THE PREVALENCE OF CLOSTRIDIUM DIFFICILE IN AIREDALE NHST HOSPITAL ENVIRONMENT. University Of Central Lancashire School Of Pharmacy And Biomedical Sciences Bsc (Hons) Biomedical Science (Part-Time) Research Project Report BL3296 The Prevalence of Clostridium Difficile at Airedale NHST Hospital Environment. Willard Erasmas Dzinyemba G20269064 1
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THE PREVALENCE OF CLOSTRIDIUM DIFFCILE AT AIREDALE NHST ENVIRONMENT
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THE PREVALENCE OF CLOSTRIDIUM DIFFICILE IN AIREDALE NHST HOSPITAL ENVIRONMENT.
University Of Central Lancashire
School Of Pharmacy And Biomedical Sciences
Bsc (Hons) Biomedical Science
(Part-Time)Research Project Report
BL3296
The Prevalence of Clostridium Difficile at Airedale NHST Hospital Environment.
Willard Erasmas Dzinyemba
G20269064
November, 2013
AUTHORSHIP DECLARATION
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THE PREVALENCE OF CLOSTRIDIUM DIFFICILE IN AIREDALE NHST HOSPITAL ENVIRONMENT.
I, Willard E. Dzinyemba confirm that this dissertation and the work presented in it are my
own achievement.
Where I have consulted published work of others, or quoted from their work, this is attributed
and the source given. With these exceptions the entire dissertation is my own work;
I have acknowledged all main sources of help and contributors to relevant previous and on-
going research projects made in this area of research.
I also confirm that I have obtained informed consent from all people I have involved
in the work in this dissertation following the School's ethical guidelines.
I have read and understood the penalties associated with Academic Misconduct.
Signed:
Date: 22nd November, 2013
TABLE OF CONTENTS
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THE PREVALENCE OF CLOSTRIDIUM DIFFICILE IN AIREDALE NHST HOSPITAL ENVIRONMENT.
1. Abstract
2. Introduction
2.1 Pathogenesis
2.2 Sources of Infection
2.3 Infection Prevention
3. Methods and Materials
3.1 Air Sampling
3.2 Environmental Surface Sampling
3.3 Soil and Cow dung
3.4 Pilot Study
3.5 Sampling and Processing Method Verification
3.6 Colony identification of Clostridium difficile
4. Results
5. Discussion
6. Conclusion
7. Sources of materials and manufacturers
8. Acknowledgements
9. References
10. Appendices
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THE PREVALENCE OF CLOSTRIDIUM DIFFICILE IN AIREDALE NHST HOSPITAL ENVIRONMENT.
1. ABSTRACT
Clostridium difficile (CD) is a normal commensal bacterium of the adult gastrointestinal tract
which under certain conditions induces diseases like pseudomembranous colitis. It is a
nosocomial pathogen that is transmissible between patients in a hospital or from exogenous
sources to patients. There are variations in the reported prevalence of CD in hospital and
domestic environments. Most of the variations are due to the differences in sensitivities and
specificities of the methods used to isolate CD. The aim of this study was to determine the
prevalence and extent of Clostridium difficile (CD) contamination in ward and hospital
environments at Airedale General Hospital and from the farms that surround it using the
methods available at the hospital laboratory.
Air samples from the ward and hospital corridors were collected and tested. Premoistened
swabs were used to collect samples from ward surfaces around known Clostridium difficile
infected (CDI) patients, in corridors and farm cattle stoles. Soil samples were collected from
the hospital grounds and farms around the hospital. Cow dung was also collected from the
farms as it forms part of the hospital environment, and tested for CD.
Out of a total of 171 samples, CD was isolated from 3 (1.75%) samples. One (5.26%) of the
19 air samples was positive for CD and 2 (2.35%) out of 85 swabs collected were CD
positive. One isolate was non-toxigenic and awaiting PCR ribotype results and two isolates
were toxigenic by C. difficile GDH testing and Polymerase Chain Reaction (PCR) ribotype
027, and PCR ribotype 002 (table 4). No Clostridium difficile was isolated from the soil and
cow dung samples from the farms, air and surfaces near CDI patients except the floor (2).
