Case Studies in Infectious Disease: Clostridium Difficile 9781136987670, 9780815341420, 0203853776, 0815341423, 1136987673

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Clostridium difficile

Peter M. Lydyard Michael F. Cole John Holton William L. Irving Nino Porakishvili Pradhib Venkatesan Katherine N. Ward

This edition published in the Taylor & Francis e-Library, 2009. To purchase your own copy of this or any of Taylor & Francis or Routledge’s collection of thousands of eBooks please go to www.eBookstore.tandf.co.uk.

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©2010 by Garland Science, Taylor & Francis Group, LLC

This book contains information obtained from authentic and highly regarded sources. Reprinted material is quoted with permission, and sources are indicated. A wide variety of references are listed. Reasonable efforts have been made to publish reliable data and information, but the author and the publisher cannot assume responsibility for the validity of all materials or for the consequences of their use. All rights reserved. No part of this book covered by the copyright heron may be reproduced or used in any format in any form or by any means—graphic, electronic, or mechanical, including photocopying, recording, taping, or information storage and retrieval systems—without permission of the publisher.

The publisher makes no representation, express or implied, that the drug doses in this book are correct. Readers must check up to date product information and clinical procedures with the manufacturers, current codes of conduct, and current safety regulations. ISBN 978-0-8153-4142-0 Library of Congress Cataloging-in-Publication Data Case studies in infectious disease / Peter M Lydyard ... [et al.]. p. ; cm. Includes bibliographical references. SBN 978-0-8153-4142-0 1. Communicable diseases--Case studies. I. Lydyard, Peter M. [DNLM: 1. Communicable Diseases--Case Reports. 2. Bacterial Infections--Case Reports. 3. Mycoses--Case Reports. 4. Parasitic Diseases-Case Reports. 5. Virus Diseases--Case Reports. WC 100 C337 2009] RC112.C37 2009 616.9--dc22 2009004968

Published by Garland Science, Taylor & Francis Group, LLC, an informa business 270 Madison Avenue, New York NY 10016, USA, and 2 Park Square, Milton Park, Abingdon, OX14 4RN, UK. Visit our web site at http://www.garlandscience.com ISBN 0-203-85377-6 Master e-book ISBN

Peter M. Lydyard, Emeritus Professor of Immunology, University College Medical School, London, UK and Honorary Professor of Immunology, School of Biosciences, University of Westminster, London, UK. Michael F. Cole, Professor of Microbiology & Immunology, Georgetown University School of Medicine, Washington, DC, USA. John Holton, Reader and Honorary Consultant in Clinical Microbiology, Windeyer Institute of Medical Sciences, University College London and University College London Hospital Foundation Trust, London, UK. William L. Irving, Professor and Honorary Consultant in Virology, University of Nottingham and Nottingham University Hospitals NHS Trust, Nottingham, UK. Nino Porakishvili, Senior Lecturer, School of Biosciences, University of Westminster, London, UK and Honorary Professor, Javakhishvili Tbilisi State University, Tbilisi, Georgia. Pradhib Venkatesan, Consultant in Infectious Diseases, Nottingham University Hospitals NHS Trust, Nottingham, UK. Katherine N. Ward, Consultant Virologist and Honorary Senior Lecturer, University College Medical School, London, UK and Honorary Consultant, Health Protection Agency, UK.

Preface to Case Studies in Infectious Disease The idea for this book came from a successful course in a medical school setting. Each of the forty cases has been selected by the authors as being those that cause the most morbidity and mortality worldwide. The cases themselves follow the natural history of infection from point of entry of the pathogen through pathogenesis, clinical presentation, diagnosis, and treatment. We believe that this approach provides the reader with a logical basis for understanding these diverse medically-important organisms. Following the description of a case history, the same five sets of core questions are asked to encourage the student to think about infections in a common sequence. The initial set concerns the nature of the infectious agent, how it gains access to the body, what cells are infected, and how the organism spreads; the second set asks about host defense mechanisms against the agent and how disease is caused; the third set enquires about the clinical manifestations of the infection and the complications that can occur; the fourth set is related to how the infection is diagnosed, and what is the differential diagnosis, and the final set asks how the infection is managed, and what preventative measures can be taken to avoid the infection. In order to facilitate the learning process, each case includes summary bullet points, a reference list, a further reading list and some relevant reliable websites. Some of the websites contain images that are referred to in the text. Each chapter concludes with multiple-choice questions for self-testing with the answers given in the back of the book. In the contents section, diseases are listed alphabetically under the causative agent. A separate table categorizes the pathogens as bacterial, viral, protozoal/worm/fungal and acts as a guide to the relative involvement of each body system affected. Finally, there is a comprehensive glossary to allow rapid access to microbiology and medical terms highlighted in bold in the text. All figures are available in JPEG and PowerPoint® format at www.garlandscience.com/gs_textbooks.asp We believe that this book would be an excellent textbook for any course in microbiology and in particular for medical students who need instant access to key information about specific infections. Happy learning!!

