Evaluation of the sporicidal activity of different chemical ... - Tristel

Evaluation of the sporicidal activity of different chemical disinfectants used
in hospitals against Clostridium difficile
S. Speight a ,A.Moy a , S. Macken a , R. Chitnis a , P.N. Hoffman b , A. Davies a , A. Bennett a , J.T. Walker a, *
a
HPA Microbiological Services Division, Porton Down, Salisbury, UK
b
HPA Centre for Infections, London, UK
article info
Article history:
Received 16 March 2011
Accepted 14 May 2011
by J.A. Child
Available online 28 July 2011
Keywords:
Clostridium difficile
Disinfection testing
Sporicides
Nosocomial infections
Hospital acquired infections
Introduction
summary
Clostridium difficile is a major cause of nosocomial diarrhoea in
the UK and worldwide. According to the UK Health Protection
Agency’s quarterly and annual reports, the number of cases of
C. difficile infection (CDI) is decreasing year on year (http://www.
hpa.org.uk/web/HPAweb&HPAwebStandard/HPAweb_C/1195733
750761). 1 However, with 3933 deaths associated with C. difficile in
2009 and 20,192 cases of CDI reported in England between 2009
and 2010 (http://www.hpa.org.uk), these numbers indicate that
hospitals still face major challenges in trying to control outbreaks
and reduce CDI cases even further (http://www.statistics.gov.uk/
pdfdir/cdif0810.pdf). 1,2
C. difficile disease occurs when the normal, healthy intestinal
bacterial flora is subdued by the use of antibiotics. This allows
C. difficile to flourish in the gut, where it produces a toxin that
causes diarrhoea, in those aged 65 years and over.
* Corresponding author. Address: HPA Microbiological Services Division, Porton
Down, Salisbury SP4 0JG, UK. Tel.: þ44 (0) 1980 612643; fax: þ44 (0) 1980 612672.
E-mail address: jimmy.walker@hpa.org.uk (J.T. Walker).
Journal of Hospital Infection 79 (2011) 18e22
Available online at www.sciencedirect.com
Journal of Hospital Infection
journal homepage: www.elsevierhealth.com/journals/jhin
Decontamination of surfaces and medical equipment is integral to the control of Clostridium
difficile transmission, and many products claim to inactivate this bacterium effectively. Thirtytwo
disinfectants were tested against spores of C. difficile in a suspension test based on
European Standard BS EN 13704:2002, with contact times of 1 and 60 min in simulations of
clean (0.3% albumin) and dirty (3% albumin) conditions. The addition of a 1-min contact time
was chosen as a more realistic simulation of probable real-life exposures in the situation being
modelled than the 60 min specified by the Standard. The manufacturer’s lowest recommended
concentrations for use were tested. Sixteen products achieved >10 3 reduction in viability after
60 min (the pass criterion for the Standard) under both clean and dirty conditions. However,
only eight products achieved >10 3 reduction in viability within 1 min under dirty conditions.
Three products failed to reduce the viability of the C. difficile spores by a factor of 10 3 in any of
the test conditions. This study highlights that the application of disinfectants claiming to be
sporicidal is not, in itself, a panacea in the environmental control of C. difficile, but that carefully
chosen environmental disinfectants could form part of a wider raft of control measures
that include a range of selected cleaning strategies.
Ó 2011 The Healthcare Infection Society. Published by Elsevier Ltd. All rights reserved.
0195-6701/$ e see front matter Ó 2011 The Healthcare Infection Society. Published by Elsevier Ltd. All rights reserved.
doi:10.1016/j.jhin.2011.05.016
C. difficile is highly anaerobic. Vegetative cells die within
approximately 15 min of exposure to air, and are readily susceptible
to heat, desiccation and commonly used disinfectants. 3 C. difficile
produces spores that are highly resistant to physical and chemical
agents, resulting in the persistence of spores on surfaces with the
potential to transmit infection. The spores are capable of surviving
for many months, and studies have demonstrated that environmental
surfaces and patient rooms become contaminated with
C. difficile spores over time. 4e9 The relationship between surface
contamination and CDI is difficult to prove, but is considered to be
likely as improved room disinfection strategies have coincided with
reduced levels of CDI. 9e12
A range of commercially available sporicides exist, with various
modes of action. These include disinfectants (liquids, gels and
wipes) for cleaning medical devices, instruments and/or surfaces, 13
and room fumigants. 14 Current guidelines of the Department of
Health (UK) recommend that all hospital trusts should have a policy
that covers ‘cleaning protocols that include increased environmental
cleaning and the use of disinfectants, e.g. chlorine-based
products, in areas where there are C. difficile-infected patients’
(http://www.dh.gov.uk/prod_consum_dh/groups/dh_digitalassets/

documents/digitalasset/dh_093218.pdf). 15 These guidelines and
the effectiveness of hypochlorites have resulted in a reliance on
chlorine-based products (e.g. sodium dichloroisocyanurate) to
reduce the viability of C. difficile spores on surfaces. However, there
are a number of drawbacks to using hypochlorites, including an
unpleasant odour, potential respiratory exposure issues, and
corrosion of metallic surfaces. As such, alternatives are sought by
users.
