IAVs emitted from a cough in typical residential and public settings over a relative humidity (RH) range of 10-90%. The model incorporates the size transformation of virus-containing droplets due to evaporation and then removal by gravitational settling, ventilation, and virus inactivation. The predicted concentration of infectious IAVs in air is 2.4 times higher at 10% RH than at 90% RH after 10 min in a residential setting, and this ratio grows over time. Settling is important for removal of large droplets containing large amounts of IAVs, while ventilation and inactivation are relatively more important for removal of IAVs associated with droplets <5 μm. The inactivation rate increases linearly with RH; at the highest RH, inactivation can remove up to 28% of IAVs in 10 min. Humidity is an important variable in aerosol transmission of IAVs because it both induces droplet size transformation and affects IAV inactivation rates. Our model advances a mechanistic understanding of the aerosol transmission route, and results complement recent studies on the relationship between humidity and influenza's seasonality. Maintaining a high indoor RH and ventilation rate may help reduce chances of IAV infection.     

Effects of humidity and other factors on the generation and sampling of a coronavirus aerosol

Suspensions of transmissible gastroenteritis virus (TGEV), a porcine coronavirus, were nebulized at rates of 0.1–0.2 ml/min into moving air using a Collison nebulizer or a plastic medical nebulizer operating at pressures ranging from 7 to 15 psi. The airborne viruses were collected on heating, ventilating, and air conditioning (HVAC) filters in an experimental apparatus and also sampled upstream of these test filters using AGI-30 and BioSampler impinger samplers. To study the effects of relative humidity (RH) on TGEV collection by the filters and samplers, the virus was nebulized into air at 30, 50, 70, and 90% RH. There were no significant changes in virus titer in the nebulizer suspension before and after nebulization for either nebulizer at any of the pressures utilized. Aerosolization efficiency – the ratio of viable virus sampled with impingers to the quantity of viable virus nebulized – decreased with increasing humidity. BioSamplers detected more airborne virus than AGI-30 samplers at all RH levels. This difference was statistically significant at 30 and 50% RH. Nebulizer type and pressure did not significantly affect the viability of the airborne virus. Virus recovery from test filters relative to the concentration of virus in the nebulizer suspension was less than 10%. The most and the least virus were recovered from filter media at 30% and 90% RH, respectively. The results suggest that TGEV, and perhaps other coronaviruses, remain viable longer in an airborne state and are sampled more effectively at low RH than at high humidity.     

Inactivation of stable viruses in cell culture facilities by peracetic acid fogging

Looking for a robust and simple method to replace formaldehyde fumigation for the disinfection of virus-handling laboratories and facilities, we tested peracetic acid fogging as a method to inactivate stable viruses under practical conditions. Peracetic acid/hydrogen peroxide (5.8%/27.5%, 2.0 mL/m3) was diluted in sufficient water to achieve ≥ 70% relative humidity and was vaporized as <10 μm droplets in a fully equipped 95 m3 laboratory unit. High titers of reovirus 3, MVM parvovirus and an avian polyomavirus were coated on frosted glass carriers and were exposed to the peracetic acid fog in various positions in the laboratory. After vaporization, a 60 min exposure time, and venting of the laboratory, no residual virus was detected on any of the carriers (detection limit <1 infectious unit/sample volume tested). The log reduction values were 9.0 for reovirus, 6.4 for MVM parvovirus, and 7.65 for the polyomavirus. After more than 10 disinfection runs within 12 months, no damage or functional impairment of electrical and electronic equipment was noted.     

High Humidity Leads to Loss of Infectious Influenza Virus from Simulated Coughs

Background

The role of relative humidity in the aerosol transmission of influenza was examined in a simulated examination room containing coughing and breathing manikins.

Methods

Nebulized influenza was coughed into the examination room and Bioaerosol samplers collected size-fractionated aerosols (<1 µM, 1–4 µM, and >4 µM aerodynamic diameters) adjacent to the breathing manikin’s mouth and also at other locations within the room. At constant temperature, the RH was varied from 7–73% and infectivity was assessed by the viral plaque assay.

Results

Total virus collected for 60 minutes retained 70.6–77.3% infectivity at relative humidity ≤23% but only 14.6–22.2% at relative humidity ≥43%. Analysis of the individual aerosol fractions showed a similar loss in infectivity among the fractions. Time interval analysis showed that most of the loss in infectivity within each aerosol fraction occurred 0–15 minutes after coughing. Thereafter, losses in infectivity continued up to 5 hours after coughing, however, the rate of decline at 45% relative humidity was not statistically different than that at 20% regardless of the aerosol fraction analyzed.