The authors March, 2009

Table of Contents The glossary for Case Studies in Infectious Disease can be found at http://www.garlandscience.com/textbooks/0815341423.asp Case 1 Case 2 Case 3 Case 4 Case 5 Case 6 Case 7 Case 8 Case 9 Case 10 Case 11 Case 12 Case 13 Case 14 Case 15 Case 16 Case 17 Case 18 Case 19 Case 20 Case 21 Case 22 Case 23 Case 24 Case 25 Case 26 Case 27 Case 28 Case 29 Case 30 Case 31 Case 32 Case 33 Case 34 Case 35 Case 36 Case 37 Case 38 Case 39 Case 40

Aspergillus fumigatus Borellia burgdorferi and related species Campylobacter jejuni Chlamydia trachomatis Clostridium difficile Coxiella burnetti Coxsackie B virus Echinococcus spp. Epstein-Barr virus Escherichia coli Giardia lamblia Helicobacter pylori Hepatitis B virus Herpes simplex virus 1 Herpes simplex virus 2 Histoplasma capsulatum Human immunodeficiency virus Influenza virus Leishmania spp. Leptospira spp. Listeria monocytogenes Mycobacterium leprae Mycobacterium tuberculosis Neisseria gonorrhoeae Neisseria meningitidis Norovirus Parvovirus Plasmodium spp. Respiratory syncytial virus Rickettsia spp. Salmonella typhi Schistosoma spp. Staphylococcus aureus Streptococcus mitis Streptococcus pneumoniae Streptococcus pyogenes Toxoplasma gondii Trypanosoma spp. Varicella-zoster virus Wuchereia bancrofti

Guide to the relative involvement of each body system affected by the infectious organisms described in this book: the organisms are categorized into bacteria, viruses, and protozoa/fungi/worms

Organism

Resp

MS

GI

H/B

GU

CNS

CV

Skin

Syst

1+

1+

L/H

Bacteria Borrelia burgdorferi

4+

Campylobacter jejuni

4+

Chlamydia trachomatis

2+ 2+

Clostridium difficile

4+

4+

Coxiella burnetti

4+

Escherichia coli

4+

4+

Helicobacter pylori

4+

4+

4+

4+

4+

Listeria monocytogenes

2+

4+

Mycobacterium leprae

4+ 4+

4+

2+ 4+

Neisseria meningitidis

2+ 4+

Rickettsia spp.

4+ 4+

Salmonella typhi

4+

4+ 1+

1+

2+

1+ 1+

4+

Streptococcus pyogenes

4+ 4+

Streptococcus mitis Streptococcus pneumoniae

2+

2+

Neisseria gonorrhoeae

Staphylococcus aureus

4+

4+

Leptospira spp.

Mycobacterium tuberculosis

2+

4+

1+

4+

3+

4+

4+ 3+

Viruses Coxsackie B virus

1+

1+

4+

1+

Epstein-Barr virus Hepatitis B virus

4+

2+

4+

4+

Herpes simplex virus 1

2+

4+

4+

Herpes simplex virus 2

4+

2+

4+

2+

Human immunodeficiency virus

Influenza virus

2+

4+

1+

Norovirus

1+

4+

Parvovirus

2+

Respiratory syncytial virus

4+

Varicella-zoster virus

2+

3+

4+ 2+

4+

2+

Protozoa/Fungi/Worms Aspergillus fumigatus

4+

Echinococcus spp.

2+

Giardia lamblia Histoplasma capsulatum

1+ 4+ 4+

3+

1+

Leishmania spp.

4+

4+ 4+

4+

4+ 4+

Toxoplasma gondii Trypanosoma spp.

4+ 4+

Plasmodium spp. Schistosoma spp.

2+

2+ 4+

Wuchereria bancrofti

4+

4+ 4+ 4+

The rating system (+4 the strongest, +1 the weakest) indicates the greater to lesser involvement of the body system. KEY: Resp = Respiratory: MS = Musculoskeletal: GI = Gastrointestinal H/B = Hepatobiliary: GU = Genitourinary: CNS = Central Nervous System Skin = Dermatological: Syst = Systemic: L/H = Lymphatic-Hematological

Clostridium difficile

An elderly lady, of no fixed abode, arrived at the hospital’s emergency department after having fallen. She was admitted to hospital for fixation of a fracture of the hip. Shortly after admission she developed signs of a chest infection and was started on a cephalosporin, which she remained on for a week. Subsequently she developed profuse watery diarrhea and abdominal pain. A feces sample was sent to the laboratory to test for the toxins of Clostridium difficile, which proved positive and she was (A)

commenced on oral vancomycin. Despite treatment the diarrhea persisted and it also failed to respond to a course of metronidazole. The condition of the patient worsened and a sigmoidoscopy was performed, revealing that she had pseudomembranous colitis (Figure 1). Her clinical condition deteriorated and she developed toxic megacolon and an emergency colectomy was performed. The patient died shortly after the operation.

