Clostridium difficile : New Challenges for an Old Foe [1 ed.] 9781780843155, 9781780843179

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Clostridium difficile: New Challenges for an Old foe

Editors Glenn S Tillotson TranScrip Partners, PA, USA Karl Weiss Maisonneuve-Rosemont Hospital, QC, Canada

Published by Future Medicine Ltd Future Medicine Ltd, Unitec House, 2 Albert Place, London N3 1QB, UK www.futuremedicine.com ISSN: 2047-332X ISBN: 978-1-78084-317-9 (print) ISBN: 978-1-78084-316-2 (epub) ISBN: 978-1-78084-315-5 (pdf) © 2013 Future Medicine Ltd All rights reserved. No part of this publication may be reproduced, stored in a retrieval system or transmitted in any form or by any means electronic, mechanical, photocopying, recording or otherwise without prior written permission of the copyright holder. British Library Cataloguing-in-Publication Data. A catalogue record for this book is available from the British Library. Although the author and publisher have made every effort to ensure accuracy of published drug doses and other medical information, they take no responsibility for errors, omissions, or for any outcomes related to the book contents and take no responsibility for the use of any products described within the book. No claims or endorsements are made for any marketed drug or putative therapeutic agent under clinical investigation. Any product mentioned in the book should be used in accordance with the prescribing information prepared by the manufacturers, and ultimate responsibility rests with the prescribing physician. Content Development Editor: Duc Hong Le Senior Manager, Production & Design: Karen Rowland Head of Production: Philip Chapman Managing Production Editor: Harriet Penny Production Editor: Georgia Patey Assistant Production Editors: Samantha Whitham, Abigail Baxter & Kirsty Brown Editorial Assistant: Ben Kempson Graphics & Design Manager: Hannah Morton

Contents Clostridium difficile: new challenges for an old foe Glenn S Tillotson & Karl Weiss Clostridium difficile infection: a global threat Nuttanun Suramaethakul & Teena Chopra Pathogenicity mechanisms of Clostridium difficile Mark M Collery, Revathi Govind, Nigel P Minton & Sarah A Kuehne Immunology and Clostridium difficile Ciarán P Kelly & Saurabh Sethi The microbiome and Clostridium difficile infection Charles Y Chiu & Dylan R Pillai Economic burden of Clostridium difficile infection Samuel L Aitken, Dhara N Shah & Kevin W Garey Laboratory diagnosis of Clostridium difficile infection Peter H Gilligan Conventional therapeutics for Clostridium difficile infection Jessica Martin & Mark Wilcox

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Infection control issues in Clostridium difficile Scott Curry

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Contents Continued Future and alternative approaches to managing Clostridium difficile infection Glenn S Tillotson & Karl Weiss

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Recent advances with Clostridium difficile Joni Tillotson & Glenn S Tillotson Index

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About the Editors Glenn S Tillotson Glenn S Tillotson has almost 30 years of pharmaceutical experience, including clinical research, commercialization, medical affairs, strategic drug development, life-cycle management and global launch programs. While at Bayer (Leverkusen, Germany), he was instrumental in the development of ciprofloxacin and moxifloxacin. He has held various medical affairs leadership roles in the past decade. He has published over 140 peer-reviewed manuscripts and is on several journal editorial advisory boards.

Karl Weiss Karl Weiss is Chief of the Department of Infectious Diseases and Medical Microbiology at Maisonneuve-Rosemont Hospital in Montreal (QC, Canada). He is also Professor of Medicine at the Faculty of Medicine at the University of Montreal (QC, Canada), Director of Pharmacological Research at Maisonneuve-Rosemont Hospital and Co-Chair of the Montreal Infection Control Board. He is also an associate member of the Division of Infectious Diseases at the Jewish General Hospital in Montreal (QC, Canada), and Adjunct Professor of Medicine at McGill University (QC, Canada). His research focuses mainly on respiratory tract infections, antibiotics and antimicrobial resistance. In addition to being President of the Quebec Committee on Antibiotics and Antimicrobial Resistance, he is a member of the executive committees of several professional associations such as the Association des Médecins Microbiologistes Infectiologues du Québec. He was a board member of the Canadian Infectious Diseases Society and then the Association of Medical Microbiology and Infectious Diseases – Canada from 1997 to 2005. He has been a member and chaired numerous advisory boards in Canada and at the international level, and has worked as a consultant for several governments. He was also the chairman of the examining board of the Royal College of Physicians and Surgeons of Canada for medical microbiology between 2002 and 2005. He received the Award of Distinction from the Association of Medical Microbiology and Infectious Diseases – Canada in 2005, and the `Prix des médecins de coeur et d’action’ from l’Association des Médecins de Langue Française du Canada in 2008.

