Efficacy of Selected Hand Hygiene Agents Used to Remove Bacillus atrophaeus (a Surrogate of Bacillus anthracis) From Contaminated Hands FREE

Anthrax is caused by Bacillus anthracis, a large gram-positive, aerobic, spore-forming bacillus.^1- 2Bacillus anthracis has nearly a worldwide distribution, exists in the soil in the form of extremely stable spores, and causes infection in farm and wild animals who have grazed on contaminated land or ingested contaminated feed.² Human infection is most commonly acquired by contact with infected animals or with contaminated animal products such as wool, hide, hair, or bone; inhalation of spores; or by ingestion of contaminated meat.² Clinical infection depends on the route of transmission and includes cutaneous anthrax, inhalation or pulmonary anthrax, and gastrointestinal anthrax.

Bacillus anthracis has been recognized as a likely agent for bioterrorism because the spores are highly stable in the environment, virtually all persons are susceptible, infection can occur as a result of inhalation of spores, and considerable morbidity and mortality result from infection.^3- 5 During October and November 2001, 22 people in the United States developed anthrax (11 cases of inhalation anthrax and 11 cases of cutaneous anthrax) as a result of the intentional exposure to B anthracis via contaminated letters.^5- 6 More recently, a laboratory worker acquired cutaneous anthrax as a result of contact with the surface of vials containing B anthracis isolates.⁷

Currently, guidelines to prevent acquisition of anthrax during a terrorist event recommend decontamination of exposed patients^5,8- 10 and potentially contaminated environmental surfaces,^8- 10 use of contact precautions for patients with cutaneous anthrax,^9- 10 and hand hygiene with a nonantimicrobial soap and water or with an antimicrobial soap and water if exposure to B anthracis is suspected or proved.¹¹ However, only limited data are available on the susceptibility of B anthracis to current antiseptics. For this reason, we studied the susceptibility of the closely related Bacillus atrophaeus (formerly B subtilis) to several hand hygiene agents. Bacillus atrophaeus has been reported to be slightly less susceptible to germicides than B anthracis, and therefore is an excellent surrogate.^12- 13

Health care workers may be exposed to B anthracis in at least 4 circumstances. First, direct contact with cutaneous lesions of anthrax has led to person-to-person transmission of anthrax including transmission from patients to health care workers.¹⁴ Second, persons exposed to B anthracis may arrive at health care facilities with contaminated clothes, and unprotected contact with such clothes may result in acquisition of anthrax.¹⁰ Third, laboratory workers culturing anthrax have acquired cutaneous infection via contact with contaminated surfaces.⁷ Finally, first responders may have contact with contaminated environmental surfaces.¹⁵ We believe that the use of hand hygiene agents with efficacy against B anthracis along with the proper use of gloves would decrease the risk of acquisition of anthrax in these circumstances. Importantly, the use of agents without efficacy (eg, alcohols) has failed to prevent acquisition of infection.⁷

The Standard Test Method for Evaluation of the Effectiveness of Health Care Professional Handwash Formulations (American Society for Testing and Materials E 1174-94) was used in our challenge study conducted June through August 2002 to measure the efficacy of various hand hygiene products.¹⁶ This method, with minor modifications, has been accepted by the US Food and Drug Administration as a validated method for submitting a label claim as an antiseptic.¹⁷ Following an initial baseline measurement of hand contamination, we assessed the efficacy of the antiseptics and control after each of 3 washes at room temperature. Separate experimental trials were conducted using 10-, 30-, or 60-second applications of an antiseptic or control. During each trial, participants were challenged 3 times by hand contamination followed by washing with the test agent.

Six participants were evaluated for each product tested, 2 participants at each time interval. All volunteers were healthy adults. The study was approved by the University of North Carolina Committee for the Protection of Human Subjects and all participants provided written informed consent. Participants were screened for skin disorders and allergies and excluded if they had any of the following: eczema, psoriasis, any other chronic skin condition; nonintact skin; and allergies to any study agent (ie, chlorhexidine gluconate, chlorine, or alcohol). All participants received nonantimicrobial hand hygiene products and were instructed to use such products for 1 week prior to the study. A single trial consisted of 3 washes with 1 product and 1 wash time (ie, 10, 30, or 60 seconds). Volunteers were randomly assigned to a specific trial and were allowed to participate in no more than 2 trials during the study. The time between trials (ie, wash-out period) for volunteers participating twice was at least 2 weeks.