Isolation of CD from air samples in hospital corridors show the sporadic contamination of air
away from symptomatic CDI patients which may be an exogenous source of CD.
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THE PREVALENCE OF CLOSTRIDIUM DIFFICILE IN AIREDALE NHST HOSPITAL ENVIRONMENT.
The findings from this study also imply that adherence of health workers to infection
prevention protocols mainly hand hygiene. The cleaning detergent used at this hospital may
indicate that it is an effective sporicide and bactericide as shown by 0% of CD isolated from
all other sites especially contact areas near a patient with Clostridium difficile infection (CDI)
except from the floor. However, the repeated isolation of CD from the floor of this ward puts
in question the thoroughness of cleaning. The results also indicate that soil and cow dung in
their (hospital) environment do not pose a potential risk for exogenous CD transmission to
patients.
Adequate, more frequent and thorough decontamination of rooms and corridors may be
needed to minimise the risk of nosocomial infection with CD.
2. INTRODUCTION
Clostridium difficile infection is the most common cause of nosocomial diarrhoea with risk
factors which include advanced age, severity of underlying illness, gastrointestinal surgery,
the use of electronic rectal thermometers, and prior use of antimicrobials (Mayfield et al.
2000, Bartlett 1994, Loo et al. 2011, Koss et al. 2006, Friedman et al. 2013). It is associated
with mild diarrhoea, pseudomembranous colitis, and toxic megacolon (Bartlett 2008, Murray
et al. 2003, Yakob et al. 2013a). The infection results in an increased length of stay in
hospital ranging from 8 to 21 days which increases the cost of healthcare (Barbut et al. 2001,
Yakob et al. 2013a). It can cause sepsis and even death (McDonald et al. 2007, Muto et al.
2007). It has been isolated from healthy adults, asymptomatic neonates, animals, water from
rivers, lakes, sea and tap water, and also from soil (Malamou-Ladas et al. 1983, Al Saif et al.
1996). The infection is generally acquired nosocomially (Al Saif et al. 1996).
The reported prevalence rates by different studies were between 2% to 12% (Best et al.
2010), 7% - 13% (Martirosian 2006, Al Saif et al. 1996) Variations in the techniques used
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THE PREVALENCE OF CLOSTRIDIUM DIFFICILE IN AIREDALE NHST HOSPITAL ENVIRONMENT.
account for these differences. Some studies used direct plating (Al Saif et al. 1996) and others
used sample enrichment methods (Akhi et al. 2011, Martirosian 2006, Vaishnavi et al.
2012). In this study, currently available techniques to the hospital laboratory for the isolation
of CD (direct plating) were used. A more preferred highly recommended and sensitive
method of enrichment using brain heart infusion with 1% sodium taurocholate (Akhi et al.
2011)was not used in this study due to budgetary constraints.
Clostridium difficile infection is a burden to health care facilities and Airedale NHS
foundation trust is no exception. Healthcare facilities have to deal with high financial costs of
morbidity and mortality related to CDI (Hill et al. 2013, Yakob et al. 2013a). While Figure 1
below shows a marked reduction of cases (25%) between 2011 and 2012 in England, Wales
and Northern Ireland, more measures need to be put in place to eradicate it and meet targets
(HPA 2012).
Figure 1: Data from (HPA 2012).
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THE PREVALENCE OF CLOSTRIDIUM DIFFICILE IN AIREDALE NHST HOSPITAL ENVIRONMENT.
The number of cases of CD as apportioned by the department of health for Airedale NHS
trust for 2012 was 12. A total of 9 of the 12 were reported by October 2012 indicating high
incidence (Charlesworth 2012b).
Clostridium difficile was first isolated in 1935 from stool samples of new-born children and
named Bacillus difficilis (Lyerly et al. 1998). It was found to exist as a commensal organism
of the digestive tract of young infants (Bartlett 2008, Barbut et al. 2001, Lyerly et al. 1998).
