Epidemiological study of hospital-acquired Clostridium difficile infection in Kuwait teaching ...

278 Pages·2013·15.53 MB·English

Epidemiological study of hospital-acquired Clostridium difficile infection in
Kuwait teaching hospitals and investigation of their virulence characteristics
Submitted by:
Wafaa Jamal
A thesis submitted to Cardiff University, Cardiff, United Kingdom in fulfillment of the
requirements for PhD degree in Medical Microbiology
Supervised by:
Professor Brian I. Duerden
Professor Vincent O. Rotimi
November 2009
UMI Number: U584378
All rights reserved
INFORMATION TO ALL USERS
The quality of this reproduction is dependent upon the quality of the copy submitted.
In the unlikely event that the author did not send a complete manuscript
and there are missing pages, these will be noted. Also, if material had to be removed,
a note will indicate the deletion.
Dissertation Publishing
UMI U584378
Published by ProQuest LLC 2013. Copyright in the Dissertation held by the Author.
Microform Edition © ProQuest LLC.
All rights reserved. This work is protected against
unauthorized copying under Title 17, United States Code.
ProQuest LLC
789 East Eisenhower Parkway
P.O. Box 1346
Ann Arbor, Ml 48106-1346
Declaration
I declare that this thesis represents my own unaided work except where otherwise acknowledged.
Wafaa Jamal
November 2009
ABSTRACT
Clostridium difficile infection (CDI) is the most common type of infectious nosocomial
diarrhoea. Overwhelming evidence indicate that the most important risk factors are prior
antibiotic use and elderly patients. The severity of the disease varies from asymptomatic carrier
to mild diarrhoea to colitis (AAC) and life threatening pseudomembranous colitis (PMC).
Because little is known about C. difficile and CDI in Kuwait, this study was undertaken to
determine the nosocomial acquisition of C. difficile by new patients admitted to the intensive
care units (ICU) of 4 teaching hospitals in Kuwait between February 2001 and January 2002
(first part) and January 2003 to December 2005 (second part) and evaluate cytotoxin (toxin B)
production by clinical isolates upon exposure to minimum inhibitory concentrations (MICs) and
sub-MICs of certain antibiotics. The first part of the study was accomplished by serially
culturing the stool specimens of 922 newly admitted patients to the ICUs; screening their stools
for toxins A/B and screening their immediate environment for C. difficile. The isolates were
typed by the PCR ribotyping technique developed in the Anaerobe Reference Unit, Cardiff. The
effects of various concentrations of antibiotics that could predispose to CDI and those used for
its therapy on the production of cell-bound and cell-free toxin B produced by C. difficile was
investigated by experiments using cell cultures of the Vero cell line. Prevalence, epidemiology and
risk factors of CDI in Kuwait hospitals was investigated during second part of the study by culturing
patients’ stool specimens, ribotyping the isolates and detection of toxin A/B in stool samples. The
susceptibility of all isolates was assessed by MIC determination to 16 antibiotics using the E test
method. During the first part of the study, 95 (10.3%) out of 922 patients with negative cultures
initially on the day of admission acquired C. difficile during their hospitalisation at various time
intervals. Of these, 65 ( %) remained symptom-free while 30 (32%) were symptomatic; 2
68
patients had PMC, 4 AAC and 24 AAD. C. difficile toxin A/B was present in 28 (93%) of 30
symptomatic patients but in only 7 (10.8%) of 65 symptom-free patients. The hospital
environments occupied by symptomatic patients as well as those occupied by asymptomatic
patients were contaminated by C. difficile. The 95 isolates from patients belonged to a total of 32
different ribotypes. Ribotypes 097 and 078 were responsible for >40% of C. difficile infections in
Kuwait ICUs. There was a heterogeneous relationship between antibiotic exposure and intra- and extra­
cellular toxin production by the toxigenic strains. Clinical strains of C. difficile when exposed to MIC and
sub-inhibitory concentrations of certain antibiotics produced high level of cytotoxin. Ampicillin and
clindamycin were the most potent inducers of cytotoxin followed by metronidazole and vancomycin.
Cefotaxime induced the least amount of the cytotoxin activity.
During the second part of the study, 73 (10.5%) out of 697 met the diagnosis of CDI. Out of these 73, 56
(76.7%) were hospital-acquired and 17 (23.3%) were from outpatient clinics. Thus, the prevalence of
hospital-acquired CDI was % over the study period. The prevalence of hospital-acquired CDI in 2003,
8
2004 and 2005 were, 9.7%, 7.8% and 7.2%, respectively. Our data showed that 42.9% of the CDI patients
were above 60 years out of which over 79% were aged 71 years and above. Patients with CDI were more
likely than the controls to have been exposed to immunosuppressive drugs and feeding via naso-gastric
tube. The most common ribotypes isolated during the second part of the study were 002 and 001. The
later was isolated only from one environmental sample in the first part of the study. PCR-ribotype 027
was not isolated during 2003-2005 study. None of our 151 C. difficile isolates were resistant to
amoxicillin-clavulanic acid, ampicillin, linezolid, metronidazole, piperacillin-tazobactam, teicoplanin or
vancomycin. Resistance to penicillin and meropenem among the clinical isolates increased from 2.4 to
16.4% and 4.8 to 21.4%, respectively while resistance to imipenem (another carbapenem) was extremely
high in both studies.
iv
ACKNOWLEDGMENTS
To Almighty God, the most merciful and most benevolent. This study was made possible by the
relentless pursue of excellence and commendable encouragement demonstrated by my
supervisor in Cardiff, Professor Brian I. Duerden and my supervisor in Kuwait, Professor
Vincent O. Rotimi, both of whom took pains to supervise my work diligently and patiently. They
read and corrected my thesis with care and concern and I wish to thank them from the bottom of
my heart for their constant encouragement and support at all times.