Hands were contaminated with 5 mL of a liquid inoculum containing ≈2.2 ×10⁶B atrophaeus spores per milliliter. The spores were poured onto cupped hands, and participants spread the inoculum over their entire hands below their wrists for 45 seconds. Hands were then allowed to air dry for 60 seconds. For the baseline measurement, the hands were sampled immediately following the first contamination and air-dry period using the glove method described below. To assess the number of spores left on the hands after washing with the test products, the hands were placed into large-sized gloves filled with a sampling and neutralizing solution. The gloves were taped around the participants' wrists and the hands were massaged for 60 seconds. Then 5 mL of glove rinseate was aseptically removed. This sample was serially diluted 10-fold and assayed by the spread plate technique in duplicate. Following the experimental run, the subject washed extensively with 4% chlorhexidine gluconate and then rubbed with 95% ethanol.

The B atrophaeus spores were stored at 4°C. The stock vial contained 4.4 ×10⁹ spores per milliliter and was diluted immediately prior to each experimental run. Antiseptics tested in this study were a waterless rub containing 61% ethyl alcohol (AvagardD, 3M Health Care, St Paul, Minn), 2% chlorhexidine gluconate (Bactoshield, Steris Corp, St Louis, Mo), and an antibacterial microfiber towel (Clorox Co, Pleasanton, Calif). This towel contains a durable and regenerable agent capable of binding chlorine (≈400 ppm) and is activated by laundering with household bleach. Just prior to use, the towel is moistened with 60 mL of tap water. The control consisted of a nonantimicrobial soap (Soft 'N Sure, Steris Corp). We did not include chlorine dioxide or hydrogen peroxide in this evaluation since they are used for disinfection of environmental surfaces and are not used as antiseptics applied to skin.

Log reduction of spore contamination was determined by calculating the difference between the log₁₀ of the baseline and the log₁₀ of each wash. The mean log₁₀ reduction was calculated by averaging the results obtained from each volunteer hand for the 2 trials using the same product and hand wash time. The 95% confidence intervals (CIs) were calculated and the t test (2-sample equal variance, 2-tailed distribution) was used to compare the results at specified times between products. Excel 97 (Microsoft Corp, Bellevue, Wash) was used for analyses, and P≤.05 was used to determine statistical significance.

A baseline measurement of the level of hand contamination prior to use of a hand hygiene product revealed that levels of contamination with B atrophaeus spores ranged from 1.6 to 2.5 ×10⁵ CFUs/mL.

Handwashing with a nonantimicrobial soap under running water was very effective in reducing the amount of B atrophaeus spore contamination on the hands (Figure 1). Washes of 10, 30, and 60 seconds produced the following reductions (95% CIs) at wash 3: 2.4 (2.2-2.5), 2.3 (2.2-2.4), and 2.1 (1.9-2.4) log₁₀ CFUs/mL. Handwashing with 2% chlorhexidine gluconate achieved the following reductions at wash 3: 2.1 (2.0-2.3), 1.8 (1.5-2.0), and 1.7 (1.5-1.9) log₁₀ CFUs/mL. At 10 seconds, there was no statistical difference between nonantimicrobial soap and chlorhexidine (P = .07) at eliminating B atrophaeus. For both the nonantimicrobial soap and chlorhexidine gluconate, the extent of spore elimination was not increased by prolonging the wash time from 10 to 60 seconds.

Handwashing with the chlorine-containing microfiber towels eliminated 1.3 (1.1-1.5) log₁₀ CFUs/mL of B atrophaeus spores at 10 seconds. Increasing wipe time increased elimination of B atrophaeus spores to 1.6 (1.2-2.0) log₁₀ CFUs/mL at 30 seconds and 2.2 (2.1-2.2) log₁₀ CFUs/mL at 60 seconds. At 10 seconds both soap and chlorhexidine were significantly better than chlorine-containing towels (P<.001) at eliminating B atrophaeus. However, at 60 seconds the chlorine-containing towels were superior to chlorhexidine (P = .008) and similar to nonantimicrobial soap and water (P = .09).