Its toxins were also identified by (Bartlett 2008) as the cause of pseudomembranous colitis
for the first time in 1978 (George et al. 1979, Tenover et al. 2011, Bartlett 2008).
Clostridium difficile belongs to the family Clostridiaceae and genus Clostridium (Murray et
al. 2003). It is a motile gram positive sub terminal spore forming rod (Howerton et al. 2011,
Barbut et al. 2001, Koss et al. 2006) measuring 3-5 µm in length and 0.5µm in width
(Murray et al. 2003). It is a heterotrophic organism with an optimal growth temperature of
37°C in an anaerobic environment with peritrichous flagella (Murray et al. 2003). Over 400
strains of Clostridium difficile have been identified to date and only 20 toxic stains are known
to be seriously pathogenic towards humans or animals (Tonna et al. 2005, Hatheway 1990).
Colonies on culture media appear flat and slightly grey in colour with a ground glass
appearance. They have a distinctive ‘elephant house’ odour due to the production of iso-
valeric acid, iso-caproic acid and p-cresol, which are the products of various metabolic
pathways within the organism. They also produce catalase which can be used for differential
diagnosis of CD (Hatheway 1990, Tenover et al. 2011, Murray et al. 2003).
2.1 Pathogenesis
Clostridium difficile bacillus exists either as a vegetative cell or an endospore (Murray et al.
2003, Poutanen et al. 2004). The spores are highly resistant to physical and chemical
treatment with some cleaning agents known to enhance their resistance (Dancer 2009,
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THE PREVALENCE OF CLOSTRIDIUM DIFFICILE IN AIREDALE NHST HOSPITAL ENVIRONMENT.
Howerton et al. 2011). CD is a nosocomial pathogen (hospital acquired) which is responsible
for Clostridium Difficile-associated diarrhoea (CDAD) and significant morbidity and
mortality amongst elderly people and patients in healthcare facilities (Roberts et al. 2008,
Tonna et al. 2005). It is also reported to be a cause of enteric diseases in animals like horses,
dogs, birds, pigs and rodents which are believed to act as reservoirs for CD (Kuijper et al.
2006). It is mainly acquired from the environment through the faecal-oral route and lives as a
commensal in the colon (Tonna et al. 2005, Cohen et al. 2010, Mulligan et al. 1979, Yakob et
al. 2013a). While most of the vegetative cells are killed by acid in the gut, spores, which are
resistant survive, establish themselves and colonise the gastrointestinal gut of people who
may be asymptomatic (Anonymous2004, Poutanen et al.2004)
The presence of up to 1012 organisms of normal flora in a gram of faeces composed of
predominantly Lactobacilli and enterococci help resist colonisation and stop multiplication of
C. Difficile in the colon (Tonna et al. 2005, Lopetuso et al. 2013). CDAD usually occurs
during or after antibiotic treatment (Bignardi 1998) by disrupting the normal gut flora
(dysbiosis), allowing CD from endogenous or exogenous origins to start multiplying and
proliferating (Barbut et al. 2001, Tonna et al. 2005, Lyerly et al.1998). Bile acids in the
stomach may also promote germination of the bacilli (Poutanen, Simor 2004).
Pathogenic strains of C. difficile produce two major glycosylating toxins; Toxin A
(enterotoxin) and Toxin B (cytotoxic) which are also its virulence factors and encoded on
pathogenicity locus 19.6 kb – PoLac(Deneve et al. 2009, Lyerly et al. 1998, Stabler et al.
2009, Voth et al. 2005). These toxins are encoded for by the genes tcdA and tcdB
respectively as seen in Figure 2 below. Both toxins are produced during the late lag and
stationary phases of growth which allows cells to become established within the host gut
before toxin production begins (Voth et al. 2005)
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THE PREVALENCE OF CLOSTRIDIUM DIFFICILE IN AIREDALE NHST HOSPITAL ENVIRONMENT.
Figure 2: Showing the genetic arrangement of the C. difficile pathogenicity locus and proposed protein domain
structures of TcdA and TcdB (Voth et al. 2005).