I cannot forget Dr J. Brazier, Dr M. Gal, Dr V. Hall, Mr. T. Morris and their colleagues in the
Anaerobe Reference Unit, Cardiff, to whom I am greatly indebted for their technical help,
encouragement and support. Much gratitude goes to Mrs Tina Verghese, Mrs May Shahin and
Ms Fatima Khodakhast, in the Anaerobe Reference Laboratory, Faculty of Medicine, Kuwait
University, for their excellent technical support. Other members of staff of the Department of
Microbiology, Faculty of Medicine, such as Dr Gyorgy Scuzs, Dr Samah Lotfi and Professor
Alex Pacsa are gratefully acknowledged for allowing me to use the tissue culture facility in their
Virology Laboratories as well as the Chairperson of the Department, Professor Eiman Mokaddas,
for allowing me free hand in her department.
Finally, I would forever be grateful to my family, especially my late father, mother and sisters
for the strong support they have always given me at all times.
Publications
Published Manuscripts
1) Jamal WY, Mokaddas EM, Verghese TL, VO Rotimi. In vitro activity of 15
antimicrobial agents against clinical isolates of Clostridium difficile in Kuwait. Int J
Antimicrob Agents 2002; 20: 270-274.
2) Rotimi VO, Jamal WY, Mokaddas EM, Brazier JS, Johny M, Duerden BI. Prevalent
PCR-ribotypes of clinical and environmental strains of Clostridium difficile isolated from
intensive-therapy unit patients in Kuwait. J Med Microbiol 2003; 52: 705-709.
3) Jamal WY, Rotimi VO, Grubesic A, Rupnik M, Brazier J, Duerden BI. Correlation of
multidrug resistance, toxinotypes and PCR ribotypes in Clostridium difficile isolates from
Kuwait. Accepted in Journal of Chemotherapy 2009; 21:
Work presented at conferences as poster presentation
1. Jamal WY, Rotimi VO, Suzcs G, Duerden BI. Influence of antibiotic exposure on cell-bound
and cell-free Clostridium difficile cytotoxin. The th Biennal Congress of Anaerobe Society of
8
the Americas ‘Anaerobe 2006’, Boise, ID., USA. July 25-28, 2006.
2. Jamal WY, Rotimi VO, Grubesic A, Rupnik M, Brazier J, Duerden BI. Correlation of
multidrug resistance, toxinotypes and PCR ribotypes in Clostridium difficile isolates from
Kuwait. Health Science Poster Day, Kuwait University, Kuwait. April 2005.
vi
LIST OF ABBREVIATIONS
AAC Antibiotic-associated colitis
AACD Toxin lethal to 50% of animals
50
AAD Antibiotic-associated diarrhea
Amox/clav Amoxicillin-clavulanic acid
AMP Ampicillin
AST Antibiotic susceptibility testing
BHI Brain Heart Infusion
CCEYA Cycloserine-cefoxitin Egg-Yolk Agar
CCFA Cycloserine-cefoxitin Fructose Agar
CDAD Clostridium *##?cz7e-associated diarrhea
CDC Centers for Disease Control and Prevention
CDI Clostridium difficile Infection
CFU Colony Forming Unit
Clin Clindamycin
CNS Central Nervous System
CNSIP Canadian Nosocomial Infection Surveillance Program
COPD Chronic Obstructive Pulmonary Disease
CT Computerized Tomography
CTX Cefotaxime
CU Cytotoxic Unit
CVA Cerebrovascular Accident
D-MEM Minimal Essential Media
Eh Redox potential
EIA Enzyme immunoassay
ELISA Enzyme linked immunoassay
FAA Fastidious anaerobe agar
FAB Fastidious anaerobe broth
FBS Foetal Bovine Serum
FDA Food and Drug Administration
Fox Cefoxitin
GLC Gas liquid chromatography
GDH Glutamate dehydrogenase
HCW Healthcare Worker
HPA Health Protection Agency
ICU Intensive Care Unit
IDSA Infectious Disease Society of America
IL-8 Interleukin-8
Imip Imipenem
ITU Intensive Therapy Unit
K Kuwaiti
KCCC Kuwait Cancer Control Centre
MDR Multi-drug resistant
MIC Minimum inhibitory concentration
MIC Concentration that kills 50% of the isolates
50
MIC Concentration that kills 90% of the isolates
90
MTZ Metronidazole
NAP North American pulsed-field
NCCLS National Committee for Clinical Laboratory Standards
NGT Nasogastric tube
NK Non-Kuwaiti
NT Non-toxigenic
PaLoc Pathogenicity locus
PBS Phosphate Buffered Saline
PCR Polymerase Chain Reaction
Pip/taz Piperacillin-tazobactam
PMC Pseudomembranous colitis
RCM Robertson Cooked Meat
RFT Renal Function Test
RR Relative risk
SHEA Society for Health Epidemiology of America
S-layer Surface-protein layer
SLE Systemic lupus erythromatosis
SS Salmonella-Shigella agar
T Toxigenic
Tox+ Toxin-positive
TcdA Toxin A gene
TcdB Toxin B gene
TcdA-,TcdB+ ta/A-negative and ta/B-positive.
TNFa Tumor necrosis factor a
Trov Trovafloxacin
TSI slant Triple Sugar Iron slant
UK United Kingdom
USA United States of America