This study was undertaken to assess the efficacy of a range of
sporicidal agents, using a variation on European Standard BS EN
13704:2002 to determine the activity of 32 commonly available
disinfectants against C. difficile spores (NCTC 11209) after 1 and
60 min of contact under clean and dirty conditions. This test is
applicable to food, industrial, domestic and institutional areas but
uses Bacillus subtilis as the test organism. As there is no equivalent
test for medical areas, the authors substituted C. difficile spores to
make the test more relevant to clinical applications. Whilst a standard
is being developed for testing products with medical applications,
using spores including C. difficile, it is only in draft form and
has not been adopted for product testing to date. 16e18 The objective
of this study was to provide healthcare workers, and those charged
with procurement of disinfectants, with information on the efficacy
of sporicidal products in order to improve the efficacy of standard
disinfection of clinical environments contaminated with C. difficile.
Methods
An adaptation of European Standard BS EN 13704:2002 was
used to assess the sporicidal activity of a range of chemical
Table I
Range of active agents and how they were prepared for use in the modified BS EN 13704 test
S. Speight et al. / Journal of Hospital Infection 79 (2011) 18e22 19
disinfectants against C. difficile spores. 19 For all products, the
manufacturer’s lowest recommended concentration was used with
contact times of 1 and 60 min (Table I). The 1-min contact time was
included to simulate a realistic time period for which the product
might be applied in a ward or care home environment during
cleaning. According to the Standard, >10 3 reduction in spore
viability is required for a product to be considered effective.
Test products
Thirty-two products voluntarily supplied by 10 manufacturers
were included in this study, with the majority being specifically targeted
towards C. difficile and sporicidal disinfection (Table I). The
active agents were as follows: chlorine dioxide (N ¼ 19, in varying
concentrations and formulations), hypochlorite solutions (N ¼ 5),
triamine (N ¼ 4), quaternary ammonium compound-based mixtures
(N ¼ 3) and peracetic acid (N ¼ 1).
Preparation of C. difficile spores
A freeze-dried ampoule of C. difficile (NCTC 11209) was obtained
from the National Collection of Type Cultures, and cultured on to
prereduced (37 2 C for 24 h under anaerobic conditions) Clostridium
(CLO) blood agar plates (bioMérieux ref. 43431, CLO Media
BioMérieux, Basingstoke, UK). A 10-mL loop was used to lift colonies
discretely from the agar surface and inoculate 10 mL thioglycollate
broth (anaerobic broth, HPA Medical Supplies, Salisbury, UK). The
broth was then incubated anaerobically (37 2 C for 48 h) in
sealed jars (Oxoid, Basingstoke, UK) using AnaeroGen sachets
Number/agent Manufacturer’s recommended formula Ingredients used by Health Protection Agency
1. Chlorine dioxide 5 mL base, 5 mL activator, activate for 15 s As per manufacturer’s recommendation
2. Chlorine dioxide 12.5 g each of Compounds A and B in 10,000 mL 1.5625 g of Compounds A and B to 1000 mL
3. Chlorine dioxide 5 mL base, 5 mL activator, activate for 15 s As per manufacturer’s recommendation
4. Chlorine dioxide 5 mL base, 5 mL activator, activate for 15 s As per manufacturer’s recommendation
5. Chlorine dioxide 3 mL base, 1.5 mL activator, activate for 15 s As per manufacturer’s recommendation
6. Chlorine dioxide 20 mL base, 20 mL activator to 960 mL, activate for 1 min 2.5 mL base, 2.5 mL activator to 96 mL, activate for 1 min
7. Chlorine dioxide 20 mL base, 20 mL activator to 960 mL, activate for 1 min 2.5 mL base, 2.5 mL activator to 96 mL, activate for 1 min