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Foreword Clostridium difficile : new challenges for an old foe

Glenn S Tillotson & Karl Weiss Clostridium difficile has emerged as one of the most problematic and challenging bacterial pathogens of the past decade. Initially discovered in the 1930s, it only became a human pathogen of note in the 1970s. However, since the 1970s, many facts about C. difficile were discovered but it became an orphan disease for many years, with no new drugs and barely any research. The arrival of the NAP1 strain in Quebec (Canada) was the perfect storm: an older population with more underlying medical conditions, more broad-spectrum antibiotics and major infection control issues due to costcutting measures. Since the early 2000s, C. difficile has taken on epidemic proportions in North America and Europe, with certain strains predominating and spreading leading to devastating outbreaks in hospitals in Canada and the UK. Our understanding of this organism has also grown remarkably in the past few years as new technologies and science have enabled the establishment of much deeper knowledge of how this organism has evolved into the major nosocomial bacterial infection in the USA. This book has been compiled with the assistance of several experts who have a deep knowledge and awareness of the many facets of this virulent species, including epidemiology, pathogenicity, immunological aspects, the role of the microbiome and C. difficile infection, the healthcare burden of this escalating disease, therapeutic management and future options in treating or preventing C. difficile. doi:10.2217/EBO.13.271

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Tillotson & Weiss We hope you enjoy this compilation of current literature and perspectives on the future of C. difficile, an evolving pathogen. Financial & competing interests disclosure GS Tillotson has served as Consultant for Summit PLC, Basilea and Astellas EU, and is an employee of TranScrip Partners US. K Weiss has received research grants from Optimer Pharma, Merck, Cubist and Novartis, and served as Speaker for Optimer Pharma and Merck. The authors have no other relevant affiliations or financial involvement with any organization or entity with a financial interest in or financial conflict with the subject matter or materials discussed in the manuscript apart from those disclosed. No writing assistance was utilized in the production of this manuscript.

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About the Authors Nuttanun Suramaethakul Nuttanun Suramaethakul is an American Board of Internal Medicine-certified physician. She finished her medical school training in Thailand, then moved to the USA and completed her internal medicine residency training in Ohio. After finishing residency, with strong interest in infectious diseases, she decided to go on with fellowship training in infectious disease at Detroit Medical Center, Wayne State University (MI, USA). She finished her fellowship training in June 2013 and will pursue her career as an infectious disease specialist after graduation.

Teena Chopra Teena Chopra is an Assistant Professor of Medicine in the Division of Infectious Diseases at Wayne State University. Her research interests include the epidemiology of healthcare-associated infections, infection prevention and antibiotic stewardship. She has published over 30 papers in various journals and book chapters. In addition, she has independently reviewed over 20  journal articles. She has a special interest in studying the epidemiology of infections, including Clostridium difficile and multidrug-resistant organisms.

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Chapter

Clostridium difficile infection: a global threat

Global epidemiology of CDI

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CDI in previously low-risk populations 13 Conclusion14

doi:10.2217/EBO.13.251

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Nuttanun Suramaethakul & Teena Chopra In recent years, the epidemiology of Clostridium difficile infection (CDI) has changed globally [1–6]. Coupled with an alarming increase in CDI incidence, there has been a steep rise in mortality and recurrence from CDI [7]. A review of English literature on CDI mortality from 2005 to 2011 showed that all-cause mortality and the attributable mortality at 30 days ranged from 9 to 38% and 5.7 to 6.9%, respectively [8]. The emergence of CDI as a major hospital-acquired infection has caused enormous economic burden to the healthcare system. A systematic review of 13 studies from the USA, Canada and Europe estimated that the incremental cost of CDI per case had ranged from US$2871 to 4846 for primary CDI in the USA and from US$5243 to 8570 in non-USA countries. For recurrent CDI, the cost ranged from US$13,655 to 18,067 in the USA and was approximately US$13,655 in non-USA countries [9]. The annual management costs for CDI was estimated to be US$800  million in the USA and €3000 million in Europe [10]. These findings support that CDI has become a serious problem worldwide. Increased awareness of the healthcare burden from CDI would encourage better infection control and treatment effort to prevent recurrence and spreading of the disease.

