the prevalence of clostridium diffcile at airedale nhst environment

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

1


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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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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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 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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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 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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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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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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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

10


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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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

12


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

13


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

Airedale NHS foundation Trust pathology services - Microbiology department’s protocol –

Appendix 1 was employed to prime a plate of commercially prepared Brazier’s Agar Medium

to ensure sterility (Crabtree 2012). A petri dish with Brazier’s media was placed on the clips

and the steel sampl’air head was placed on top. The sampl’air is run for 2 minutes.

The air sampler was then moved to different locations where samples were collected for 2

minutes (100 L/minute) at a velocity of 16.8 m/s. The samples were collected direct onto a

fresh commercially prepared Brazier’s Clostridium difficile Selective medium PB1055A,

Oxoid Limited, Basingstoke, United Kingdom. Plates were transported to the laboratory and

incubated anaerobically at 370C for 48 hours initially and read every 24 hours for the next 3

days if no growth was observed. The numbers of colonies grown on each plate were counted

and Appendix 2 was used to find N. This was used to calculate the level of airborne

contamination using the formula:

( N ÷V )CFU /m3where N is equal to n (the number of colonies counted on a plate) used to

find N on appendix 1, V= volume derived by multiplying the sampling dilution by 0.1 m3

/minute.

3.2 Environmental Surface Swab Sampling

Two swabs from environmental sites were collected from door handles, floors, commodes,

bedrails, sinks, toilets, walls and bathrooms. One swab was transferred into a brain heart

infusion broth for the recovery of CD. They were vortexed for 1 minute and incubated

anaerobically for 48 hours at 370C. They were then subcultured onto Brazier’s CD selective

medium and incubated anaerobically at 370C for 48 hours and every 24 hours for the next 72

14


hours if no growth was observed. This was done for the pilot study only. The second swab

was inoculated direct onto Brazier’s media and processed as the first swab.

Sterile premoistened blue cellulose swabs (6) – (Polywipes) form Medical wire & equipment

Co. (Bath) Ltd, Wiltshire, United Kingdom as those used by (Best et al. 2010) were used for

collection of samples from surface areas of ward 6. For comparison, all the sites which were

sampled using a premoistened swab had a Polywipe swab collected as well. After collection,

the Polywipes were put in a sterile bag and transported to the laboratory where they were

placed in contact with the surface of Brazier’s media for one minute and the plate incubated

as described in section 3.2. A few free samples were used for this purpose.

3.3 Soil and Cow dung

Soil and cow dung samples were collected in a sterile screw capped bijou bottle. The sample

was alcohol shocked by adding an equal volume of absolute alcohol, vortexed and incubated

at room temperature for 30 – 60 minutes as done by (Best et al. 2010). The samples were then

centrifuged at 300 rpm for 5 minutes and two drops of the deposit were inoculated onto

Brazier’s media and incubated at 35 ±2 0 C in an anaerobic condition, for 48 hours initially

and read every 24 hours for the next 72 hours if no growth was observed.

3.4 Pilot Study

Comparing the use of brain heart infusion as an enrichment step with direct plating onto

Brazier’s media; the following procedure was used:

Duplicate samples were collected from different sites in ward 2 which had a colonised

patient at the time using pre-moistened swabs (Physiological buffered saline)

One swab was placed in brain heart infusion and processed following the proposed

procedure above for environmental surface swabs.

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The second swab was inoculated directly onto Brazier’s media and processed

following the proposed procedure above for environmental surface swabs.

3.5 Sampling and Processing Method Verification

(i) A Clostridium difficile positive sample was used for verification of the method

proposed in this study. It was used to test the ability of the two methods i.e. direct

plating and enrichment in brain heart infusion to recover C.D. from the spores on

the plate. CD was inoculated onto Brazier’s media, incubated at 35 ±2 0 C in an

anaerobic environment for 48hours. The plate was then placed on the open at

room temperature to allow it to dry and sporulate. After one week, two swabs

were collected from the plate and processed as per procedure for environmental

surface sampling above. During the process of inoculating the media, an air

sampler placed less than a metre below the bench was collecting air samples onto

Brazier’s media for 2 minutes. It was processed following the air sampling

procedure above.

(ii) This part of the study was conducted to show the ability of the method proposed

to recover C. difficile from surfaces at different concentrations. This was

performed following the procedure as done by (Buggy et al. 1983) with changes

as below.