The toxins A and B cause inflammation and damage to the mucosa and fluid secretions as its
characteristic pathology (Barbut et al. 2001, Poxton et al. 2001). They cause damage by
opening tight junctions between the cells of the intestine that result in increased vascular
permeability and haemorrhage. They also induce the production of tumour necrosis factor-
alpha (TNF-alpha) and pro inflammatory interleukins that cause a large inflammatory
response and ultimately the formation of pseudo membranes (Voth et al. 2005). The
pathogenesis of CD can be seen on fig. 3 below.
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THE PREVALENCE OF CLOSTRIDIUM DIFFICILE IN AIREDALE NHST HOSPITAL ENVIRONMENT.
Figure 3: Pathogenicity of Clostridium difficile in the gut (Poutanen et al. 2004)
Other virulence factors also used by CD are the capsule (used as an antiphagocytic factor),
proteolytic enzymes (used to enhance mucus penetration), adhesins (involved in mucus and
cell adhesion)(Hennequin et al. 2001), fimbriae and flagella for penetration of mucus layer
(Deneve et al. 2009) . Lower levels of anti-toxin A IgG are associated with the severe form of
the disease (Tonna et al. 2005, Loo et al. 2011). People with a weakened immunity like HIV
infection may be prone to CD infection. Another factor is the emerging virulent strains of CD
as reported by (Cohen et al. 2010). The strain PCR ribotype 027 with genes encoding for
toxins A and B is an epidemic strain with an 18 base pair deletion in tcdC and is highly
virulent. It also has binary toxins called CDT (McDonald et al. 2005, Deneve et al. 2009)
which potentiates the toxicity of TcdA and TcdB leading to a more severe disease (Deneve et
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THE PREVALENCE OF CLOSTRIDIUM DIFFICILE IN AIREDALE NHST HOSPITAL ENVIRONMENT.
al. 2009) . Within two weeks of colonisation with CD, cells of CD are shed in stool (Yakob et
al. 2013b).
2.2 Sources of Infection
Most studies have reported that people infected with CD shed up to 107 of CD per gram of
faeces into the environment. This is believed to be a source of CD infection (Best et al.
2010). The isolation of CD from skin sites of patients of CDAD even after the resolution of
diarrhoea is well documented (Rutala et al. 2013). These sites are the potential sites of
transmission between nurses, housekeepers and other patients (Rutala et al. 2013, Bobulsky
et al. 2008). Roberts et al. 2005 demonstrated that the environment is contaminated by the
use of nebulisers, the movement of people and bed making among others which liberate
aerosols into the environment as summarised in Figure 4 below (Roberts et al. 2006). This
means that these activities can lead to the contamination of air, food and fomites with CD if
present (Best et al. 2010, Al Saif et al. 1996). (Al Saif et al. 1996) noted the presence of CD
on vegetables - 2.4%, soil samples - 21%, river and lake water - 40 – 81.2%, hospital
environment – 20%, and nursing homes at 2.2%.
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THE PREVALENCE OF CLOSTRIDIUM DIFFICILE IN AIREDALE NHST HOSPITAL ENVIRONMENT.
Figure 4: Overview of potential sources of Clostridium difficile transmission. (Donskey 2010)
2.3 Infection Prevention
Infection control teams and other healthcare workers are faced with a challenge to control CD
infection. Prevention of CDI is delivered by preventing or stopping patient exposure to the
organism or ensuring that the patient’s gut flora is not disrupted and left susceptible to CDI
(HPA 2006). Several recommendations and guidelines have been rolled out over the years
which most healthcare providers including Airedale NHS trust have adopted (Gerding et al.