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Suramaethakul & Chopra This chapter focuses on the change in epidemiology of Clostridium difficile infection (CDI) around the world in the recent years: the factors contributing to this dramatic change, the emergence of new strains of CDI and rising incidence of CDI in previously low-risk populations.

Global epidemiology of CDI In North America, the increasing incidence of CDI has been observed since the late 1980s. A review conducted in the intensive care units of the National Nosocomial Infections Surveillance System hospitals in the USA between 1987 and 2001 showed an upward trend of CDI incidence [1]. Likewise, the National Hospital Discharge Survey showed the discharge diagnosis of C. difficile had increased from 31 per 100,000 population in 1996 to 61 per 100,000 population in 2003, with a markedly rising rate during 2000–2003 and in patients older than 65 years of age [11]. A similar increasing trend was also observed in the child population. A large retrospective cohort study conducted in 22 children’s hospitals in the USA between 2001 and 2006 showed that the incidence of CDI had increased from 2.6 to four cases per 1000 admissions [12,13]. Several other reports in children have also showed a rise in incidence of CDI among this population [14–16]. In the early 2000s, many hospitals throughout the USA and Canada reported outbreaks of CDI. Due to increased incidence and severity of CDI in these areas, a large retrospective chart review of all cases of CDI from 1991 to 2003 was conducted in a hospital in Quebec (Canada). This review identified 1721 new cases of CDI and showed that the annual incidence of CDI had increased dramatically from 35.6 cases per 100,000 population in 1991 to 156.3 cases per 100,000 population in 2003, especially in patients aged 65 years or more where the incidence rate had increased tenfold [2]. The rise in the rate of CDI was also simultaneously associated with an increase in severity of illness and mortality [2,17–19]. Preliminary data from the US National Vital Statistics Reports showed that death from C. difficile enterocolitis had increased from 793 in 1999 to 7483 in 2008. The age-adjusted death rate increased 15% from two deaths per 100,000 population in 2007 to 2.3 deaths per 100,000 population in 2008. Approximately 93% of deaths from CDI occurred in patients aged  65  years or older [7]. Furthermore, a recent prospective surveillance data from 28 community hospitals in the southeastern USA showed that C. difficile has replaced methicillin-resistant Staphylococcus aureus as the most common healthcare-associated infection [20]. This further emphasizes the significance of the CDI problem in this region. One of the factors that contributed to the rapid increase in incidence of CDI in North America and Canada is the emergence of a new hypervirulent strain of C. difficile that was first reported in the early 2000s. A study of 187 C. difficile

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Clostridium difficile infection: a global threat Toxinotype: a group of strains with identical isolates from six states in the USA where the changes in a chromosome region called the outbreaks occurred between 2001 and 2003 pathogenicity locus. (including Maine, Pennsylvania, New Jersey, PCR ribotyping: a typing method based on patterns of Georgia, Illinois and Oregon), found a PCR products of the 16S–23S ribosomal RNA common strain of C. difficile that infected the intergenic spacer region. majority of patients in the hospitals [21]. This same strain was also observed as a single predominant strain circulating among 12  hospitals in Quebec during an outbreak in 2004 [22]. This new highly virulent epidemic strain was characterized as toxinotype III, restriction endonuclease ana­lysis group BI, pulse-field gel electrophoresis North American PFGE type 1 (NAP1) and PCR ribotype 027, or known as BI/NAP1/027 strain [21,23]. This strain causes more severe clinical conditions and higher mortality rates due to increased toxin A and B production [24]. An in vitro study to measure C. difficile toxin production showed that this BI/NAP1/027 strain had a 16- and 23-times higher peak median concentration of toxin A and B, respectively, than other studied strains [25]. Binary toxin is another toxin found to be associated with this C. difficile strain [22,24]. The role of this binary toxin as a virulence factor has not been well established. However, recent reports found that it can affect the actin cytoskeleton of the epithelial cells, thus enhancing adherence and colonization of the organism [26,27]. In addition, the resistance to fluoroquinolones was observed to be significantly more common with this BI/NAP1/027 strain than other C.  difficile strains [21,22]. The wide use of fluoroquinolones may select for and cause spreading of the disease with this particular strain.

