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Clostridium difficile continues to cause infections in healthcare and other settings. Its spores survive well indoors and require sporicidal chemicals for infection control. However, proper testing of disinfectants is impeded due to difficulties in obtaining viable spores of high enough quality and titers to meet current regulations for sporicidal claims. A new liquid medium (Clospore) has been developed, based on a systematic review of the compositions of 20 other available media. C. difficile spores grown in the new medium and treated with a mixture of lysozyme and trypsin yielded final suspensions with >109 CFU/mL of viable spores, with a purity of >91 as tested by spore-staining and phase-contrast microscopy. The spores showed a biological decay rate of about 0.1 log10/month when dried on metal disks and stored indoors (air temperature 23 2C; relative humidity 52.76 15.08). Heating the purified spore suspensions to 70C for 10 min to inactivate any vegetative cells showed no spore activation or inactivation. The spores could be stored for at least 14 months either refrigerated (4C) or frozen (20 or 80C) in 50 (v/v) ethanol with virtually no loss in viability. The resistance of the enzyme-treated spores to three levels of sodium hypochlorite (1000, 3000, and 5000 ppm), using a standardized quantitative carrier test, was almost identical to that of the spores concentrated by centrifugation alone. The described procedure has been successfully applied to four standard (ATCC) and six clinical strains of C. difficile.

Human noroviral infections are generally more common during winters in temperate regions. This study used a murine norovirus (MNV) as a human norovirus surrogate to test the effect of water temperature (4 and 25°C) on virus survival and its susceptibility to the levels of monochloramine (~1.89 ppm) to terminally disinfect municipally treated potable waters. The titre of MNV remained essentially unchanged for at least 24 h in raw river water at both temperatures. The virus became undetectable in

Microbicides for reprocessing heat-sensitive medical devices, such as flexible endoscopes, must be mycobactericidal to reduce the risk of nosocomial infections. Suspension test methods currently used for efficacy evaluation lack the stringency required for assessing inactivation of mycobacteria on surfaces. The quantitative carrier test method reported here is based on mycobacteria-contaminated reference carrier disks of brushed stainless steel. Each disk was contaminated with 10 L of a suspension of Mycobacterium terrae containing a soil load. Each disk with a dried inoculum was placed in a glass or Teflon vial, and then overlaid with 50 L of the test formulation or 50 L saline for the control carriers. Five test and 3 control disks were used in each run. At the end of the contact time, each vial received 9.95 mL neutralizer solution with 0.1% Tween-80 to stop the reaction and perform the initial microbicide dilution. The inoculum was eluted by mixing on a Vortex mixer for 60 s, and the eluates and saline used to subsequently wash the vials and the funnels were membrane-filtered. Filters were placed on plates of Middlebrook 7H11 agar and incubated at 37C for at least 30 days before colonies were counted and log10 reductions were calculated in colony-forming units. Tests with a range of commercially available products, having claims against mycobacteria, or believed to be broad-spectrum microbicides, showed that the method gave reproducible results. Products used included oxidizing agents (sodium hypochlorite and an iodophore), a phenolic, a quaternary ammonium compound, and ortho-phthalaldehyde. This method represents a much more realistic evaluation than the currently used quantitative suspension test method for the evaluation of mycobactericidal formulations for registration and, when performed at different product concentrations, allows an assessment of any safety margin or risks in using the test formulation in the field.