A CD suspension of 0.5 Mcfarland standard equal to 1.5x108CFU/ml in normal

saline from a known Clostridium difficile positive sample was diluted by

transferring 1ml of the suspension into 9mls of normal saline and into the next

subsequent test tube to make a 1:10 dilution as shown in table 1 below.

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Tube # 1 2 3 4 5 6 7Conc. of CD 1.5x108CFU/ml 1.5x107CFU/ml 1.5x106CFU/ml 1.5x105CFU/ml 1.5x104CFU/ml 1.5x103CFU/ml 1.5x102CFU/ml

Smear volume

100µl 100µl 100µl 100µl

Table 1: Concentrations of CD diluted 1:10 from test tube 1 to 7 and slide preparations for

method verification study.

A volume of 100µls from each suspension in the 1st, 3rd, 5th and 7th test tubes as

shown in table 1 were placed on glass slides and left at room temperature for a

week to dry and sporulate. Two pre-moistened swabs were used to collect a

sample from each glass slide and inoculated on to Brazier’s media. The swabs

were processed following the procedure above for processing environmental

surface swabs.

During the process of inoculating the media, an air sampler placed less than a metre below

the bench was collecting air samples onto Brazier’s media for 2 minutes. It was processed

following the air sampling procedure above. This was to isolate any aerosols (CD) liberated

during sample collection from surfaces where different concentrations of CD were placed.

3.6 Colony Identification of C. Difficile

Identification of CD was done by studying colony morphology, gram stain and biochemical

methods with the aid of a table 2 below.

C. difficile C. innocuum C. glycolicum C. sordelli/ bifementans

Odour + - - -Lecithinase - - - +UV fluorescence + + - -API test

Table 2: differential tests for recognition of colonies of Clostridium difficile.

17


Colonies on Brazier’s medium appear yellowish to white, circular to irregular and flat with

rhizoid edge and a ground-glass appearance as described by (Beaugerie et al. 2003, Al Saif,

Brazier 1996). Screening colonies subcultured on Fastidious Anaerobe Agar (FAA) under

long-wave ultra violet light (365nm), shows yellow-green fluorescence. They produced

distinctive horse manure like odour (Murray et al. 2003). Gram stain showed gram positive to

variable thin rods 3-5µm by 0.5 µm. Colonies that fit this criteria were further tested by

API(Analytical Profile Index) test (a commercial system used to identify bacteria using

biochemical tests and a database) (Janda, Abbott 2002) called rapid ID 32 A V3.2 -

BioMérieux, Basingstoke. All isolates of CD positive samples by API test were also tested to

detect Clostridium difficile glutamate dehydrogenase antigen and toxins A and B in a single

reaction using C. Diff Quik Chek Complete – Alere limited, Cheshire, United Kingdom as

per manufacturer’s instructions. This was performed by modifying the method as performed

by (Friedman, Pollard et al. 2013) using a suspension of colonies instead of faeces. A sample

of the isolates grown on chocolate agar slopes was sent to Leeds general infirmary laboratory

for confirmation of C. difficile by PCR (Polymerase Chain Reaction) Ribotyping (Rupnik et

al. 2001) in keeping with the ANHST Microbiology protocol.

Analysis of the results was done using Microsoft Excel 2010.

4. RESULTS

(1) In the pilot study which was meant to evaluate the need for enrichment step with brain

heart infusion to be included or not, there was no CD isolated using the two methods – Direct

plating or enrichment with brain heart infusion. The next pilot study as in section 3.5 (i)

showed that brain heart infusion broth only isolated 50% of the know positive samples and

was thus its use in the study was stopped.

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Clostridium difficile was recovered from all levels of concentrations using the proposed

method during method verification study with the lowest tested being 1.5x102 as shown in

table 3.

Tube # 1 2 3 4 5 6 7Conc. of

CD 1.5x108CFU/ml 1.5x107CFU/ml 1.5x106CFU/ml 1.5x105CFU/ml 1.5x104CFU/ml 1.5x103CFU/ml 1.5x102CFU/ml

Smear volume

100µl

-100µl

-100µl

-100µl

Recovery of CD >100 colonies - 80 colonies - 10 colonies - 3 colonies

API Test CD recovered - CD recovered - CD recovered - CD recovered

Table 3: Results of CD recovery for both direct plating and air sampling for method verification.

(2) The overall results of the study analysed using Excel 2010 are shown in figure 5 and 6

below.