As the incidence of CDI and the healthcare costs continued to rise in the USA, from January 2013, the Centers for Medicare and Medicaid Services required all acute-care hospitals in the USA to report CDI using the National Healthcare Safety Network [101]. The authors anticipate that this would help to identify target population and follow the future trend of CDI in a more systematic way. In Europe, a similar increasing trend of CDI was observed in many countries. A surveillance of CDI conducted in Germany found that the incidence of CDI had increased from 1.7 to 3.8 cases per 100,000 population between 2002 and 2003 to 14.8 cases per 100,000 population in 2006 [3]. In 2005, a 2-month prospective surveillance study in 38 hospitals from 14 different European countries showed that the incidence of CDI had ranged from 0.13 to 7.1 per 10,000 patient-days with the mean incidence of 2.45 per 10,000 patient-days. A higher incidence and more severe disease were observed in the countries that had recent outbreaks of the BI/NAP1/027 strain, such as The Netherlands, Belgium and France [28].

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Suramaethakul & Chopra Owing to the difference in reporting systems and the diagnostic methodology among the European countries, to study the epidemiology of CDI in Europe is somewhat difficult. In order to better understand the overview of the current CDI situation in Europe, a pan-European hospital-based survey of CDI, enrolling 97 hospitals from 34 European countries, was conducted in November 2008. The survey showed that 80% of cases were healthcare associated, 16% were community acquired and the rest were undetermined. The incidence of CDI varied widely between countries. The mean healthcareassociated CDI rate was 4.1 cases per 10,000 patient-days, ranging from 0 to 36.3 per 10,000 patient-days, which was higher compared with earlier years. The countries that had relatively high incidence of CDI were Finland, Poland, the UK and Sweden. Most patients had risk factors previously known to be associated with CDI, including severe comorbidities, previous antibiotics exposure and advanced age. Almost two-thirds of patients in this study were aged 65 years or over. The most common PCR ribotypes were 014 and 020 and the number of hypervirulent 027 strain isolates was relatively low in this study, which is different from what was observed in North America [10,29]. The C. difficile BI/NAP1/027 strain was first reported in Europe from an outbreak in England in 2005 [30]. Later, CDI was made a reportable condition in the UK in 2007. It was estimated that there were more than 55,000 cases of C. difficile in England between 2007 and 2008 [4]. A review of 12,603 fecal specimens from the C. difficile Ribotyping Network in England from 2007 to 2010 found that the proportion of C. difficile isolates had increased and the BI/NAP1/027 strain was the most common isolate. However, the prevalence of this particular strain had markedly declined during the 3 years of the study period and a significant increase in other C. difficile strains was observed [4]. By 2007, C. difficile BI/NAP1/027 strains had been detected in 11 countries in Europe [31]. In The Netherlands, 35 healthcare facilities were affected with BI/NAP1/027 strain in 2007 compared with 22 healthcare facilities in 2006. Patients infected with this strain were significantly older and more patients were exposed to fluoroquinolones, compared with patients who were infected with non-027 ribotypes [32]. In Belgium, BI/NAP1/027 strain was the most common isolate which accounted for 17.6% of C. difficile cases. In France, the first BI/NAP1/027 strain was reported in April 2006 and accounted for 27.5% of the C. difficile cases. The majority of these isolates were resistant to erythromycin and moxifloxacin but not to clindamycin. However, as of June 2008, clindamycin-resistant BI/NAP1/027 strains had been reported from three European countries [33], which raises a concern over the use of clindamycin as it may lead to spreading of this particular strain.