Background: Clostridium difficile is an increasingly common nosocomial pathogen, and its spores are resistant to common environmental surface disinfectants. Many high-level disinfectants (eg, aldehydes) are unsuitable for environmental decontamination because they need several hours of contact to be sporicidal. This study tested the potential of selected oxidative microbicides to inactivate C difficile spores on hard surfaces in relatively short contact times at room temperature. Methods: The spores of a clinical isolate of C difficile were tested using disks (1 cm diameter) of brushed stainless steel in a quantitative carrier test. The spores of C sporogenes and Bacillus subtilis, common surrogates for evaluating sporicides, were included for comparison. The clostridia were grown separately in Columbia broth (CB), and B subtilis was grown in a 1:10 dilution of CB. Each disk received 10 mL test spores with an added soil load, and the inoculum was dried. One disk each was placed in a glass vial and overlaid with 50 mL test formulation; controls received an equivalent volume of normal saline with 0.1% Tween 80. At the end of the contact time the microbicide was neutralized, the inoculum recovered from the disks by vortexing, the eluates were membrane filtered, and the filters placed on plates of recovery medium. The colony-forming units (CFU) on the plates were recorded after 5 days of incubation. The performance criterion was $6 log10 ($99.9999%) reduction in the viability titer of the spores. The microbicides tested were domestic bleach with free-chlorine (FC) levels of 1000, 3000, and 5000 mg/L; an accelerated hydrogen peroxide (AHP)-based product with 70,000 mg/L H2O2 (Virox STF); chlorine dioxide (600 mg/L FC); and acidified domestic bleach (5000 mg/L FC). Results: Acidified bleach and the highest concentration of regular bleach tested could inactivate all the spores in #10 minutes; Virox STF could do the same in #13 minutes. Regular bleach with 3000 mg/L FC required up to 20 minutes to reduce the viability of the all the spores tested to undetectable levels; chlorine dioxide and the lowest concentration of regular bleach tested needed approximately 30 minutes for the same level of activity. Conclusions: Acidified bleach, Virox STF, and regular bleach (3000-5000 mg/L FC) could inactivate C difficile spores on hard environmental surfaces in approximately 10 to 15 minutes under ambient conditions. All of these products are strong oxidizers and should be handled with care for protection of staff, but acidified and regular bleach with high levels of FC also release chlorine gas, which can be hazardous if inhaled by staff or patients. (Am J Infect Control 2005;33:320-5.)

For well over a decade, many deficiencies have been identified in current AOAC methods used to assess the microbicidal activities of chemical disinfectants on medical devices and environmental surfaces. This report discusses the development of quantitative carrier tests (QCT) designed to address these concerns. Decontamination of surfaces with dried inocula is invariably more difficult than when microorganisms are in suspension. For medical device as well as environmental decontamination, microbicides are used on contaminated surfaces, thus making it necessary to evaluate their microbicidal action on representative carrier materials contaminated with a dried challenge microorganism(s). Our approach is a 2-tiered QCT. The first tier (QCT-1) uses relatively ideal conditions to assess performance of the microbicide for screening purposes; the test uses smooth glass surfaces and quantities of disinfectant in excess of those likely to be experienced in the field. The second tier of testing (QCT-2) is more stringent because it uses (1) disks of brushed stainless steel as carriers, (2) only 50 μL of the test formulation on each carrier as compared to 1 mL in QCT-1, and (3) an added soil load to simulate the presence of residual body fluids or accumulated surface dirt. This review also discusses the factors that affect disinfection of medical devices and environmental surfaces in the context of the methodology used to evaluate the potency of microbicides. Specific recommendations for discussion are included, and performance criteria are suggested based on a risk-reduction approach for different classes of disinfectants. The focus is on improving the relevance of the test methodology to actual field use of disinfectants for devices and facilities in health care, and potentially in other settings. It is hoped that this review and its recommendations will initiate needed discussion and resolution of the many issues identified.

Enteric and respiratory viruses are among the most frequent causes of human infections, and hands play an important role in the spread of these and many other viral diseases. Regular and proper hand hygiene by caregivers and food handlers in particular is essential to decontaminate hands and potentially interrupt such spread. What would be considered a proper decontamination of hands? Handwashing with regular soap and water is often considered sufficient, but what of hygienic handwash and handrub antiseptic products? Are they more effective? The evidence suggests that some clearly are. Activity against bacteria may not reflect the ability of hygienic hand antiseptics to deal with viruses, especially those that are nonenveloped. In spite of the acknowledged importance of hands as vehicles for viruses, there is a lack of suitable regulatory mechanism for handwash or handrub products to make claims of efficacy against viruses. This is in contrast with the ability of general-purpose disinfectants to make antiviral claims, although transmission of viruses from surfaces other than those of reusable medical devices may play only a minor role in virus transmission. This review discusses the (1) recent information on the relative importance of viruses as human pathogens, particularly those causing enteric and respiratory infections; (2) the survival of relevant viruses on human hands in comparison with common gram-negative and gram-positive bacteria; (3) the potential of hands to transfer or receive such contamination on casual contact; (4) role of hands in the spread of viruses; (5) the potential of hygienic measures to eliminate viruses from contaminated hands; (6) relative merits of available protocols to assess the activity of hygienic hand antiseptics against viruses; and (7) factors considered crucial in any tests to assess the activity of hygienic hand antiseptics against viruses. In addition, this review proposes surrogate viruses in such testing and discusses issues for additional consideration by researchers, manufacturers, end-users, and regulators. (Am J Infect Control 2002;30:355-72)