(B)

Figure 1. Endoscopic view (A) of a patient with pseudomembranous colitis showing plaques of pseudomembranes. Histologically (B) the pseudomembranes are composed of mushroom-shaped collections of neutrophils, cellular debris, and fibrin.

1. What is the causative agent, how does it enter the body and how does it spread a) within the body and b) from person to person? Causative agent Clostridium difficile is an anerobic spore-forming motile gram-positive rod measuring 0.5–1.0 ¥ 3.0–16.0 mm (Figure 2). It produces irregular white colonies on blood agar (Figure 3). The species was named ‘difficile’ because initially it was hard to culture. It is saccharolytic and produces three exotoxins. Toxin A (TcdA) and B (TcdB) induce glucosylation of G-proteins (Rho, Rac, and Cdc42) and ultimately affect actin polymerization leading to loss of tissue integrity. These two toxins, particularly toxin B, are associated with disease in humans. The toxin A-negative strains are mentioned in Section 2 below. The third toxin is a binary toxin that has ADP-ribosylating activity. The clinical correlates of this toxin are currently unclear, but it may be associated with increased virulence. The structure of the toxin gene is shown in Figure 4. Toxin A and B production occurs maximally in stationary phase and during nutrient limitation. Carbohydrate limitation results in a switch to amino acid fermentation, which in turn results in a shortage of amino acids and toxin production.

Figure 2. Gram stain of Clostridium difficile showing that it is a gram-positive rod. The spore can be clearly seen as a subterminal clear area in most of the bacilli.

2

CLOSTRIDIUM DIFFICILE

Low levels of biotin also increase toxin production. During exponential growth, TcdC is high and it is believed to be an anti-sigma factor negatively regulating toxin production. TcdC mutants have been described. These strains produce increased amounts of toxins A and B in vitro, particularly in stationary phase. These mutant strains appear to be particularly virulent. TcdD is not an exotoxin per se, but a positive regulator of transcription of toxin genes and is also responsible for the temperaturedependent regulation of toxin production. The sigma factor TcdR is also a positive regulator, although it is itself under some form of regulation. Additionally, C. difficile has a luxS-type quorum-sensing signaling system, although evidence suggests that addition of the inducer (AI-2) has no effect on toxin production. Several methods of typing C. difficile are available, the most frequently used being pulse field gel electrophoresis, and ribotyping (Figure 5), although multilocus enzyme electrophoresis (MLEE) and analysis of variable number of tandem repeats (VNTR) may also be used. In the UK the common ribotypes are 001, 106, and more recently 027, these three accounting for 70% of all isolates sent to the reference laboratory.

Figure 3. Colonies of Clostridium difficile showing the typical appearance of white colonies with crenated edges. Under UV light the colonies fluoresce a greenishyellow color.

Phylogenomic studies of C. difficile using micro-arrays demonstrate four clades: a hypervirulent clade, a TcdA-negative/TcdB-positive clade, and two other human and animal clades. Differences between the clades are related to virulence, ecology, antibiotic resistance, motility, adhesion, and metabolism. The hypervirulent clade is characterized by a single nucleotide deletion in a regulatory gene (TcdC) resulting in a truncated protein and increased toxin production.

Entry into the body Following the ingestion of spores from contaminated feces or contaminated environment, the organism colonizes mainly the large intestine of the gastrointestinal tract. The dynamics of the complex ecosystem that is the microflora of the gastrointestinal tract is little understood. Many studies have demonstrated that the microflora in an individual is rather stable, although variations have been shown in relation to diet and stress. Figure 4. This figure shows the arrangement of the operon for toxins A and B of Clostridium difficile. The expression of the toxin genes (TcdA and TcdB) is controlled by positive (TcdD) and negative (TcdC) regulators, which respond to changes in the environment. The operon also carries a gene, TcdE, which produces a protein that is thought to assist in the release of the toxins across the bacterial cell wall. Toxin A is a 308 kDa protein and toxin B is a 270 kDa protein. Each protein has an enzyme active domain E (glucosyltransferase); a domain responsible for binding B, which consists of multiple oligopeptide repeat units; and a domain T that is involved in translocation of the toxin across the host cell membrane.

TcdD

TcdB

TcdE

TcdA

TcdC

DNA

TcdB 270kD protein

E

T

B TcdA 308kD E

T

B

genes

domains

TcdD positive regulator TcdC negative regulator TcdE enzyme that may assist release of toxins from the bacterial cell TcdB toxin TcdA toxin

E enzyme activity glucosyltransferase T translocation across host membrane B oligopeptide repeat units involved in binding to host receptors

CLOSTRIDIUM DIFFICILE

3

Figure 5. Ribotyping of Clostridium difficile – photograph of a gel showing some different common ribotypes of C. difficile. The pattern of bands produced by the restriction enzyme is characteristic for the different ribotypes and the patterns can be used to determine if there is crossinfection or a common source outbreak where the bands would be identical. Because some ribotypes are currently very common, e.g. 027, a more discriminatory typing system, e.g. variable number of tandem repeats (VNTR) may be used to subdivide the common ribotypes.