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Clostridium difficile infection: a global threat Another new emerging C. difficile ribotype, 078, was also increasingly reported in many countries in Europe, including Belgium, The Netherlands, Northern Ireland, Scotland and Spain. This 078 strain has similar mechanisms to the BI/NAP1/027 strain, which increase the production of the toxins [33]. Further surveillance studies are needed to follow the epidemiology of this strain. Data on the incidence of C. difficile in other parts of the world, including Asia, Africa, Central and South America, Australia, New Zealand and the Middle East are limited. Most published data are reports from one healthcare facility or individual case reports. It is difficult to evaluate the changing trend of CDI in these regions. However, most of these reports indicate a rising trend of CDI [5,6,34]. In Asia, the majority of C.  difficile strains that are isolated are not the hypervirulent strain, BI/NAP1/027. A study in a Korean tertiary-care hospital between September 2008 and January 2010 showed that the most common strain isolated was the toxin  A-positive/toxin  B-positive strain, which accounted for 77.5% of cases [5]. High prevalence of toxin A negative/toxin B positive has also been reported in many studies from Asia [35–37]. To date, only a few cases of the 027 strains have been reported in some Asian countries, including Japan, Hong Kong and Singapore, and the susceptibility pattern of the isolates from Singapore was actually more consistent with the 027 historical strain than the epidemic strain that occurred in North America and Europe [34]. In Australia, the first recognized case of CDI from the ribotype 027 was reported in 2009 in a patient who was thought to have acquired the infection in the USA [38]. In 2011, the second case of hypervirulent strain CDI was reported and this was thought to be the first local acquisition case in Australia [39]. In South America, a recent study in an intensive care unit setting in a tertiary-care hospital in Brazil showed a threefold increase in the rate of CDI from 1.8 to 5.5 per 1000 patient-days between December 2007 and August 2008 [40]. In other regions of the world, there are currently not enough comparative data available and the new BI/NAP1/027 strain has not been reported. Data on CDI in cancer, transplantation and neutropenic patients are very scant. Cancer and transplantation patients tend to have a higher chance of developing CDI than the general population due to more hospitalization and more exposure to antibiotic prophylaxis or treatment. In addition, chemotherapeutic agents are recognized as independent risk factors for CDI. Recent data showed the incidence of CDI in cancer patients who received

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Suramaethakul & Chopra chemotherapy had ranged from 2.3 to 7%. More data are needed to better understand epidemiology of CDI in this patient population [41]. Although the emergence of a new, virulent C. difficile strain seems to play an important role in the change of epidemiology in North America, in other parts of the world where the incidence of this new strain is not very high other factors such as an increasing number of high-risk populations should be taken into consideration. Interesting data from the WHO shows that the percentage of the world’s population who are over 60 years of age will double from 11 to 22% between 2000 and 2050 and the absolute number of people over 60 years of age is expected to increase from 605 million to 2 billion over that period of time [42]. Advanced age is one of the wellestablished risk factors for CDI and with this estimated change in the elderly populations; it could be predicted that the incidence of CDI might continue to go up in the next few decades. Another important concern is the spreading of CDI in nursing home or long-term care facilities, as well as in dialysis centers. Since infected patients frequently get transferred from one facility to another, a lack of concern or inadequate precautions could lead to rapid spreading or even outbreaks among those facilities. Increased awareness, appropriate hand hygiene and contact precaution are the mainstays to control the spreading of the disease and should be strongly emphasized to all healthcare personnel in the facilities. Apart from the infection control, the world should promote reasonable use of antimicrobial therapy. As previous antibiotic exposure remains a major risk factor for CDI, improvement of antibiotic policies and clear indications for antibiotic treatment are critically needed. Variation in diagnostic methods used among different countries can influence the epidemiologic data. Many cases of CDI currently remain undiagnosed due to lack of the use of sensitive diagnostic tests [43]. Enzyme immunoassays (EIAs) for toxin A and B has been used for diagnosis of CDI in many laboratories for many years [44,45]; however, the sensitivity of this test appears to be low. An evaluation of six commercially available EIAs showed the sensitivity of EIAs was only 75% (range: 60–86%) compared with that of the toxigenic culture [46]. Therefore, EIA is currently considered a suboptimal diagnostic method [46,47]. Although some laboratories have now changed to use PCR testing to diagnose CDI – as it appears to be rapid, more sensitive and specific [47,48] – many laboratories, especially in resource-limited areas, continue to use low-sensitivity tests. Comparing epidemiologic data from Enzyme immunoassay: a method that uses an enzyme linked to an antibody or antigen as a different laboratories or from different time marker to detect a specific protein. This method is periods requires careful interpretation.

used to detect the presence of Clostridium difficile toxins A and B in stools.

