Virucidal activity of 2 alcohol-based

formulations proposed as hand rubs

by the World Health Organization
Jochen Steinmann, PhD,a Britta Becker, PhD,a Birte Bischoff,a Dajana Paulmann, PhD,a Martina Friesland,c Thomas
Pietschmann, PhD,c Jörg Steinmann, MD, PhD,b and Eike Steinmann, PhDc
Bremen, Essen, and Hannover, Germany

The virucidal activity of 2 hand rubs proposed by the World Health Organization was studied in a quantitative suspension test for
chemical disinfectants and antiseptics in human medicine (EN 14476). These formulations are recommended if no hand rubs with
declared microbiological activity are available in health care settings. Formulation I, based on ethanol, inactivated bovine viral
diarrhea virus (BVDV), hepatitis C virus (HCV), adenovirus, and murine norovirus as a surrogate for human norovirus. Formulation
II, based on isopropyl alcohol, was active only against adenovirus and enveloped viruses, such as BVDV and HCV. Both formulations
failed to inactivate poliovirus by 4 log10 steps within 300 seconds.
Copyright ª 2010 by the Association for Professionals in Infection Control and Epidemiology, Inc. Published by Elsevier Inc. All rights
reserved. (Am J Infect Control 2010;38:66-8.)

Hygienic hand antisepsis is one of the most impor-
tant measures in preventing nosocomial infections
caused by viruses. Guidelines published by the Centers
for Disease Control and Prevention and the World
Health Organization (WHO) emphasize the need for
hand hygiene using an alcohol-based hand rub.1,2
Ready access to a fast-acting hand rub with a proven
antimicrobial efficacy is a key prerequisite for proper
and frequent hand hygiene.
Recently, the WHO proposed 2 alcohol-based for-
mulations for hygienic hand antisepsis as well as for
surgical hand preparations for use in health care set-
tings in which commercial hand rubs are not available
or are too costly.2 Formulation I contains 80% v/v eth-
anol, 1.45% v/v glycerol, and 0.125% v/v hydrogen
peroxide (H2O2), whereas formulation II contains
75% v/v isopropyl alcohol, 1.45% v/v glycerol, and
0.125% v/v H2O2. H2O2 and glycerol are included to
eliminate bacterial spores during storage and increase
acceptability.2 The components of these formulations
From MikroLab GmbH, Bremen, Germanya; Institute of Medical Micro-
biology, Essen University Hospital, Essen, Germanyb; and Division of
Experimental Virology, TWINCORE, Centre for Experimental and
Clinical Infection Research, Hannover, Germany.c
Address correspondence to Dr Jochen Steinmann, MikroLab GmbH,
Norderoog 2, D-28259 Bremen, Germany. E-mail: jochen-steinmann@
t-online.de.
0196-6553/$36.00
Copyright ª 2010 by the Association for Professionals in Infection
Control and Epidemiology, Inc. Published by Elsevier Inc. All rights
reserved.
doi:10.1016/j.ajic.2009.07.009
were chosen based mainly on cost constraints and
microbiological efficacy, including activity against
bacteria, fungi, and viruses.
Data on the virucidal activity of these hand rubs are
not yet available, however. The aim of the present study
was to examine both formulations against the test
viruses as mentioned in EN 14476:2007-02,3 as well
as other important viral pathogens causing nosocomial
infections, including their surrogates.
The most common method for testing hand rubs
against viruses in Europe is a quantitative suspension
test according to EN 14476:2007-02.3 In this test, a
4 log10 reduction is necessary to demonstrate virucidal
activity.
In our study, EN 14476:2007-02 was chosen as a
first-step method to evaluate the activity of the 2 for-
mulations against the nonenveloped poliovirus type
1 (strain LSc-2ab) and adenovirus type 5 (strain Ade-
noid 75). The nonenveloped murine norovirus (MNV),
the enveloped hepatitis C virus (HCV), and enveloped
bovine viral diarrhea virus (BVDV) as a surrogate for
HCV were included as well.
Poliovirus was cultivated with BGM cells, whereas
A-549 cells were used for titration of adenovirus.
MNV (strain Berlin isolate S99) was included as surro-
gate for human norovirus (NoV). In contrast to human
NoV, MNV can be cultivated in cell cultures4 and is
considered a suitable surrogate for the human patho-
gen.5 Virus suspensions were prepared with RAW
264.7 cells.
Two enveloped viruses were included as well. BVDV
strain NADL was passaged and cultured in embryonic
bovine lung cells. Like HCV, this pestivirus belongs to