8. Chlorine dioxide 25 mL agent, 75 mL diluent 31.25 mL agent, 68.75 mL diluent
9. Didecyl-dimethylammonium Use neat Used neat
10. Chlorine dioxide 50 mL base, 50 mL activator to 1000 mL, activate for 1 min 50 mL base, 50 mL activator to 800 mL, activate for 1 min
11. Chlorine dioxide 245 mL base, 5 mL activator, activate for 20 min As per manufacturer’s recommendation
12. Chlorine dioxide 50 mL base, 50 mL activator to 5000 mL, activate for 1 min 6.25 mL base, 6.25 mL activator to 500 mL, activate for 1 min
13. Chlorine dioxide 10 mL base, 10 mL activator to 1480 mL, activate for 1 min 1.25 mL base, 1.25 mL activator to 148 mL, activate for 1 min
14. Triamine 5 mL agent, 95 mL diluent 6.25 mL agent, 93.75 mL diluent
15. Chlorine 1 tablet in 1000 mL water 1.6 g (c. ½ tablet) in 400 mL water
16. Chlorine 1 tablet in 1000 mL water 1.6 g (c. ½ tablet) in 400 mL water
17. Chlorine 1 tablet in 2500 mL water 1 tablet in 2000 mL water
18. Hypochlorite-based mixture Use neat Used neat
19. Hypochlorite 1 tablet in 1000 mL water 1 tablet in 800 mL water
20. Chlorine dioxide 4.5 mL base, 4.5 mL activator to 600 mL, activate for 1 min 5.625 mL base, 5.625 mL activator to 600 mL, activate for 1 min
21. Chlorine dioxide 50 mL base, 50 mL activator to 5000 mL, activate for 1 min 6.25 mL base, 6.25 mL activator to 500 mL, activate for 1 min
22. Chlorine dioxide 50 mL base, 50 mL activator to 5000 mL, activate for 1 min 6.25 mL base, 6.25 mL activator to 500 mL, activate for 1 min
23. Chlorine dioxide 50 mL base, 50 mL activator to 5000 mL, activate for 1 min 6.25 mL base, 6.25 mL activator to 500 mL, activate for 1 min
24. Chlorine dioxide 50 mL base, 50 mL activator to 5000 mL, activate for 1 min 6.25 mL base, 6.25 mL activator to 500 mL, activate for 1 min
25. Chlorine dioxide 50 mL base, 50 mL activator to 5000 mL, activate for 1 min 6.25 mL base, 6.25 mL activator to 500 mL, activate for 1 min
26. Triamine (wipes) Expressed fluid used neat Expressed fluid used neat
27. Peracetic acid (wipes) Expressed fluid used neat Expressed fluid used neat
28. Triamine 5 mL agent, 95 mL diluent 6.25 mL agent, 93.75 mL diluent
29. Triamine 2.5 mL agent, 97.5 mL diluent 3.125 mL agent, 96.875 mL diluent
30. Chlorine dioxide Use neat Used neat
31. Benzalkonium chloride,
didecyldimonium chloride,
bronopol, polyaminopropyl
biguanide hydrochloride
Use neat Used neat
32. Benzalkonium chloride,
didecyldimonium chloride,
Bronopol, polyaminopropyl
biguanide hydrochloride
Use neat Used neat

20
Table II
Active agents demonstrating the log reduction achieved at 1 and 60 min under clean
(0.3% albumin) and dirty (3% albumin) conditions
No. Active agent Log10 reduction from 10 6 challenge
(Oxoid, Basingstoke, UK) to generate the anaerobic atmosphere.
Anaerobic indicators (Oxoid) were used in the jar as a visual check
that anaerobic conditions were achieved. The resulting C. difficile
suspension was used to inoculate 15e100 prereduced CLO agar
plates, which were incubated anaerobically at 37 2 C for three to
five days. The growth was scraped from the surface of the agar into
10 mL hard water (as per BS EN 13704:2002) and vortexed until the
mixture appeared homogenous. The suspension was stored at
4e8 C for three to five days and then heat shocked at 70 C for
20 min. The concentration of viable spores was determined by
carrying out serial 10-fold dilutions, inoculating 100 mL on to prereduced
CLO blood agar plates in duplicate and incubating anaerobically
at 37 2 C for three to five days. Microscopic examination
(400 ) of an aliquot of the suspension was carried out after preparation
to ascertain the presence of spores and the absence of
vegetative cells.