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Clostridium difficile infection: a global threat Fecal microbiota transplantation: a treatment With rapid change in the epidemiology of method that infuses fecal microflora from a CDI, effective treatment is very important, healthy individual into a patient’s bowel to help not only to cure the disease but also to restore colonic flora. prevent the recurrence. In the last several years, many new treatment modalities have been developed. Fidaxomicin is one of the new treatment options that has shown promising results in achieving a higher cure rate in patients receiving concomitant antibiotics compared with oral vancomycin [49]. Fecal microbiota transplantation has been increasingly used in refractory cases or in recurrent CDI and has shown encouraging results [50,51]. Other new treatment modalities such as monoclonal antibodies, probiotics or C. difficile vaccine, are under investigation. With these promising effective treatments, improvement in disease control and a positive impact on epidemiology of CDI in the future can be anticipated.

CDI in previously low-risk populations Patients with previous antibiotic exposure, older adults and those with prior exposure to a hospital, nursing home or long-term care facility are at increased risk for CDI. However, during the past decade, an increasing number of patients with CDI without known risk factors has been reported with severe CDI. These include pregnant women, healthy healthcare workers and healthy people in the community with no previous antibiotic exposure. A case report and review of English literature of CDI in peripartum patients from 1966 to 2007 identified 24 cases of peripartum CDI, including four new cases that were reported in this review. Of those 24 cases, one patient had no previous record of antibiotic exposure. Two of the four new cases were infected with a hypervirulent strain BI/NAP1/027 and developed severe disease. These two patients were previously healthy and one of them had no hospital admission within the past 90 days. Impaired cell-mediated immunity during pregnancy could be one of the contributing factors that increase the risk of CDI in pregnancy [52]. Community-acquired CDI has also been increasingly reported [53]. Data from the USA, Canada and Europe suggest that community-acquired CDI accounts for 20–27% of all CDI with an incidence of 20–30  cases per 100,000 persons [7]. A review of CDI in residents of Olmsted County (MN, USA) between 1991 and 2005 found that 41% of 381 C.  difficile cases were community acquired. These patients were younger and more likely to be female. Fewer patients in the community-acquired group had recent antibiotic exposure or developed severe disease compared with the hospitalacquired group and the recurrent rate was not different between the two groups [54]. Between 2003 and 2005, 23 cases of community-acquired CDI

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Suramaethakul & Chopra were reported from the greater Philadelphia area (PA, USA). The estimated annual incidence rate of the community-acquired CDI in Philadelphia during that time was 7.6  cases per 100,000 population. These patients were generally young with a mean age of 26 years [55]. A few other outbreaks of community-acquired CDI were also later reported in other areas in the USA, including Connecticut [56] and North Carolina [57]. Another recent report from Kaiser Permanente Colorado and Kaiser Permanente Northwest members between June 2005 and September 2008 showed that 56% of 3067 CDI cases were identified in an outpatient setting; of these, 14% did not receive antibiotics in the 180  days before CDI and only 27% had a history of hospitalization in the previous 30 days [58]. In Europe, a retrospective study in the south of England between April 2008 and March 2009 showed an estimated prevalence of community-acquired CDI of 1.29 cases per 10,000 population. Cases were more likely to be female between 31 and 40 years of age and less than 50% of cases had established risk factors [59]. This new emergence of CDI in low-risk populations is worrisome and should alert physicians to consider this diagnosis and provide appropriate diagnostic tests in patients with diarrhea even without traditional risk factors.

Conclusion CDI has become a serious global problem, not only in hospitalized patients, but also in healthy people from the community. With emergence of more virulent strains and infection in patients without traditional risk factors, increasing awareness along with rapid and accurate diagnosis are extremely important, thus allowing prompt initiation of appropriate treatment and infection control to prevent serious complications and spread of the disease. Appropriate use of antibiotics only when indicated would further prevent developing of new cases. The new emerging hypervirulent strain, BI/NAP1/027 is a major concern especially in North America and Europe; however, the incidence remains low in other parts of the world. Given the lack of good surveillance and reporting system in many countries, it is a challenge to study the epidemiology of CDI. The use of low-sensitivity diagnostic tests could also lead to missed diagnosis and, hence underestimation of the true incidence of CDI. Improvement of diagnostic and surveillance methods should be promoted to better understand and follow the true spread of CDI. Finally, since the world has become more connected, the lack of infection control and stewardship programs, especially in the developing countries, would increase the risk of global transmission. More education is crucially required in many countries. Improved understanding of current global burden of the disease will increase

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Clostridium difficile infection: a global threat awareness, attention and effort to improve early diagnosis, treatment and control of spreading of CDI. Financial & competing interests disclosure T Chopra is a speaker/consultant for Optimer, Pfizer, Merck and Forest. The authors have no other relevant affiliations or financial involvement with any organization or entity with a financial interest in or financial conflict with the subject matter or materials discussed in the manuscript apart from those disclosed. No writing assistance was utilized in the production of this manuscript.