Hepatitis B virus (HBV) and hepatitis C virus (HCV) are the most prevalent bloodborne pathogens. Infections caused by these organisms can become chronic and may lead to liver cirrhosis and carcinoma. Limited chemotherapy is now available, but only HBV can be prevented through vaccination. Both viruses are enveloped and relatively sensitive to many physical and chemical agents; their ability to survive in the environment may not be as high as often believed. As a result, their spread occurs mainly through direct parenteral or percutaneous exposure to tainted body fluids and tissues. Careful screening of and avoiding contact with such materials remain the most effective means of protection. Nevertheless, the indirect spread of these viruses, although much less common, can occur when objects that are freshly contaminated with tainted blood enter the body or contact damaged skin. Germicidal chemicals are important in the prevention of HBV and HCV spread through shared injection devices, sharps used in personal services (such as tattooing and body piercing), and heat-sensitive medical/dental devices (such as flexible endoscopes) and in the cleanup of blood spills. Microbicides in vaginal gels may also interrupt their transmission. General-purpose environmental disinfection is unlikely to play a significant role in the prevention of the transmission of these viruses. Testing of low-level disinfectants and label claims for such products against HBV and HCV should be discouraged. Both viruses remain difficult to work with in the laboratory, but closely related animal viruses (such as the duck HBV) and the bovine viral diarrhea virus show considerable promise as surrogates for HBV and HCV, respectively. Although progress in the culturing of HBV and HCV is still underway, critical issues on virus survival and inactivation should be addressed with the use of these surrogates.

Infectious diseases continue to exert a heavy toll on human health even in industrialized countries. Recent data from the World Health Organization suggests that infectious diseases are the leading cause of death in the world. Many changing trends in our society have a known or potential impact on infectious disease spread and may have an impact on the normal routine of home hygiene. Important amongst these societal trends are increasing population and life expectancy, changes in urbanization, grouping of susceptibles, increased ambulatory and home care, increased immunosuppression, increased and faster travel, changes in technology, increasing antibiotic resistance as a result of misuse of antibiotics, changes in food and water consumption, and changes in personal cleaning, washing, and laundry practices. This review will highlight these factors and their impact on home hygiene and steps that may be needed to reduce the risk from infections.

Clostridium difficile continues to cause infections in healthcare and other settings. Its spores survive well indoors and require sporicidal chemicals for infection control. However, proper testing of disinfectants is impeded due to difficulties in obtaining viable spores of high enough quality and titers to meet current regulations for sporicidal claims. A new liquid medium (Clospore) has been developed, based on a systematic review of the compositions of 20 other available media. C. difficile spores grown in the new medium and treated with a mixture of lysozyme and trypsin yielded final suspensions with10(9) CFU/mL of viable spores, with a purity of91% as tested by spore-staining and phase-contrast microscopy. The spores showed a biological decay rate of about 0.1 log10/month when dried on metal disks and stored indoors (air temperature 23 +/- 2 degrees C; relative humidity 52.76 +/- 15.08%). Heating the purified spore suspensions to 70 degrees C for 10 min to inactivate any vegetative cells showed no spore activation or inactivation. The spores could be stored for at least 14 months either refrigerated (4 degrees C) or frozen (-20 or -80 degrees C) in 50% (v/v) ethanol with virtually no loss in viability. The resistance of the enzyme-treated spores to three levels of sodium hypochlorite (1000, 3000, and 5000 ppm), using a standardized quantitative carrier test, was almost identical to that of the spores concentrated by centrifugation alone. The described procedure has been successfully applied to four standard (ATCC) and six clinical strains of C. difficile.

