Weber DJ, Sickbert-Bennett E, Gergen MF, Rutala WA. Efficacy of Selected Hand Hygiene Agents Used to Remove Bacillus atrophaeus (a Surrogate of Bacillus anthracis) From Contaminated Hands. JAMA. 2003;289(10):1274–1277. doi:10.1001/jama.289.10.1274
Author Affiliations: Division of Infectious Diseases, Department of Medicine, University of North Carolina at Chapel Hill (Drs Weber and Rutala); and Department of Hospital Epidemiology, University of North Carolina Health Care System, Chapel Hill (Drs Weber and Rutala, and Mss Sickbert-Bennett and Gergen).
Context The intentional use of Bacillus anthracis transmitted
via the US mail in October-November 2001 resulted in 22 people developing
inhalation or cutaneous anthrax. Glove use with handwashing prior to and after
contact with potential contaminated environmental surfaces and cutaneous lesions
has been recommended. However, only limited data are available on the susceptibility
of B anthracis to antiseptics.
Objective To evaluate the efficacy of several hand antiseptics (interventions)
and soap and water (control) against Bacillus atrophaeus, a surrogate of B anthracis.
Design, Setting, and Participants Challenge study conducted among healthy adult volunteers, using the
Standard Test Method for Evaluation of the Effectiveness of Health Care Professional
Handwash Formulations (American Society for Testing and Materials E 1174-94)
to determine the efficacy of various hand hygiene products at wash times of
10, 30, and 60 seconds. Volunteers were excluded if they had eczema, psoriasis,
or other chronic skin conditions; nonintact skin; or allergies to any study
agent. Study agents were a waterless rub containing 61% ethyl alcohol, a 2%
chlorhexidine gluconate preparation, and an antibacterial microfiber towel
that releases hypochlorite. A nonantimicrobial soap was used as a control.
Main Outcome Measure Reduction of B atrophaeus spores (log10 CFU/mL) on contaminated hands.
Results Washes of 10, 30, and 60 seconds with either soap and water or 2% chlorhexidine
gluconate eliminated 1.5 to 2.0 log10 CFUs/mL of B atrophaeus spores at wash 3. Mean reductions (95% confidence intervals)
with 10-, 30-, and 60-second washes with soap and water were 2.4 (2.2-2.5),
2.3 (2.2-2.4), and 2.1 (1.9-2.4) log10 CFUs/mL, respectively; and
with 2% chlorhexidine gluconate, 2.1 (2.0-2.3), 1.8 (1.5-2.0), and 1.7 (1.5-1.9)
log10 CFUs/mL, respectively. Handwashing with chlorine-containing
towels was increasingly effective as the wipe time increased; reductions at
10, 30, and 60 seconds were 1.3 (1.1-1.5), 1.6 (1.2-2.0), and 2.2 (2.1-2.2)
log10 CFUs/mL, respectively. A waterless rub containing 61% ethyl
alcohol was ineffective in eliminating B atrophaeus spores
at all times tested (0 [−0.1 to 0.1], −0.2 [−0.3 to −0.1],
and 0 [−0.2 to 0.2] log10 CFUs/mL).
Conclusions In this evaluation of hand hygiene agents, handwashing with soap and
water, 2% chlorhexidine gluconate, or chlorine-containing towels reduced the
amount of B atrophaeus spore contamination, whereas
use of a waterless rub containing ethyl alcohol was not effective in removing
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 patients5,8- 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
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
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