Floor

Patien

t Tab

leSin

k

Door Han

dle

CommodeTo

ilet

Bathroom

Air Sam

ple

Soil S

ample

Walls

Cow Man

ureTo

tal0

20

40

60

80

100

120

140

160

180

Number of Samples per location versus Clostridium difficile positives

Total# of samples# of Positive samples

Location

Tota

l num

ber o

f sam

ples

Figure 5: a graph of Clostridium difficile isolates and the total number of samples collected from different

locations of Airedale NHS hospital and surrounding farms.

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Out of 171 samples collected for the study, Clostridium difficile was isolated from 3 samples

(1.75%). Out of Nineteen air samples, one (5.26%) was positive for CD. The level of air

contamination was calculated as below:

Number of colonies (n) = 1; N (from appendix 1) = 1, total time = 2 minutes, volume=0.1;

Using (N ÷ V );(1 ÷ 2× 0.1) CFU / m3 = 5 CFU / m 3

The other 2 (2.35%) isolates out of 85 swabs were from ward 6 floor with a CD infected

patient.

During the comparative study of Polywipes against normal swabs (6), 1 colony of CD was

isolated using Polywipes from the floor. No CD was isolated from all the other surfaces by

Polywipes or normal swabs.

As seen from the data on figure 6 below, there was no CD isolated from cow manure and soil

samples.

Air samples Swab samples

Soil samples Cow dung Total0

20

40

60

80

100

120

140

160

180

19

85

3928

171

1 2 0 0 3

Number of Samples tested versus Clostridium difficile pos-itives

Total # of samples# of Pos. samples

Sample type

Tota

l num

ber o

f sam

ples

Figure 6: showing different types of samples and the number of CD isolates.

20


The three isolates were tested for Clostridium difficile glutamate dehydrogenase antigen and

toxins A and B and PCR Ribotyping was tested at Leeds general infirmary Hospital. Isolates

were assigned novel ribotypes (RT) according to Brazier’s nomenclature.

The results are shown in table 4 below.

Sample I.D. API test GDH TestAntigen Toxin

PCR Ribotyping

069-08-01 CD Isolated + ± 002111-01-01 CD Isolated + + 027155-01-01 CD Isolated + - pending

Table 4: Results of C. Difficile isolates:

No C. difficile was seen on any of the control (QC) air samples from ward and corridors

(n=4).

5. DISCUSSION

The purpose of the study was to determine the prevalence of C. difficile in the hospital

environment in order to understand the extent of CD contamination which other patients may

be exposed to during hospital stay.

The hospital environment is a major source of infection where C. difficile spores can be found

for months, due to their resistance to heat and some disinfectants (Otter et al. 2011, Weber et

al. 2010, McFarland et al. 1989). While spores may remain in a dormant state for up to 40

days(Mulligan et al. 1979), providing a reservoir for new infections, vegetative forms of C

difficile survives for up to 15 minutes on dry surfaces in room air, or may remain viable for

up to 6 hours on moist surfaces (Otter et al. 2011). Infected patients and asymptomatic

carriers may serve as reservoir for the organism which may act as an exogenous source of CD

to susceptible individuals (Roberts et al. 2008, Vaishnavi, Singh 2012). Medical devices

including portable bed commode and electronic rectal thermometers have been linked to the

21


transmission of CD in the hospital (Weber et al. 2010). Health workers’ hands have also been

reported to help in the transfer of contamination between patients or inanimate objects (Otter

et al. 2011, McFarland et al. 1989, Rutala, Weber 2013).

The prevalence of Clostridium difficile in a hospital environment is now well documented

with reported surface contamination rates ranging from 2% -59%(Kaatz et al. 1988, Kim et

al. 1981, McFarland et al. 1989, Al Saif, Brazier 1996, Martirosian 2006, Malamou-Ladas et

al. 1983, Titov et al. 2000, Vaishnavi, Singh 2012) air contamination from 0%-12%(Best et

al. 2010, Roberts et al. 2008, Kim et al. 1981), soil contamination of 21%-37%(Al Saif,

Brazier 1996, Simango 2006), and CD contamination in cow and its environment between

1%-3.4% (Al Saif, Brazier 1996, Simango 2006, Koene et al. 2012).