Previous studies of microbial behaviour in situ have used dialysis sacs and diffusion chambers to permit contact of contained microorganisms with the external aqueous milieu. Their main limitation is a slow response to changes in water quality, preventing adequate simulation of field conditions. This study used the ECODYNE system™, a new device responsive to real time changes in water quality. It consists of a hollow fibre membrane module and fluid reservoir which separates the test microorganisms from the external environment. Use of a mixture of Escherichia coli, Enterococcus dwans, MS-2 phage, poliovirus type 1 (Sabin) and hepatitis A virus (HM-175) allowed direct comparison of their survival in river water under identical conditions over a period of at least 24 hours. The results can be summarised as follows: a) survival patterns of E. coli in nutrient rich river water were highly variable, b) a ten-fold or greater increase in numt)ers of E. coli was sometimes observed in twth latwratory and field tests, c) no regrowth was ever observed with E. durons, and its titre declined faster than that of E. coli, and d) over the first 24 hours, the phage survival was similar to that of the human pathogenic viruses. The observations regarding the variable survival of E. coli and its potential for regrowth raise questions about its suitability as a water quality indicator. MS-2 phage shows promise as an indicator of viral pollution, but requires further study. The ECODYNE system proved highly suitable in this study, and shows potential for other environmental interaction/environmental fate studies including ecotoxicological investigations and monitoring.

Chemical disinfection is widely practiced as a means of controlling and preventing the spread of infectious diseases. Although disinfection of bacteria has been widely studied, much less attention has been paid to the virucidal potential of commonly used disinfectants in spite of the low infective dose of many human pathogenic viruses. This review considers what is known about the disinfection of viruses and the virucidal properties of different classes of disinfectant chemicals. It focuses on virus disinfection from a practical viewpoint and also critically evaluates the testing techniques currently used for examining the efficacy of disinfectant products.

Microbicides for reprocessing heat-sensitive medical devices, such as flexible endoscopes, must be mycobactericidal to reduce the risk of nosocomial infections. Suspension test methods currently used for efficacy evaluation lack the stringency required for assessing inactivation of mycobacteria on surfaces. The quantitative carrier test method reported here is based on mycobacteria-contaminated reference carrier disks of brushed stainless steel. Each disk was contaminated with 10 microL of a suspension of Mycobacterium terrae containing a soil load. Each disk with a dried inoculum was placed in a glass or Teflon vial, and then overlaid with 50 microL of the test formulation or 50 microL saline for the control carriers. Five test and 3 control disks were used in each run. At the end of the contact time, each vial received 9.95 mL neutralizer solution with 0.1% Tween-80 to stop the reaction and perform the initial microbicide dilution. The inoculum was eluted by mixing on a Vortex mixer for 60 s, and the eluates and saline used to subsequently wash the vials and the funnels were membrane-filtered. Filters were placed on plates of Middlebrook 7H11 agar and incubated at 37 degrees C for at least 30 days before colonies were counted and log10 reductions were calculated in colony-forming units. Tests with a range of commercially available products, having claims against mycobacteria, or believed to be broad-spectrum microbicides, showed that the method gave reproducible results. Products used included oxidizing agents (sodium hypochlorite and an iodophore), a phenolic, a quaternary ammonium compound, and ortho-phthalaldehyde. This method represents a much more realistic evaluation than the currently used quantitative suspension test method for the evaluation of mycobactericidal formulations for registration and, when performed at different product concentrations, allows an assessment of any safety margin or risks in using the test formulation in the field.

For well over a decade, many deficiencies have been identified in current AOAC methods used to assess the microbicidal activities of chemical disinfectants on medical devices and environmental surfaces. This report discusses the development of quantitative carrier tests (QCT) designed to address these concerns. Decontamination of surfaces with dried inocula is invariably more difficult than when microorganisms are in suspension. For medical device as well as environmental decontamination, microbicides are used on contaminated surfaces, thus making it necessary to evaluate their microbicidal action on representative carrier materials contaminated with a dried challenge microorganism(s). Our approach is a 2-tiered QCT. The first tier (QCT-1) uses relatively ideal conditions to assess performance of the microbicide for screening purposes; the test uses smooth glass surfaces and quantities of disinfectant in excess of those likely to be experienced in the field. The second tier of testing (QCT-2) is more stringent because it uses (1) disks of brushed stainless steel as carriers, (2) only 50 microL of the test formulation on each carrier as compared to 1 mL in QCT-1, and (3) an added soil load to simulate the presence of residual body fluids or accumulated surface dirt. This review also discusses the factors that affect disinfection of medical devices and environmental surfaces in the context of the methodology used to evaluate the potency of microbicides. Specific recommendations for discussion are included, and performance criteria are suggested based on a risk-reduction approach for different classes of disinfectants. The focus is on improving the relevance of the test methodology to actual field use of disinfectants for devices and facilities in health care, and potentially in other settings. It is hoped that this review and its recommendations will initiate needed discussion and resolution of the many issues identified.