C. difficile is present in the intestines of 2–3% of asymptomatic adults, but may increase to 30% in hospital, and in between 60 and 70% of neonates. C. difficile is a commensal and normally does not cause any disease. In neonates it is believed, based mostly on animal data, that they do not have the necessary adhesion to allow the toxin to bind. In adults who develop the illness the usual history is of an elderly patient coming into hospital, either already carrying C. difficile or acquiring it in hospital, who is given antibiotics and develops diarrhea associated with the C. difficile. The toxin is then produced causing cell damage and leading to disease. Current thinking is that first antibiotics are given, which disturb the normal flora and appear to create an ecological niche where C. difficile can proliferate. Those who arrive in the hospital already carrying the organism appear relatively protected from disease. Thus, arriving as a carrier may represent prior exposure with subsequent development of some type of immunity, as yet poorly defined. The environmental factors that stimulate the toxin genes to be switched on and cause disease are unknown. In experimental systems it is known that consumption of some amino acids or the production of volatile fatty acids by the indigenous microflora inhibits the proliferation of C. difficile. It is also known that viable intestinal microflora is important in inhibition of outgrowth of C. difficile, thus supporting some metabolic function. Whether these mechanisms occur in humans is unknown but it is possible. It is certainly known that patients can be symptomatic with C. difficile isolated from the feces many days before the detection of toxin in the feces. Why some patients get mild disease and some get severe disease is not known. Why disease affects the elderly more commonly than younger patients is also not known, although the disease is occurring increasingly in the younger age group, which might be associated with more virulent strains (see Section 3).

Spread within the body The organism remains within the gastrointestinal tract and only rarely is extra-intestinal disease reported. Spread from person to person Patients with diarrhea, especially if it is severe or accompanied by incontinence, may unintentionally spread the infection to other patients in hospital. In addition, the ability of C. difficile to form spores enables it to

4

CLOSTRIDIUM DIFFICILE

survive for long periods in the environment, for example on floors and around toilets, and protects it against heat and chemical disinfectants.

Epidemiology C. difficile is a well-recognized hospital ‘superbug’ that has received considerable media attention and generated much public concern. It is the commonest cause of hospital-acquired diarrhea in the USA, with 250000 cases annually at a cost to the health-care system of $1 billion. In the UK, cases are subject to mandatory reporting and in 2004 the total number of cases was 44 488, which was an increase of 23% on 2003 figures. In 2007 the number of cases recorded in patients over 65 years was 49 785 and since mandatory reporting of cases less than 65 years old was introduced in April 2007 the number of cases reported in this age group to the end of 2007 was 7597. In the UK the hypervirulent strain ribotype 027 in particular has been associated with hospital outbreaks such as in Stoke Mandeville Hospital and several other hospitals in the South East of England. In Canada there is a similar epidemiology with the newly recognized hypervirulent strain (NAP1, ribotype 027). This strain produces large amounts of toxin A and B and in some strains a binary toxin. This strain has been isolated across Europe, North America, and globally, and has been associated with severe disease that is refractory to treatment, both in hospitals and in communityacquired cases – in the latter apparently in the absence of prior antibiotic exposure. However, severe disease can also occur with other ribotypes.

2. What is the host response to the infection and what is the disease pathogenesis? Immune responses Although some information has accumulated on the development of immune responses during infection with C. difficile, their role is unclear in both controlling the tissue damage and protection against relapses, commonly seen in adult patients following initial treatment with antibiotics. Both TcdA and B toxins induce an inflammatory response involving a number of factors including interleukin (IL)-8, IL-6, tumor necrosis a (TNF-a a), leptin, and substance P. The precise pathways are not factor-a clear. TcdA stimulates the vanilloid receptor, VR1, which leads to the release of substance P from sensory neurons (and increases the expression of its receptor – neurokinin 1-R), which in turn leads to the release of neurotensin and mast cell degranulation. TcdA also increases the release of endocannabinoids, which increase the release of more substance P via stimulation of VR1. Both toxins also induce an inflammatory response with infiltration of polymorphonuclear leukocytes (PMNs) into the lamina propria by up-regulation of IL-8 secretion from sensory neurons and colonocytes. The polymorphism within the IL-8 gene may be related to the presentation of more severe disease. Surface layer proteins from the organism induce a mixed pro-/anti-inflammatory cytokine response with the release of IL-1b, IL-6, IL-10, and IL-12, but the significance of this is unclear. Systemic and mucosal antibodies (IgG and IgA) are produced by the host to the A and B toxins in some patients following C. difficile diarrhea and

CLOSTRIDIUM DIFFICILE

5

antibodies to surface layer proteins of the organism are also found. Some of these also appear to be neutralizing antibodies. Low levels of antibodies with low avidity for A and B toxins, typically acquired early in life, have been found in the serum of up to 70% of asymptomatic adults, which are probably not effective in preventing the initial infection. Understanding the immune response to this commensal is clearly of importance in protection against the severe consequences of infection.