Suspension test
Clean
(0.3% albumin)
Dirty
(3% albumin)
1 min 60 min 1 min 60 min
1 Chlorine dioxide >4 >4 >4 >4
2 Chlorine dioxide >4 >4 >4 >4
3 Chlorine dioxide >4 >4 >4 >4
4 Chlorine dioxide >4 >4 >4 >4
5 Chlorine dioxide >4 >4 3 >4
6 Chlorine dioxide >4 >4 3 >4
7 Chlorine dioxide >4 >4 3 4
8 Chlorine dioxide 3 >4 3 >4
9 Didecyldimethylammonium 3 >4 4
10 Chlorine dioxide 3 >4 4

their target cells in order to inactivate them; with disinfectants
applied in solution (as opposed to gaseous agents), this will only
occur whilst the product remains wet. Drying after application of
a disinfectant as a wipe will occur in far less than the 60 min of
exposure in a suspension test, and even 1 min could be thought of
as a generous approximation of what would occur in reality. There
are a number of ongoing debates on the development of standards
to evaluate the efficacy of sporicides. 16,17,20 European Standard BS
EN 13704 is currently being used to validate the claims of many
sporicides used in health care, but it has not been developed for
such an assessment, and tests agents against spores of B. subtilus
rather than more clinically relevant spore-forming microbes.
This study set out to assess the effectiveness of a range of
sporicides, under clean and dirty conditions, for short (1 min) and
long (60 min) contact times against C. difficile in a suspension test
based on the BS EN method (EN 13704). Only eight products achieved
a 10 3 -fold reduction in 1 min under dirty conditions
(3% albumin), illustrating why caution should be applied when
selecting disinfectants for clinical areas and near-patient medical
equipment that may be contaminated with C. difficile spores.
Longer exposure times may still have relevance in some environmental
decontamination contexts. Based on the evidence that
a single clean can physically reduce contamination by around 90%,
presumably compounded on sequential cleans, there may be no
need to incorporate a sporicidal agent for decontamination of
surfaces which undergo routine cleaning. 21 This still leaves the
problem that the act of cleaning may itself redistribute and spread
spores. The presence of a sporicidal disinfectant in the cleaning
fluid would have longer to act on spores acquired on the cleaning
equipment (e.g. a mop), and thus reduce the re-application of
contamination on to surfaces by the cleaning process itself. This
would not apply to wipes, where the use of an individual wipe is
more limited both in duration (single use) and area of application.
However, these current tests do not replicate how wipes are used in
a ward, and actual efficacy protocols of how the wipes would
perform in practice are recommended.
Current guidelines 15 and publications, based on in-vitro and insitu
studies, advocate chlorine-based disinfection to reduce the
viability of C. difficile spores in the clinical setting. 9,10,22 None of the
hypochlorite products tested in the present study achieved
adequate disinfection in the likely exposure time in either clean or
dirty conditions.
Whilst chlorine dioxide was generally the most effective active
agent for reducing the viability of C. difficile spores, not all of the
products based on chlorine dioxide were effective over short
contact times or under dirty conditions. To some extent, this may
have reflected the manufacturer’s dilution instructions (e.g.
Product Nos 21e25 were diluted more than the other chlorine
dioxide products, Table I).
The short contact time of 1 min was used to represent the likely
time that a product may remain in contact with a surface prior to
evaporation, although the actual contact time may be shorter than
that in many instances. Only a limited number of disinfectants were
effective in the 1-min contact time.
It is recognized that, for many disinfectants, organic matter
reduces activity by reacting with the disinfectant or by preventing
the disinfectant from accessing its microbial target; this study
reinforces these previous findings. 23e25 Such studies reinforce the
message that basic standard nursing practices, including cleaning
in the ward and its environs, are paramount in the strategy to
reduce healthcare-associated infection rates.
The purpose of decontamination is to break chains of transmission
of infection by reducing the number of viable microbes.
Whilst the BS EN method used (EN 13704) aims to achieve
>10 3 -fold reduction, it has been estimated that a patient with CDI
S. Speight et al. / Journal of Hospital Infection 79 (2011) 18e22 21
can excrete between 1 10 4 and 1 10 7 C. difficile/g faeces. 26 The
infectious dose of C. difficile can be very small, and murine studies
have shown that consumption of as few as one to two spores may
be sufficient to establish colonization and CDI in clindamycintreated
mice. 27 There does, therefore, appear to be an inherent
problem with disinfectant testing that, for pragmatic reasons, the
microbial reductions are less than the challenge in practice (e.g. 10 5
with vegetative bacteria, 10 4 with fungi/yeasts and 10 3 with
spores). These microbial reductions appear to be thresholds for
passing a test, rather than numbers that are meaningful in practical
applications.
Taking all these findings into consideration, there is a degree of
onus on healthcare workers and supplies staff to ask questions
about the products that are being considered for use in frontline
situations. Equally, is it not time for those formulating disinfection
standards to consider a more realistic sporicidal test for the medical
arena that has a higher reduction over a shorter contact time as the
pass criterion?
Conflict of interest statement
None declared.
Funding source
The authors would like to acknowledge the NHS Supply Chain
for funding the study and providing the products. The views
expressed in this manuscript are those of the authors and not
those of the funding agency.
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