Summary. ƒƒ With an increased incidence of Clostridium difficile infection (CDI) globally and the development of a new hypervirulent strain, CDI has become a serious worldwide problem during the last decade that requires attention from all healthcare facilities. ƒƒ Increased incidence of CDI in previously low-risk populations should prompt physicians to consider this diagnosis in patients with diarrhea even without traditional risk factors. ƒƒ An antibiotic stewardship program to promote reduced antimicrobial use is the key to prevent the emergence of new diseases and is needed in both acute and alternate healthcare settings. ƒƒ More and better education in infection control and antimicrobial stewardship in developing countries is crucially needed to control transmission of the disease not only within, but also between the countries. ƒƒ Improvement of diagnostic method is important to help diagnose CDI in a timely fashion and hence prevent transmission.

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Disease Control and Prevention. Healthcare Facility Reporting via NHSN to Comply with CMS Rules. www.cdc.gov/nhsn/PDFs/ commup/NHSN-CMS-RulesSept-27-2011.pdf

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About the Authors Mark M Collery Mark M Collery is a Research Fellow in the Clostridia Research Group at the University of Nottingham (UK). He received his PhD from the University of Dublin, Trinity College (Ireland) in 2008. His main research interests focus on understanding the pathogenesis of Clostridium difficile.

Revathi Govind Revathi Govind is an Assistant Professor in the Division of Biology at Kansas State University in Manhattan (USA). She received her PhD in Medical Microbiology at the Texas Tech University Health Sciences Center (USA) in 2006. Her research focus is on understanding C. difficile metabolisms that are important for the establishment of infection in its host.

Nigel P Minton Nigel Minton is Head of the Clostridia Research Group at the University of Nottingham. He focuses on improving scientific understanding of the biology of members of the genus Clostridium that are either of medical importance or those species with beneficial properties in terms of cancer therapy or in the generation of fuels and chemicals from biorenewables.

Sarah A Kuehne Sarah A Kuehne is a Senior Research Fellow in the School of Life Sciences at the University of Nottingham. She works in the Clostridia Research Group. Her main research interest focuses on understanding the important pathogenic bacterium C. difficile, paving the way for new and better diagnostics and therapeutics.

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Chapter

2 Pathogenicity mechanisms of Clostridium difficile

Toxins A & B

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The pathogenicty locus

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Binary toxin23 The spore23 Sporulation

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Germination

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S-layer & cell wall proteins25 Flagella & virulence

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Other factors

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Regulation of virulence

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Role of phage in C. difficile virulence

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Conclusion29

Mark M Collery, Revathi Govind, Nigel P Minton & Sarah A Kuehne Long before it was identified as the causative agent of antibiotic-associated disease, the effects of Clostridium difficile infection were well known to healthcare practitioners. Progress in understanding the molecular basis of pathogenesis was, however, impeded by the dearth of genetic tools available. Thankfully, a variety of procedures have now been formulated that allow both the directed and random mutational ana­lysis of this important human pathogen, and the characterization and discovery of its virulence factors. This chapter reviews the current state of knowledge of those factors and how they contribute to the disease, from the most significant and best understood to those whose roles are less clearly defined but may in time be shown to be equally as important.

doi:10.2217/EBO.13.187

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Collery, Govind, Minton & Kuehne The clinical symptoms of Clostridium difficile infection are caused by the activity of either of the two large toxins, toxins A and B.