Pathogenesis TcdA binds to carbohydrate Gal-b 1,4N-Acetyl Glucosamine by repeat units at the C-terminal end of the toxin. Both TcdA and B are taken up into the cytosol of host colonocytes and are glucosyltransferases that inactivate Rho, Rac, and Cdc42, thereby affecting actin polymerization. This leads to impairment of tight junctions, cell rounding up, and cell death (Figure 6). TcdB activates Ca influx, which is necessary for the disintegration of the actin cytoskeleton. TcdA inhibits stimulated mucin exocytosis. Both TcdA and B induce fluid loss.

3. What is the typical clinical presentation and what complications can occur? C. difficile is associated with antibiotic-associated diarrhoea (CDAD) at one extreme to pseudomembranous colitis (PMC) at the other. The latter has a significant mortality. C. difficile is largely linked to hospital admission and has been associated with hospital outbreaks. Infection has usually occurred in those aged over 65 years and usually in association with antibiotics, bowel surgery or chemotherapeutic agents. The illness may follow antibiotics taken briefly or even several weeks previously. Recently community

Ca++ (calcium)

–Gal␤1–4GlcNAc– receptor

host cell cytoplasmic membrane

receptor-mediated endocytosis

TcdB Ca++

UDP

UDP-glucose morphology

glucose G GDP

TcdA TcdB

G

actin cytoskeleton

GTP release of substance P mucin exocytosis release of cytokines

fluid loss

production of reactive oxygen species recruitment of polymorphonuclear leukocytes by interleukin-8

key G UDP GDP(GTP) TcdA/B

Rho, Rac, Cdc42 uridine diphosphate guanosine di(tri)phosphate Clostridium difficile toxins A/B

Figure 6. Mode of action of Clostridium difficile toxins A/B. This figure shows the action of the toxins A and B on the host cell. The toxins are taken up by receptormediated endocytosis after binding to specific glycoprotein receptors. The toxins inhibit the normal functioning of G proteins Rho, Rac, and Cdc42 by glucosylating them. This inactivation affects the actin cytoskeleton, which in turn affects cell morphology and the integrity of the tight junctions between the cells, thereby increasing fluid loss. The toxins also stimulate the release of cytokines, e.g. IL-8, which recruits PMNs and establishes an inflammatory response and the production of reactive oxygen radicals. The toxins increase the release of substance P, a peptide that enhances fluid loss from the intestines and decreases the exocytosis of the protective mucus barrier.

6

CLOSTRIDIUM DIFFICILE

cases of C. difficile-associated disease have been reported in a younger age group who have not necessarily been taking antibiotics. These relatively rare cases have been caused by hypervirulent strains such as ribotype 027. Patients present with colicky abdominal pain, bloating, and watery diarrhea with an almost characteristic smell for C. difficile. In severe cases (PMC) there may be blood in the feces and the colon may perforate. Toxic megacolon may occur, which can paradoxically cause diarrhea to stop or in some cases not to appear at all, and colectomy may be required. Thus C. difficile with little or no diarrhea, and pain predominating may be an ominous sign. Severe damage to the large bowel can result in rupture or perforation. The rare patients with extra-intestinal disease almost always have longterm carriage of the organism or severe gastrointestinal disease. Diseases reported include bacteremia, abscess, and prosthetic hip infection.

4. How is this disease diagnosed and what is the differential diagnosis? The routine diagnosis of C. difficile-associated disease is detection of the toxins in the feces by enzyme immunoassays (ELISA). An additional assay that is likely to be useful is the detection of glutamate dehydrogenase in the feces as a surrogate marker for the organism. Culture is also available and is important for the investigation of suspected outbreaks where the isolates can be ribotyped. The organism may be grown on cycloserine/cefoxitin/egg yolk agar and a variety of other more sensitive media, always anaerobically producing 2–3 mm gray-white colonies. Polymerase chain reaction (PCR) for toxin-related genes is also being used in some laboratories. Pseudomembranous colitis is diagnosed by colonoscopy where raised yellowish plaques may be seen on the mucosa. If severe disease is suspected limited sigmoidoscopy may be used as colonoscopy may result in perforation. The differential diagnosis includes other infective causes of diarrhea, inflammatory bowel disease, and diverticulitis.