Conclusion

At low relative humidity, influenza retains maximal infectivity and inactivation of the virus at higher relative humidity occurs rapidly after coughing. Although virus carried on aerosol particles <4 µM have the potential for remaining suspended in air currents longer and traveling further distances than those on larger particles, their rapid inactivation at high humidity tempers this concern. Maintaining indoor relative humidity >40% will significantly reduce the infectivity of aerosolized virus.     

Inactivation of caliciviruses

The viruses most commonly associated with food- and waterborne outbreaks of gastroenteritis are the noroviruses. The lack of a culture method for noroviruses warrants the use of cultivable model viruses to gain more insight on their transmission routes and inactivation methods. We studied the inactivation of the reported enteric canine calicivirus no. 48 (CaCV) and the respiratory feline calicivirus F9 (FeCV) and correlated inactivation to reduction in PCR units of FeCV, CaCV, and a norovirus. Inactivation of suspended viruses was temperature and time dependent in the range from 0 to 100 degrees C. UV-B radiation from 0 to 150 mJ/cm(2) caused dose-dependent inactivation, with a 3 D (D = 1 log(10)) reduction in infectivity at 34 mJ/cm(2) for both viruses. Inactivation by 70% ethanol was inefficient, with only 3 D reduction after 30 min. Sodium hypochlorite solutions were only effective at >300 ppm. FeCV showed a higher stability at pH <3 and pH >7 than CaCV. For all treatments, detection of viral RNA underestimated the reduction in viral infectivity. Norovirus was never more sensitive than the animal caliciviruses and profoundly more resistant to low and high pH. Overall, both animal viruses showed similar inactivation profiles when exposed to heat or UV-B radiation or when incubated in ethanol or hypochlorite. The low stability of CaCV at low pH suggests that this is not a typical enteric (calici-) virus. The incomplete inactivation by ethanol and the high hypochlorite concentration needed for sufficient virus inactivation point to a concern for decontamination of fomites and surfaces contaminated with noroviruses and virus-safe water.     

Influence of relative gas humidity on the inactivation efficiency of a low temperature gas plasma

Aims:  To investigate the effect of relative gas humidity on the inactivation efficiency of a cascaded dielectric barrier discharge (CDBD) in air against Aspergillus niger and Bacillus subtilis spores on PET foils.
Methods and Results:  The inactivation kinetics as a function of treatment time were determined using synthetic air with different relative humidity as the process gas. Spores of A. niger and B. subtilis respectively were evenly sprayed on PET foils for use as bioindicators. In the case of A. niger, increased spore mortality was found at a high relative gas humidity of 70% (approx. 2 log₁₀). This effect was more evident at prolonged treatment times. In contrast, B. subtilis showed slightly poorer inactivation at high gas humidity.
Conclusions:  Water molecules in the process gas significantly affect the inactivation efficiency of CDBD in air.
Significance and Impact of the Study:  Modifying simple process parameters such as the relative gas humidity can be used to optimize plasma treatment to improve inactivation of resistant micro-organisms such as conidiospores of A. niger .     

Effect of Temperature and Relative Humidity on Survival of Airborne Columbia SK Group Viruses

Three strains of the Columbia SK (Col-SK) group of viruses [Mengo, Maus Elberfeld (ME), and Col-SK viruses] have been studied in the airborne state. All three strains were found to give identical aerosol decay patterns at 16 or 26 C, when held at the same relative humidity (RH). During the first 5 min of aerosol storage time at 16 C, virus inactivation was RH-dependent, with survival maximal at either high (greater than 80%) or low (less than 5%) RH. After 5 min at 16 C, further inactivation, regardless of RH, was insignificant. At 26 C, the effect on survival of RH between 40 and 60% was even more pronounced than at 16 C, and continued after 5 min through 6 hr. Results of this study indicated that the inactivation of airborne Col-SK group viruses was similar to that of other ribonucleic acid (RNA) viruses, particularly poliovirus. Since members of the Col-SK group are picornaviruses, they may well serve as an aerosol model representative of small, ether-resistant, single-stranded RNA viruses.     

Thermal inactivation of nonproteolytic Clostridium botulinum type E spores in model fish media and in vacuum-packaged hot-smoked fish products