2008, Dancer 2009, Cohen et al. 2010, Stuart et al. 2011). Amongst the strategies
implemented are those aimed at targeting the environment, hospital personnel hand hygiene,
prevention of ingestion of spores and minimising antimicrobial exposure (good antimicrobial
stewardship) (Stuart et al. 2011, MacLeod-Glover et al. 2010). The use of C. difficile toxoid
vaccine is known to give high toxin A IgG in humans and confers immunity against diarrhoea
due to CD (Tonna, Welsby 2005, Kyne et al. 2001, Aboudola et al. 2003). It has been
extensively reported that CD spores survive the use of hand hygiene alcohol based gels and
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THE PREVALENCE OF CLOSTRIDIUM DIFFICILE IN AIREDALE NHST HOSPITAL ENVIRONMENT.
cleaning detergents (Gerding et al. 2008). The spores also survive exposure to heat, acids
and most antibiotics (Rutala, Weber 2013). At Airedale NHS trust, the infection prevention
team monitor the severity of CDI, laboratory results, adherence to current antibiotic policy,
management of faecal contaminated laundry, hand hygiene and general and deep cleaning of
wards and other facilities (Charlesworth 2012a).
Isolation of suspected cases of CD infection helps to minimise the spread of the infection to
other wards and patients. This also helps the team to confine the infection (Charlesworth
2012a, Cohen et al. 2010).
The use of chlorine based cleaning agents is significant in reducing the contamination of
surfaces with CD (Wilcox et al. 2003). (Dancer 2009) reported traces of CD being found
after disinfection with bleach. These remnants of CD could be potential sources of infection
for patients being admitted afterwards (Wilcox et al. 2003, Rutala, Weber 2013) . Other
detergents in use in different hospitals are those whose active agents are acidified nitrite, par
acetyl ions, glutaraldehyde, alcohol, most of which are hazardous, and known to cause
asthma and dermatitis (Faise et al. 2010, MacLeod-Glover, Sadowski 2010). Therefore,
consideration of safety, effectiveness and cost needs to be made in choosing a suitable and
reputable detergent to use. For this reason, Airedale General Hospital changed from using a
hypochlorite based detergent to a chlorine dioxide one called Tristel. It is non-toxic, non-
flammable, and sporicidal (Faise et al. 2010).
3 MATERIALS AND METHODS
3.1 Air Sampling
Air samples were collected from wards with colonised or CDI patients before and/or after a
deep cleaning exercise and hospital corridors using a portable air sampler - AES Sampl’air
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THE PREVALENCE OF CLOSTRIDIUM DIFFICILE IN AIREDALE NHST HOSPITAL ENVIRONMENT.
Lite from BioMérieux, Basingstoke, United Kingdom. Information on the CDI of ward
occupants at the time of sampling was obtained through infection prevention control team.
Following air sampler charging and head cleaning using 70% alcohol, the procedure from
THE PREVALENCE OF CLOSTRIDIUM DIFFICILE IN AIREDALE NHST HOSPITAL ENVIRONMENT.
8. ACKNOWLEDGEMENTS
This project was undertaken as part of the BSc honours degree project in Biomedical
Sciences, conducted at Airedale NHS General Hospital between July and September, 2013
and funded by the Trust with supplement funding from the University of Central
Lancashire. I thank God for the wisdom and perseverance that he has bestowed upon me
during this research project, and indeed, throughout my life: I can do anything through him
who gives me strength.
I would like to express my thanks to Suz Donald, who gave me the chance to work on this
research as part of my honours degree project.
I would also like to express sincere appreciation to my supervisor, Dr Laura McShane, for her
guidance throughout this project. Our discussions always helped me to focus my mind in
making some of the key decisions, Kathryn Moorhouse and Danielle North for their technical
support, guidance, and advice throughout the research project, as well as their pain-staking
effort in proof reading the drafts. I am indebted to most of biomedical scientists in the
department of Microbiology, for being there for me when my supervisors were on holiday.
James Stickland and the Infection prevention team for the information and coordination of
ward sampling. I thank Medical wire & equipment for providing us with free samples of
highly recommended Polywipes for use in this study. I also would like to thank farm mangers
of farms around Airedale general hospital and especially Barry for letting us into their farms
to collect samples used in this study.
Lastly, I offer my regards and blessings to all of those who supported me in any respect
during the completion of the project.
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10. APPENDICES
Appendix1: Table for calculating the air contamination from the number of colonies isolated
from air samples.
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THE PREVALENCE OF CLOSTRIDIUM DIFFICILE IN AIREDALE NHST HOSPITAL ENVIRONMENT.