In this study, we established the overall prevalence rate of Airedale General Hospital

environment as 1.75% (3/171) with 5.26% (1/9) prevalence of CD in air samples, 2.35%

(2/85) of CD isolation from environmental surfaces and 0% from soil (=39) and Cow dung

(=28). The prevalence of CD in this study is consistent with previous studies but a few. The

difference between this study and a few of those studies with ridiculously high prevalence

rates lies in the different sampling and culturing methods used for the study and that sampling

in their case was done during a CDI outbreak. The rate of CD isolation increases when

samples are collected in areas where carriers have diarrhoea (outbreak) and is reduced in

those with non-known carriers (asymptomatic) (Kim et al. 1981, Faires et al. 2013).(Roberts

et al. 2008) used an enrichment media for air samples and had high rates of isolation of CD.

The use of enrichment broth with 1% sodium taurocholate increases the isolation of CD from

the samples as reflected in the results of their isolation rate (Martirosian 2006, McFarland et

al. 1989, Vaishnavi, Singh 2012, Howerton et al. 2011, Buggy et al. 1985). We did not use

enrichment broth with 1% sodium taurocholate due to budgetary constraints. We however

used enrichment broth (general) in our pilot study (section 3.4 and 3.5 i) and discontinued

22


due to results which indicated that it did not have an added advantage over direct plating. It

also resulted in overgrowth of other bacteria due to its luck of selective advantage (Buchanan

1984) which made it difficult to study a colony of interest. For these reasons, we only used

direct plating.

We isolated one toxigenic CD ribotype 027(reported using Brazier’s nomenclature)(Brazier

1998, Rousseau et al. 2011) from swab samples from ward 6 surfaces where the patient was

diagnosed with non-toxigenic CD ribotype 027 common to Airedale and the Yorkshire region

as shown in figure 7 below.

Figure 7: the distributions of C. difficile ribotypes within Yorkshire and Humber region in England (April 2007-2011)

(Wilcox 2012).

We also isolated a non-toxigenic CD with results of ribotype yet to come back from leads

general hospital. The isolates were both from swabs collected from ward 6 floor in a space of

two weeks when the patient was still having diarrhoea. While the one isolates was of the

same ribotype (patient and environment), we could not conclude that they were linked to each

23


other due to the discordant toxigenic results. Since the patient was diagnosed with non-

toxigenic CD before coming into ward 6, toxigenic isolates from ward 6 floor may not be

from the patient. There is a possibility that the room may have been contaminated before and

not been decontaminated successfully. Some studies used multi-locus variable-number

tandem-repeat analysis (MLVA) to discriminate or link the isolates from the environment and

patients or animals (Best et al. 2010, Indra et al. 2008, van den Berg et al. 2007, Hota 2004).

Our isolates were not tested by MLVA because of the high cost of the test and that it was

beyond the scope of this study. However, the second isolate which was non-toxigenic may

have come be the same strain to that of the patient in which case it may indicate

environmental contamination from the symptomatic patient. In the absence of PCR ribotype

result, this conclusion cannot be made.

There was no CD isolated from any of the surfaces near other symptomatic patients except

the floor in ward 6 presumably due to good infection prevention practices. Compliance to

hand hygiene practices by health workers and visitors in patients’ rooms may have resulted in

less contamination transfer of CDI from patients to inanimate objects like door handles. It

may also indicate that the disinfectant (Tristel) being used on these surfaces is effective

against vegetative and spore forms of CD and used consistently as also reported by (Rutala,

Weber 2013). Even though the compliance with environmental cleaning was not measured,

the isolation of CD from the floor may indicate lack of better and more direct cleaning which

reduces the burden of microorganisms (Dancer 2009). Disinfectants do have a minimum

exposure time (Faise et al. 2010) and that these results may indicate that this was not met

especially in this case that CD was isolated twice from the floor of the same ward.

Our method verification study (section 3.5 ii) verified that the method was able to isolate CD

from surfaces with a lowest CD concentration of 1.5x102CFU/ml which was tested. However

we did not establish the lowest concentration that the method could isolate CD from. It is

24


possible that the method failed to isolate CD from the surfaces with lower contamination

levels than the minimum that we could have established. This could explain our lower

prevalence rate reported. We managed to obtain for free the highly recommended Polywipes

(Best et al. 2010) for sample collection which we used in parallel with swabs. We isolated 1/6

Polywipes and 0/6 from swabs. The Polywipes showed that they were more sensitive and that

they sampled a much wider surface which increased the chances of isolating CD. We could

not purchase any for purposes of this study due to excessive cost, but managed to use the free

ones as a small comparison to obtain and use them for this study due to budgetary constraints.