5. How is the disease managed and prevented? Management For patients who are on antibiotics and who develop diarrhea, one should stop the antibiotics if at all possible. Such a decision is on a case by case basis. The first-line treatment of CDAD is either metronidazole or vancomycin for 14 days (Table 1). Vancomycin and metronidazole must be given orally, although metronidazole may also be given parenterally. Pulsed or tapering doses of both antibiotics have been given at the end of the course of treatment and vancomycin has also been given by enema. In mild to moderate disease both are equally effective but in severe disease vancomycin was more effective in a controlled trial. In vitro studies, on the

CLOSTRIDIUM DIFFICILE

Table 1. Treatment of C. difficile-associated disease First-line

Vancomycin 125–500 mg four times daily ODS 14 days OR Metronidazole 250–500 mg four times daily QDS 14 days

Alternatives

Cholestyramine 4 g TDS 14 days Bacitracin-fuscidic acid/rifaximin Probiotic/fecal enema Intravenous immunoglobulin

other hand, indicate that metronidazole has a more rapid bactericidal effect compared with vancomycin. Other antibiotics that have been used are bacitracin, fusidic acid, nitazoxanide, and rifaximin. Tolevamer and difimicin also show some promise in the treatment. Rifampin, which had been recommended, has not been effective in a controlled trial. Alternative treatments are also available including cholestyramine (which binds the toxin but also binds vancomycin, so the two should not be given simultaneously); monoclonal anti-toxin antibodies; intravenous immune globulin (although there are no controlled trials and reported results are contradictory); probiotics such as Saccharomyces boulardiae (which is really a strain of S. cerevesiae) have been used in conjunction with antibiotics; and fecal enema (or fecal administration by oral–duodenal tube) from a close relative, to re-establish a ‘normal’ flora.

Prevention In hospitals and nursing homes, prevention is by strict control-of-infection procedures. Contact precautions for any patients in whom CDAD is suspected are essential. The patient should be isolated in a single room with dedicated toilet facilities and the use of personal protective equipment (PPE), that is gloves and apron. If more than one patient has the illness the patients should be nursed in a dedicated ward. A decision whether to close the ward/unit must be made on an individual basis but if there is evidence of widespread cross-infection then the unit should be closed. Horizontal and ‘high touch’ surfaces and other items within the immediate environment, particularly toilet facilities, should be regularly cleaned with a hypochlorite disinfectant. All items and rooms after discharge of the patient(s) should be cleaned in the same manner. Attention to hand-washing practices is important and should be undertaken with soap and water rather than alcohol, as the latter has no effect on the spores of C. difficile. In fact, the spores of C. difficile are highly resistant to many of the generic disinfectants. There is currently no vaccine although research is ongoing with at least two candidate vaccines.

7

8

CLOSTRIDIUM DIFFICILE

SUMMARY 1. What is the causative agent, how does it enter the body and how does it spread a) within the body and b) from person to person? ●

Clostridium difficile is a gram-positive sporulating anaerobe.

●

Several typing methods are available including ribotyping and PFGE.

●

The spore is acquired by feco-oral spread or from the environment and infects colonocytes of the host.

●

Up to 70% of neonates may be colonized without disease.

●

Asymptomatic colonization may occur in up to 30% of patients in hospital.

●

A hypervirulent strain (ribotype 027) first isolated in Canada produces large amounts of toxin and has spread across Europe, North America, and globally.

●

C. difficile produces three toxins: toxin A and B and sometimes a binary toxin.

●

Important risk factors for disease are increased age (> 60 years) and taking antibiotics.

●

The organism only rarely causes extra-intestinal disease.

3. What is the typical clinical presentation and what complications can occur? ●

The patient presents with abdominal pain and diarrhea.

●

Frequently the patient is aged > 60 years, in hospital, and is on antibiotics.

●

Community-acquired disease in younger patients who have not been on antibiotics is relatively rare and associated with hypervirulent strains.

●

The disease may be mild to moderate CDAD or severe PMC.

●

Complications include perforation or toxic megacolon.

4. How is this disease diagnosed and what is the differential diagnosis? ●

The disease is routinely diagnosed by detection of the toxins in the feces using enzyme immunoassays.

●

Culture is important for typing the organism in identifying and managing outbreaks.

●

PMC is diagnosed by colonoscopy or sigmoidoscopy.

●

Differential diagnosis includes other causes of gastroenteritis, diverticulitis and inflammatory bowel disease, and antibiotic-associated diarrhea not due to C. difficile.

2. What is the host response to the infection and what is the disease pathogenesis? ●

Antibodies to toxins A and B are produced following C. difficile diarrhea, some of which are neutralizing.

●

The toxins bind to host receptors on colonocytes and are taken up into the cell.

●

Toxins A/B induce disaggregation of the actin cytoskeleton by inactivating GTPase proteins.

●

Impairment of tight junctions and cell death occurs leading to fluid loss and a localized inflammatory response.

●

Both the toxins and the inflammatory response itself are thought to be involved in tissue damage.

5. How is the disease managed and prevented? ●

The first-line treatment is oral metronidazole or vancomycin for 14 days.