66
www.ajicjournal.org
Vol. 38 No. 1
Fig 1. Virucidal activity (reduction factors) of WHO formulations I (white columns) and II (black columns) against
BVDV (A), HCV (B), poliovirus (C), adenovirus (D), and MNV (E) as a surrogate for human NoV following EN
the family Flaviviridae. For HCV, a cell culture system
fully permissive for HCV replication and thus reproduc-
ing the complete viral replication cycle was developed
recently.6 We used the HCV chimera Jc1 in a quantita-
tive suspension assay, cultured in the human hepatoma
cell line Huh7.5.
All tests were conducted in accordance with EN
14476:2007-02 and performed in duplicate at 20 8C.3
Eight parts by volume of the formulations were mixed
with 1 part by volume of the test virus suspension and
1 part by volume of phosphate-buffered saline, result-
ing in an 80.0% concentration of the test product.
Activity of the antiseptic agents was immediately stop-
ped at the end of the chosen exposure time by serial
dilution. Virus controls were included after the longest
exposure time.
Infectivity was determined for all viruses in a micro-
procedure by endpoint dilution titration (TCID50). Titer
reduction, calculated as the difference between the
virus titer of the water control and the test product, is
presented as reduction factor (RF). The results of these
assays are displayed in Figure 1.
Both WHO formulations sharply differ in their be-
havior against the nonenveloped test viruses, due to
the type of alcohol used as an active ingredient.
Whereas formulation I, with 80% v/v ethanol as a
main ingredient, inactivated the hydrophilic poliovirus
Steinmann et al. 67
14476:2007-02.
by about 3 log10 after 5 minutes of exposure, formula-
tion II, with 75% v/v isopropyl alcohol, was completely
ineffective against this virus. The EN 14476 requires a
4 log10 reduction within defined exposure times of 0.5
or 1 minute (obligatory) and 3 minutes (additional).
Our data for poliovirus are in accordance with
results from a previous study. Products based on etha-
nol with concentrations , 95% (v/v) were not active,
whereas a formulation with reduced ethanol content
(55%) in combination with 10% propan-1-ol, 5.9%
propan-1.2-diol, 5.7% butan-1.3-diol, and 0.7% phos-
phoric acid was able to inactivate the strong hydro-
philic poliovirus.7
Differences in the inactivating properties of both
formulations also were observed with the adenovirus
and MNV as nonenveloped test viruses with some lipo-
philic character. With formulation I, . 99.99% of both
viruses were inactivated after 30 seconds of exposure
time. In contrast, with formulation II, a RF of only
2.74 was observed for MNV after 2 minutes. Sufficient
inactivation of adenovirus was achieved within 120
seconds with the isopropyl-based formulation.
The superiority of ethanol in the inactivation of
MNV has been demonstrated previously.8 Confirming
our findings from applying formulation I with ethanol
as an active ingredient, 2 ethanol-based hand rubs
were able to reduce MNV by 4 log10 within 30 seconds.9
68 Steinmann et al.
Both formulations were able to inactivate enveloped
viruses like HCV and BVDV, a frequently used surrogate
for HCV. No residual infectivity was observed within the
shortest exposure time of 15 seconds. These data are
the first findings concerning the stability of HCV toward
alcohol-based formulations in a suspension test.
In conclusion, both formulations demonstrated ac-
tivity against enveloped viruses. In addition, formula-
tion I, with ethanol, was able to reduce the titers of
adenovirus and MNV by . 4 log10 within 30 seconds,
whereas this hand rub failed to inactivate poliovirus
by 4 log10 steps within short exposure times, indicating
that it has insufficient activity against enteroviruses.
Additional studies with contaminated fingerpads may
elucidate the efficacy against selected viruses following
such protocols as ASTM E-1838.10
Finally, we strongly recommend using WHO formu-
lation I in the setting of frequent nosocomial viral
infections in absence of products with recognized
microbiological activity. In addition, because of its
broader spectrum against viral pathogens, formulation
I also should be used in outbreak situations with
known and unknown viruses.
References
1. Boyce JM, Pittet D. Guideline for hand hygiene in health-care settings:
recommendations of the Healthcare Infection Control Practices
American Journal of Infection Control
February 2010
Advisory Committee and the HICPA/SHEA/APIC/IDSA Hand Hygiene
Task Force. MMWR Morb Mortal Wkly Rep 2002;51:1-45.
2. World Health Organization. WHO Guidelines on Hand Hygiene in
Health Care. First Global Patient Safety Challenge: Clean Care is Safer
Care 2009.
3. EN 14476:2007–02. Chemical disinfectants and antiseptics. Virucidal
quantitative suspension test for chemical disinfectants and antiseptics
used in human medicine. Test method and requirements (phase 2,
step 1); 2007.
4. Wobus CE, Karst SM, Thackray LB, Chang K-O, Sosnovtsev SV, Belliot
G, et al. Replication of norovirus in cell culture reveals a tropism for den-
dritic cells and macrophages. PLoS Biol 2004;2:ed432.
5. Wobus CE, Thackray LB, Virgin HW. Murine norovirus: a model
system to study norovirus biology and pathogenesis. J Virol 2006;80:
5104-12.
6. Bartenschlager R, Sparacio S. Hepatitis C virus molecular clones and
their replication capacity in vivo and in cell culture. Virus Res 2007;
127:195-207.
7. Kramer A, Galabov AS, Sattar SA, Dohner L, Pivert A, Payan C, et al.
Virucidal activity of a new hand disinfectant with reduced ethanol
content: comparison with other alcohol-based formulations. J Hosp
Infect 2006;62:98-106.
8. Steinmann J, Becker B, Bischoff B, Paulmann D, Steinmann E. Effec-
tiveness of alcohols, hand rubs and scrubs against murine norovi-
rus: a surrogate of human norovirus. Am J Infect Control 2009;37:
E21-2.
9. Belliot G, Lavaux A, Souihel D, Agnello D, Pothier P. Use of murine
norovirus as a surrogate to evaluate resistance of human norovirus
to disinfectants. Appl Environ Microbiol 2008;74:3315-8.
10. ASTM E1838-02. Standard test method for determining the virus-
eliminating effectiveness of liquid hygienic handwash and handrub
agents using the fingerpads of adult volunteers.