Thermal inactivation of nonproteolytic Clostridium botulinum type E spores was investigated in rainbow trout and whitefish media at 75 to 93 degrees C. Lysozyme was applied in the recovery of spores, yielding biphasic thermal destruction curves. Approximately 0.1% of the spores were permeable to lysozyme, showing an increased measured heat resistance. Decimal reduction times for the heat-resistant spore fraction in rainbow trout medium were 255, 98, and 4.2 min at 75, 85, and 93 degrees C, respectively, and those in whitefish medium were 55 and 7.1 min at 81 and 90 degrees C, respectively. The z values were 10.4 degrees C in trout medium and 10.1 degrees C in whitefish medium. Commercial hot-smoking processes employed in five Finnish fish-smoking companies provided reduction in the numbers of spores of nonproteolytic C. botulinum of less than 10(3). An inoculated-pack study revealed that a time-temperature combination of 42 min at 85 degrees C (fish surface temperature) with >70% relative humidity (RH) prevented growth from 10(6) spores in vacuum-packaged hot-smoked rainbow trout fillets and whole whitefish stored for 5 weeks at 8 degrees C. In Finland it is recommended that hot-smoked fish be stored at or below 3 degrees C, further extending product safety. However, heating whitefish for 44 min at 85 degrees C with 10% RH resulted in growth and toxicity in 5 weeks at 8 degrees C. Moist heat thus enhanced spore thermal inactivation and is essential to an effective process. The sensory qualities of safely processed and more lightly processed whitefish were similar, while differences between the sensory qualities of safely processed and lightly processed rainbow trout were observed.     

Evaluation of hydrogen peroxide vapour as a method for the decontamination of surfaces contaminated with Clostridium botulinum spores

The aim of this study was to evaluate the efficacy of hydrogen peroxide vapour (HPV) against spores of Clostridium botulinum, for use as a method for decontaminating environments where this pathogen has been handled. Spores were dried onto stainless steel slides and exposed to HPV in a sealed glovebox enclosure, transferred to a quenching agent at timed intervals during the exposure period, before survivors were cultured and enumerated. D-values were calculated from graphs of log₁₀ survivors plotted against time and were found to range from 1.41 to 4.38 min. HPV was found to be effective at deactivating spores of toxigenic Cl. botulinum, non-toxigenic Clostridium spp. and Geobacillus stearothermophilus dried onto stainless steel surfaces. HPV could be used to decontaminate cabinets and rooms where Cl. botulinum has been handled. The cycle parameters should be based on studies carried out with relevant spores of this organism, rather than based on inactivation data for G. stearothermophilus spores, which have been used in the past as a standard biological challenge for disinfection and sterilisation procedures. HPV could provide an attractive alternative to other decontamination methods, as it was rapid, residue-free and did not give rise to the health and safety concerns associated with other gaseous decontamination systems.     

Heat inactivation of mammalian cell cultures for biowaste kill system design

A biowaste kill system was implemented to treat biological waste generated from a clinical manufacturing and R&D antibody facility. To confirm that design parameters of this continuous decontamination system are sufficient to inactivate mammalian cell culture waste, bench-scale experiments were conducted. The biowaste kill system heat inactivates mammalian cell cultures before they are piped to a neutralization tank and subsequently released to the sewage system. Heat inactivation of cells is accomplished by exposing cells to 80 degrees C for 1 min. Small-scale heat inactivation studies were performed on CHO, 293-HEK, and hybridoma cells. Cells at 1 x 10(6) cells/mL or 1 x 10(7) cells/mL were exposed to 37, 60, 70, or 80 degrees C for 0, 30, 60, and 120 s. Viability based on trypan blue exclusion method and ability to proliferate was assessed after exposure to heat. Data suggest that exposure of cells to 80 degrees C for 60 s is sufficient to inactivate these cultures before they are released to the sewage system.     

Effect of fixation on demonstration of phosphatases of Eimeria tenella grown in chick kidney cell cultures.

The effects of fixation with various concentrations of glutaraldehyde or formaldehyde, acetone or ethanol, and freeze-drying on 5 phosphatases of Eimeria tenella and chick kidney cell cultures were demonstrated in situ. Gultaraldehyde inactivated the phosphatases more than did the formaldehyde, but the effect of the combination of the 2 (Karnovsky's fixative) was greater than that of either glutaraldehyde or formaldehyde alone. The higher the concentration of aldehyde and the longer the duration of exposure, the greater the inactivation. The order of sensitivity to aldehyde fixation of the enzymes tested was glucose-6-phosphatase greater than thiamine pyrophosphatase greater than 5'-nucleotidase greater than adenosine triphosphatase greater than acid phosphatase. Cytologic detail was preserved more efficiently with glutaraldehyde than with formaldehyde. Optimal preservation of enzyme activity for cytochemistry was with 2% glutaraldehyde for 30 min or 2% formaldehyde for 1 hr for G-6-Pase, TPPase, and 5'-nucleotidase, and with 2% glutaraldehyde or 2% formaldehyde for 2 hr with ATPase and AcPase. Quenching with subsequent fixation in cold acetone or ethanol resulted in complete inactivation of G-6-Pase, TPPase, and 5'-nucleotidase; although cells fixed in this manner yielded large amounts of reaction product for ATPase and AcPase, the distribution was diffuse, and some of it appeared to be artifactual. Quenching with subsequent freeze-drying was unsatisfactory because nearly all of the cell layers rolled off the cover glasses.