We isolated toxigenic CD ribotype 002 from air sample in the hospital corridors. This strain

is common to Airedale general hospital and the Yorkshire region as shown in figure 7

(Wilcox 2012). Figure 7 also shows that the strain was first reported in this region in 2008

and figures have stayed consistent. At a contamination level of 5 CFU / m3 isolated from the

corridor, it was the lowest detectable contamination level which resulted from 1 colony. In

our sample verification method study (section 3.5 ii) the lowest air contamination level of 15

CFU / m3 (table 3) was verified. However the method managed to isolate CD at a much

lower air contamination level (5 CFU / m3) showing that it was highly sensitive.

No CD was isolated from air samples from wards with CDI patients or outside the hospital.

Contrary to the findings of other studies (Best et al. 2010, Roberts et al. 2008) where they

reported isolating CD from air samples sampled close to CDI or symptomatic patients, we

isolated CD away from the CDI patients (corridors) and did not isolate it from air close to

CDI patients. Failure to identify CD from air samples close to symptomatic patient may

indicate that the air was not contaminated with CD.

Activities like bed making, movement of people, swinging doors open and close do help to

disperse the spores around (Best et al. 2010, Roberts et al. 2006). It is possible that the air at

25


sampling time had undetectable levels of CD contamination due to little activity by the bed

ridden immobile patients. We noted that an increase in sampling time for more than 2

minutes could have boosted the isolation rate.

We concluded that the isolation of CD from air samples in the hospital corridors indicated a

risk of CD contamination through air at a distance away from symptomatic patients even

though the level of air contamination at 5 CFU / m3 was low presumably due to good

ventilation.

The results also showed that soil and cow dung from part of the hospital environment

including the farm pose no potential risk as an exogenous source of CDI for the patients. Our

results are, in many ways, consistent with those of previous studies that have examined the

prevalence of CD in cow (Al Saif, Brazier 1996, Kim et al. 1981) who found no CD in them

and their environment. However, there is a possibility that vegetative bacilli of CD may have

been present in some of our fresh samples of Cow dung. The procedure of alcohol shock used

in processing them may have killed the CD as noted by (Chankhamhaengdecha et al. 2013)

that alcohol kills all bacteria including vegetative cells of CD except spores. This then may be

the reason reports of CD prevalence in cows may be reported lower. It may be necessary to

carry out a two arm study to test treated and untreated cow dung to confirm this hypothesis.

This study also demonstrated the inefficacy of standard cleaning and disinfection procedures

against CD spores more so from the floor.

6. CONCLUSION

It is evident from the study results that inanimate objects in an environment of a person with

CDI can get contaminated by CD. Cleaning and decontamination of these areas with the right

detergents eradicate or reduce the contamination. The isolation of CD from air samples in the

hospital corridors also underlines the sporadic presence of CD in the air away from a CDI

26


patient. There was an indication that the cleaning detergents (Triste) in use at Airedale

hospital were effective and that there is need of intensifying the cleaning protocol in order to

be consistent and thorough. This conclusion was made due to the all-clear results from ward 2

where the CDI patient was admitted to before moving to ward 6 and that no CD was isolated

from all other sites except the floor in ward 6. More studies need to be carried out to include

the use of enrichment broth with 1% sodium taurocholate for better recovery of CD to help

report accurate prevalence of CD as recommended by a number of studies (Akhi et al. 2011,

Martirosian 2006). The use of Polywipes for sampling environmental surfaces would also be

recommended as it samples a lager surface area and is better than normal swabs (Best et al.

2010).

7. SOURCES OF MATERIALS AND MANUFACTURERS

1. C. Diff. Quik Check Complete, Alere limited, Stockport, Cheshire, SK7 5BW,

England.

2. Brazier’s medium, Fastidious Anaerobe Agar (FAA), Chocolate agar slopes,

Brain heart infusion, Oxoid Limited, Wade road, Basingstoke, England.

3. API rapid ID 32 A, AES Sampl’air Lite, Biomerieux UK ltd, Grafton Way,

Basingstoke, RG22 6HY, Hampshire, England.

4. Polywipes, Medical Wire & Equipment Co. Limited, Wiltshire, SN13 9RT,

England.

5. Swabs,

6. Gram stain kits, Pro-Lab diagnostics,unit 7 westwood court, Cheshire, CH64

3UJ.

27


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.

28


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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 diffcile at airedale nhst environment

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