●

Alternative treatments include agents to bind the toxin, probiotics, or fecal enema.

●

In hospital, prevention is by standard control of infection methods.

CLOSTRIDIUM DIFFICILE

9

FURTHER READING Cimolai N. Laboratory Diagnosis of Bacterial Infections. Marcel Dekker, NY, 2001: 731–734. Mandell GL, Bennet JE, Dolin R. Principles & Practice of Infectious Diseases, 6th edition, Vol 3. Elsevier/Churchill Livingstone, 2005: 1249–1259.

Mayhall GC. Hospital Epidemiology & Infection Control, 3rd edition. Philadelphia, Lippincott Williams & Wilkins, 2004: 623–631. Murphy K, Travers P, and Walport M. Janeway’s Immunobiology, 7th edition, Garland Science, New York, 2008.

REFERENCES Ausiello CM, Cerquetti M, Fedele G, et al. Surface layer proteins from Clostridium difficile induce inflammatory and regulatory cytokines in human monocytes and dendritic cells. Microbes Infect, 2006, 8: 2640–2646. Durai R. Epidemiology, pathogenesis and management of Clostridium difficile infection. Dig Dis Sci, 2007, 52: 2958–2962. Matamouros S, England P, Dupuy B. Clostridium difficile toxin expression is inhibited by the novel regulator TvdC. Mol Microbiol, 2007, 64: 1274–1288. McMaster-Baxter NL, Musher DM. Clostridium difficile: recent epidemiological findings and advances in therapy. Pharmacotherapy, 2007, 27: 1029–1039.

Owens RC. Clostridium difficile associated disease: changing epidemiology and implications for management. Drugs, 2007, 67: 487–502. Peled N, Pitlik S, Samra Z, Kazakov A, Bloch Y, Bishara J. Predicting Clostridium difficile toxin in hospitalized patients with antibiotic associated diarrhoea. Infect Control Hosp Epidemiol, 2007, 28: 377–381. Smith JA, Cooke DL, Hyde S, Borriello SP, Long RG. Clostridium difficile toxin binding to human intestinal epithelial cells. J Med Microbiol, 1997, 46: 953–958. Voth DE, Ballard JD. Clostridium difficile toxins: mechanism of action and role in disease. Clin Microbiol Rev, 2005, 18: 247–263.

WEB SITES Centers for Disease Control and Prevention (CDC), Division of Healthcare Quality Promotion, 2008, United States Public Health Agency: http://www.cdc.gov/ncidod/dhqp/id_Cdiff.html Centre for Infections, Health Protection Agency, HPA Copyright, 2008: http://www.hpa.org.uk/infections/topics_az/ clostridium_difficile/C_diff_faqs.htm Department of Health, National Health Service, Crown Copyright 2008: http://www.dh.gov.uk/en/Policyandguidance/ Healthandsocialcaretopics/Healthcareacquiredinfection/ Healthcareacquiredgeneralinformation/DH_4115800

Infection Control Services, Ltd, 2007, consultant microbiologists at University College London Hospitals providing advice about infection control to hospitals in the private sector: http://www.infectioncontrolservices.co.uk/ Institute of Biomedical Sciences, 2005, the professional body for biomedical scientists in the United Kingdom: http://www.ibms.org/pdf/bs_articles_2005/clostridium_difficile.pdf

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MULTIPLE CHOICE QUESTIONS The questions should be answered either by selecting True (T) or False (F) for each answer statement, or by selecting the answer statements which best answer the question. Answers can be found in the back of the book.

4. Which of the following are important in the differential diagnosis of Clostridium difficile-associated disease? A. Leptospirosis. B. Toxic megacolon.

1. Which of the following are true of Clostridium difficile? A. It is an anaerobic bacterium. B. It produces spores. C. It produces a zinc metalloprotease as the only toxin. D. Toxin production is controlled by a sigma factor. E. A phage typing system is most useful in differentiating strains. 2. Which of the following are true concerning the epidemiology of Clostridium difficile?

C. Giardia gastroenteritis. D. Inflammatory bowel disease. E. Duodenal ulcer. 5. Which of the following are true statements concerning the diagnosis of Clostridium difficile? A. The Gram stain of biopsy material is useful. B. Detection of toxins in the blood is the main way of diagnosis. C. Culture of the organism is useful for epidemiological investigation.

A. Asymptomatic carriage in hospital is uncommon.

D. Colonoscopy is important.

B. Commonly associated with outbreaks in hospital.

E. Ribotyping is important.

C. Rarely causes extra-intestinal disease. D. A recognized hypervirulent strain is ribotype 027. E. Neonates may be colonized but do not acquire disease.

6. Which of the following are used in the treatment of Clostridium difficile-associated disease? A. Ampicillin.

3. Which of the following are known to be important in the pathogenesis of Clostridium difficile-associated disease? A. Attaching and effacing lesions. B. Production of glucosyltransferases by the organism. C. Vacuolating cytotoxin. D. Taking antibiotics. E. Disruption of the actin cytoskeleton.