Clostridium difficile is an increasingly common nosocomial pathogen, and its spores are resistant to common environmental surface disinfectants. Many high-level disinfectants (eg, aldehydes) are unsuitable for environmental decontamination because they need several hours of contact to be sporicidal. This study tested the potential of selected oxidative microbicides to inactivate C. difficile spores on hard surfaces in relatively short contact times at room temperature.The spores of a clinical isolate of C. difficile were tested using disks (1 cm diameter) of brushed stainless steel in a quantitative carrier test. The spores of C. sporogenes and Bacillus subtilis, common surrogates for evaluating sporicides, were included for comparison. The clostridia were grown separately in Columbia broth (CB), and B. subtilis was grown in a 1:10 dilution of CB. Each disk received 10 microL test spores with an added soil load, and the inoculum was dried. One disk each was placed in a glass vial and overlaid with 50 microL test formulation; controls received an equivalent volume of normal saline with 0.1% Tween 80. At the end of the contact time the microbicide was neutralized, the inoculum recovered from the disks by vortexing, the eluates were membrane filtered, and the filters placed on plates of recovery medium. The colony-forming units (CFU) on the plates were recorded after 5 days of incubation. The performance criterion wasor = 6 log(10) (or = 99.9999%) reduction in the viability titer of the spores. The microbicides tested were domestic bleach with free-chlorine (FC) levels of 1000, 3000, and 5000 mg/L; an accelerated hydrogen peroxide (AHP)-based product with 70,000 mg/L H2O2 (Virox STF); chlorine dioxide (600 mg/L FC); and acidified domestic bleach (5000 mg/L FC).Acidified bleach and the highest concentration of regular bleach tested could inactivate all the spores inor = 10 minutes; Virox STF could do the same inor = 13 minutes. Regular bleach with 3000 mg/L FC required up to 20 minutes to reduce the viability of the all the spores tested to undetectable levels; chlorine dioxide and the lowest concentration of regular bleach tested needed approximately 30 minutes for the same level of activity.Acidified bleach, Virox STF, and regular bleach (3000-5000 mg/L FC) could inactivate C. difficile spores on hard environmental surfaces in approximately 10 to 15 minutes under ambient conditions. All of these products are strong oxidizers and should be handled with care for protection of staff, but acidified and regular bleach with high levels of FC also release chlorine gas, which can be hazardous if inhaled by staff or patients.

Suspension tests for virucidal activity of chemical germicides are easier to perform, but they normally do not present the test product with a strong enough challenge. In contrast, carrier tests, where the test virus is dried on an animate or inanimate surface, offer the test formulation a higher level of challenge because it first has to penetrate successfully the inoculum to gain access to and inactivate the target organism on the carrier. Since pathogens in nature are normally found adsorbed to surfaces and/or embedded in organic or cellular debris, the results of carrier tests are more relevant to predicting the activity of chemical germicides under field situations. The method described below uses discs (1 cm in diameter) of brushed stainless steel discs as carriers. Ten micro l of the test virus in a soil load is placed on each disc and the inoculum dried under ambient conditions. The dried inoculum is then exposed to 50 micro l of the test formulation or a control solution for a defined contact time at the specified temperature. EBSS (0.95 ml) is added to each carrier holder to dilute/neutralize the germicide, the inoculum eluted and the eluates titrated in cell cultures to determine the degree of loss in virus viability. At least five test and three control carriers are used in each test. Controls are also included to test for toxicity of the test formulation to the host cells and any interference sub-cytotoxic levels of the formulation may have on the ability of the virus to infect the cells. The method has been used with several types of human and animal pathogenic viruses to test the activity of all major classes of chemical germicides against them.