The use of alcohol did not produce a statistically significant (ie, P>.05) reduction of spores at any time tested. Use of the alcohol rub for 10, 30, and 60 seconds produced the following reductions (95% CIs) at wash 3: 0 (−0.1 to 0.1), −0.2 (−0.3 to −0.1), and 0 (−0.2 to 0.2) log₁₀ CFUs/mL. Thus, hand hygiene with a waterless wash containing 61% ethyl alcohol was ineffective in removing or inactivating B atrophaeus spores at all times tested.

The recent bioterrorism-related anthrax outbreak in the United States resulted in 11 cases of inhalation anthrax and 11 cases of cutaneous anthrax.⁶ The inhalation cases may have resulted from either inhalation of aerosolized spores or resuspension of spores that had contaminated environmental surfaces. Extensive environmental contamination inside the Brentwood mail processing and distribution center has been described.¹⁸ Health care workers, emergency medical personnel, and first responders are at risk for acquiring anthrax via direct contact with a contaminated surface, contact with exposed persons' contaminated clothes, or direct unprotected contact with the open lesions of cutaneous anthrax.¹⁰ To minimize these risks, decontamination of exposed persons and contaminated environmental surfaces has been advised. Decontamination of exposed persons with soap and water has been recommended.^5,10,19 Persons with extensive exposure should follow current recommendations that include removal of clothes, storage of clothes in a sealed plastic bag, and showering with soap and water.¹⁰ If possible, showers for decontamination should be available in the field.

Although the use of multiple gloves has been recommended when performing environmental cultures for anthrax, hand hygiene following glove removal has not been mentioned.²⁰ Hand hygiene following glove use is recommended as an essential part of the appropriate use of protective equipment.¹¹ The appropriate agents for handwashing following possible exposure to anthrax spores are not discussed in current anthrax management guidelines.^5,8- 9 The recent hand hygiene guideline from the US Centers for Disease Control and Prevention recommends that health care workers exposed to B anthracis should wash their hands with either nonantimicrobial soap and water or with antimicrobial soap and water.¹¹ This is listed as a category II recommendation, "suggested for implementation and supported by suggestive clinical or epidemiologic studies or a theoretical rationale." An alternative method for hand hygiene that does not require running water is not provided.

The susceptibility of microbes to germicides varies depending on the type of microbe. Only prions are less susceptible to germicides than are bacterial spores.²¹ Limited data are available regarding the susceptibility of B anthracis to germicides.^12- 13,22Bacillus anthracis has been demonstrated to be inactivated by chlorine,^12- 13 4% formaldehyde,²¹ 2% glutaraldehyde,¹³ and 0.025% peracetic acid,¹³ but not 70% ethanol.²²Bacillus atrophaeus spores have been reported to be slightly more resistant to germicides than are B anthracis spores,^12- 13 making B atrophaeus an excellent surrogate for B anthracis. Chlorine at a concentration of 100 ppm will kill more than 99.9% of B atrophaeus spores in 5 minutes.^23- 24 Alcohol is not sporicidal and should not be used either for hand hygiene or environmental decontamination.^25- 26 Despite the attempted decontamination with alcohol of the outside of vials containing B anthracis, in one instance these surfaces remained contaminated, resulting in cutaneous infection in a laboratory worker.⁷

Our data suggest that current recommendations for decontamination of exposed persons with soap and water is likely adequate. Chlorhexidine gluconate, an agent with excellent antimicrobial activity against vegetative bacteria and viruses, did not provide improved elimination of spores compared with soap and water. Our study did not allow us to distinguish between physical removal and bacterial inactivation.

Health care workers should wear gloves for touching potentially contaminated surfaces and wash their hands after glove removal with nonantimicrobial or antimicrobial soap and water as recommended by the US Centers for Disease Control and Prevention guideline for hand hygiene.¹¹ Our study provides the first direct evidence to support this recommendation, and based on our data, we believe this recommendation should now be classified as a category IB recommendation (ie, "strongly recommended for implementation and supported by certain experimental, clinical, or epidemiologic studies and a strong theoretical rationale").

When soap and running water are not available in the field, waterless rubs containing ethyl alcohol should not be used, because they are ineffective in inactivating or removing spores. Rather, small amounts of water should be carried in rescue vehicles, allowing the use of chlorine-containing towels for handwashing.