B. Cephalosporins. C. Metronidazole. D. Vancomycin. E. Clindamycin.

Answers to Multiple Choice Questions 1. Which of the following are true of Clostridium difficile? A. It is an anaerobic bacterium. TRUE: it is a gram-positive rod. B. It produces spores. TRUE: they can remain in the environment for long periods and act as a source of infection. C. It produces a zinc metalloprotease as the only toxin. FALSE: it produces three toxins: TcdA, TcdB, and a binary toxin. TcdA/B are glucosyltransferases and the binary toxin is an ADP ribosyltransferase. D. Toxin production is controlled by a sigma factor. TRUE: TcdD (positive regulator, sigma factor) and TcdC (negative regulator). E. A phage typing system is most useful in differentiating strains. FALSE: typing systems include ribotyping, PFGE, and MLEE. 2. Which of the following are true concerning the epidemiology of Clostridium difficile? A. Asymptomatic carriage in hospital is uncommon. FALSE: it can be up to 30%. B. Commonly associated with outbreaks in hospital. TRUE: although community-acquired infection is becoming increasingly recognized. C. Rarely causes extra-intestinal disease. TRUE: usually abscess, bacteremia. D. A recognized hypervirulent strain is ribotype 027. TRUE: although severe disease can also be caused by other ribotypes. E. Neonates may be colonized but do not acquire disease. TRUE: colonization can occur in up to 70% and it is thought that the neonate does not have the receptor for the toxin. 3. Which of the following are known to be important in the pathogenesis of Clostridium difficile-associated disease? A. Attaching and effacing lesions. FALSE: this occurs in E. coli. B. Production of glucosyltransferases by the organism. TRUE: they inactivate Rho, Rac Cdc42. C. Vacuolating cytotoxin. FALSE: this is produced by H. pylori. D. Taking antibiotics. TRUE: this is a risk factor, as is age > 60 years and hospital admission. E. Disruption of the actin cytoskeleton. TRUE: this causes loss of the tight junction holding cells together and cell death.

4. Which of the following are important in the differential diagnosis of Clostridium difficile-asssociated disease? A. Leptospirosis. FALSE: this presents with meningitis, jaundice or renal failure. B. Toxic megacolon. TRUE: this is a complication of CDAD and requires colectomy. C. Giardia gastroenteritis. TRUE: any infectious cause of diarrhea is in the initial differential diagnosis, although in an elderly patient in hospital on antibiotics CDAD is the most likely diagnosis. D. Inflammatory bowel disease. TRUE. E. Duodenal ulcer. FALSE: this presents with epigastric pain or evidence of perforation but not diarrhea and is caused by Helicobacter pylori or taking NSAIDs/steroids. 5. Which of the following are true statements concerning the diagnosis of Clostridium difficile? A. The Gram stain of fecal material is useful. FALSE: Gram stain of feces has no use. B. Detection of toxins in the blood is the main way of diagnosis. FALSE: detection of the toxin in the feces is the routine way of making a diagnosis. C. Culture of the organism is useful for epidemiological investigation. TRUE: as the organism can then be typed. D. Colonoscopy is important. TRUE: to detect pseudomembranes in PMC. E. Ribotyping is important. FALSE: it is not used in diagnosis of the illness but for epidemiological studies once the diagnosis has been made. 6. Which of the following are used in the treatment of Clostridium difficile-associated disease? A. Ampicillin. FALSE: it may cause CDAD. B. Cephalosporins. FALSE: it is one of the commonest antibiotic groups to cause CDAD. C. Metronidazole. TRUE. D. Vancomycin. TRUE. E. Clindamycin. FALSE: it may cause CDAD.

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Figure Acknowledgements Figure 1a. Reprint permission kindly granted by Science Photo Library. Additional photographic credit given as David M. Martin, MD and Science Photo Library (M130/559). Figure 1b. Reprint permission kindly granted by Jae Lim , Calvin Oyer and Murray Resnick, editors and photographic contributors to the Digital Pathology website associated with Brown Medical School, Providence, Rhode Island. Figure 2. Reprint permission kindly given by the Centers for Disease Control, Atlanta, Georgia. Image is found in the Public Health Image Library #3876. Additional photographic credit is given to Dr. Gilda Jones who took the photo in 1980.

Figure 3. Reprint permission kindly given by the Centers for Disease Control, Atlanta, Georgia. Image is found in the Public Health Image Library #3211. Additional photographic credit is given to Dr. Gilda Jones who took the photo in 1980. Figure 4. This figure is the creation of the case study author, Professor John Holton, and was produced specifically for this publication. Figure 5. Reprint permission kindly given by the Windeyer Institute of Medical Sciences, University College London, UK. Figure 6. This figure is the creation of the case study author, Professor John Holton, and was produced specifically for this publication.