Background: There is mounting concern regarding the efficacy of many germicides on the market because officially recognized germicidal tests for various classes of microorganisms vary widely and often lack reproducibility and proper quantitation. We report here a carrier method for simultaneously and quantitatively assessing the efficacy of liquid chemical germicides against a mixture of microorganisms of varying degrees of resistance. Methods: In the test, each small glass cup (10 mm wide × 14 mm long) was contaminated with 10 μl of a standardized mixture of Staphylococcus aureus , Mycobacterium bovis bacille Calmette-Guerin, Trichophyton mentagrophytes spores, Sabin poliovirus type 1, and Bacillus stearothermophilus spores in 5% fetal bovine serum. The inoculum was dried for 60 minutes under ambient conditions and covered with 60 μl of the disinfectant under test or a balanced salt solution control for the desired contact time. The carrier was then placed in 2940 μl of an eluent and the eluates assayed separately for the five microorganisms. Tap water was used to dilute the test product as needed. Results: Of the 11 products tested, 2% alkaline glutaraldehyde, 0.6% sodium hypochlorite (about 5000 ppm free chlorine), and a 0.4% quarternary ammonium compound containing 23% hydrochloric acid were effective against all five challenge organisms. A hard-surface spray containing 0.1% o -phenylphenol with 79% ethanol was effective against all but bacterial spores; 70% (volume/volume) ethanol alone and povidone-iodine (1% available iodine) were effective against S. aureus , the mycobacterium, and the fungus; a 3% solution of peroxygen compounds was effective only against S. aureus and the poliovirus; 1.5% chlorhexidine gluconate, 0.06% quaternary ammonia compound, and 0.03% o -phenylphenol + 0.03% p -tertiary amylphenol could inactivate nothing but S. aureus ; and 3% hydrogen peroxide was ineffective in all tests. Conclusions: This method shows promise for use with various classes of microorganisms, individually or as mixtures. Its application should enable the classification of germicides according to spectrum of activity.

We compared the efficiency of paper, cloth, and electric warm air drying in eliminating rotaviruses and Escherichia coli remaining on finger pads washed with 70% isopropanol, a medicated liquid soap, an unmedicated liquid soap, or tap water alone. The contaminated area on the finger pads of a volunteer was exposed to the hand-washing agent for 10 seconds and then rinsed in 40 degrees C tap water. The washed areas were dried for 10 seconds by one of the three methods. Irrespective of the hand-washing agent used, electric air drying produced the highest and cloth drying the lowest reduction in the numbers of both test organisms. These findings indicate the importance of selecting the right means for drying washed hands, particularly when less effective hand-washing agents are used.

Rotaviruses are among the major causes of acute gastroenteritis in man as well as a variety of animals. Although not much is known about the mechanisms of transmission of these viruses in nature, fecally-contaminated water appears to play a role in this regard. This study was, therefore, aimed at determining how well rotaviruses could survive in water. Calf rotavirus (strain C-486) grown in MA-104 cells was used as a model in this study. Samples of raw (RW) and municipally-treated tap water (TW) from the Ottawa River (Ontario, Canada) were contaminated with the virus to give a final concentration of approximately 5.0 × 104 plaque forming units (PFU)/ml. The virus-contaminated water samples were held either at 4°C or 20°C in the dark for a total of 64 days. In TW held at 4°C, there was no significant drop in the virus titre even after 64 days, whereas, at 20°C the titre was reduced by nearly 2-logl0 over the same period. In RW a 3-10gl0 drop in PFU occurred in 16 and 32 days at 20°C and 4°C, respectively. These data indicate that rotaviruses could survive long enough in the water environment to give water a considerable potential as a vehicle for their spread.

Enterovirus 70 (EV-70) has caused at least two pandemics and several major epidemics of acute hemorrhagic conjunctivitis (AHC) in the past 18 years, with the largest number of cases occurring in the coastal areas of the tropics. The exact means of its spread are not known, but fomites and hands contaminated by them are the most likely vehicles. We, therefore, tested EV-70 survival under different environmental conditions using stainless steel disks (1 cm diameter). Each disk received 10 microliter of the virus in phosphate-buffered saline (PBS). The disks were held at various temperatures with the relative humidity (RH) at the low (20 +/- 5%), medium (50 +/- 5%), high (80 +/- 5%), or ultrahigh (95 +/- 5%) level. The virus was eluted from the disks with tryptose phosphate broth and the eluate assayed in LLC-MK2cells. Inactivation rates (Ki), expressed as hourly loss of virus plaque forming units (PFU) in log10, were then calculated. At 20 degrees C, virus survival was proportional to the RH level, with the highest virus survival at the ultrahigh RH; at this level nearly 5% of the input virus was detectable even after 24 hr. Virus inactivation rates were only slightly higher at the ultrahigh RH when the temperature was raised to 33 degrees C or 35 degrees C. However, at 80% RH, increasing the temperature from 20 degrees C to 33 degrees C resulted in a dramatic rise in virus inactivation. The finding that high levels of RH prolong EV-70 survival on fomites may help explain the epidemiology of AHC resulting from EV-70.