Toxins A & B The clinical symptoms for which Clostridium difficile is best known (diarrhea, inflammation and damage to the colonic mucosa) are caused by two large toxins, toxin A (TcdA) and B (TcdB), 308 and 270 kDa in size, respectively [1,2]. These toxins belong to the family of large clostridial cytotoxins, or clostridial glucosylating toxins, since their toxicity depends on their glucosyltransferase activity. Both toxins are cytopathic to cultured cells owing to disruption of the cytoskeleton, although TcdB is thought to be up to 1000-times more potent [3]. In addition to damaging or killing cells, both toxins can also affect cells in other ways, such as disruption of cell–cell junctions by the inactivation of Rho GTPases. The disruption of these junctions has been suggested to be responsible for the increased permeability and fluid accumulation associated with C. difficile infection (CDI) [4]. The pathogenicty locus TcdA and TcdB are encoded by two genes that form part of a five-gene locus known as the pathogenicity locus (PaLoc) (Figure 2.1). In addition to tcdA and tcdB, the locus contains genes tcdC, tcdE and tcdR. Until recently, TcdC was thought to be a negative regulator of toxin production, and has been shown to sequester TcdR in vitro as an antisigma factor [5]. Subsequently, studies showing apparently opposing results led to the current conclusion that TcdC may be involved in modulating toxin expression, but that a nonfunctional TcdC is not necessarily associated with elevated toxin production [6,7]. The role of TcdC in vivo remains unclear. TcdE is a holin-like protein that has been suggested to be involved in the secretion of TcdA and TcdB, although data in support of this hypothesis are conflicting [1,8]. TcdR is an alternative sigma factor that positively regulates transcription of the tcdA and tcdB genes [9]. The respective roles in disease of the two toxins are also controversial. It had been shown previously that when administered intragastrically

Figure 2.1. The arrangement of genes within the pathogenicity locus of Clostridium difficile, including the tcdA and tcdB genes encoding the two major toxins, as well as ancillary genes tcdC, tcdE and tcdR. tcdR

tcdB

tcdE

tcdA

tcdC

Modified from [11].

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Pathogenicity mechanisms of Clostridium difficile to hamsters, TcdB was incapable of causing disease whereas TcdA caused hemorrhage, diarrhea and death [4] . As a consequence, TcdA was regarded as the principal cause of CDI symptoms. Subsequently, Lyras et al. demonstrated that hamsters infected with a TcdA-positive [10], TcdB-negative mutant strain of C.  difficile did not develop CDI. By contrast, Kuehne et al. demonstrated that both toxins are independently capable of causing disease in hamsters [11]. This paradox has yet to be resolved.

Binary toxin In addition to TcdA and TcdB, up to 35% of strains of C. difficile produce a third binary toxin known as C. difficile toxin (CDT), which consists of two subunits, CDTa and CDTb. These constitute the enzymatic and binding/ translocation components, respectively [12]. CDT is part of the family of actin-ADP-ribosylating toxins, which includes the C2 toxin from Clostridium botulinum and iota toxin from Clostridium perfringens. Despite the similarity to these other clostridial toxins, the precise role of CDT in disease has yet to be elucidated. CDT has been associated with the so-called ‘hypervirulent’ strains of C. difficile. These strains, classified as NAP1/restriction endonuclease assay type BI/PCR-ribotype 027 have been reported to produce increased amounts of both TcdA and TcdB and to be associated with increased disease severity as well as morbidity and mortality [13]. These characteristics have been attributed to a mutation within the tcdC gene, although this has since been called into question (as discussed above), as have claims that these strains display different patterns of sporulation to other strains [7,14].

The spore While disease symptoms are primarily attributable to TcdA and TcdB, endospores represent perhaps the most important C. difficile virulence factor. Sporulation is pivotal to disease transmission, while without germination there would be no toxin production, and therefore no disease. Endospores are formed in response to adverse environmental conditions. They are resistant to high temperatures, radiation and many chemicals traditionally used as antibacterial disinfectants [15] and, therefore, allow the survival of C. difficile on surfaces in a healthcare setting, including skin. Following their inadvertent ingestion, spores germinate in the anaerobic conditions of the human gut, thus reverting to vegetative, toxin-producing cells [16]. Upon egestion, the cells return to their spore form and, thus, the cycle of Spores: endurance form of C. difficile. infection continues.

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Collery, Govind, Minton & Kuehne Spores are the agents of infection and endurance. They allow the survival of C. difficile in the aerobic environment and transmission from host to host potentially via surfaces, skin or medical equipment.

Sporulation While the mechanisms involved in sporulation in Bacillus species are well understood, comparatively little is known of how spores are formed in C. difficile [17], although the two processes are believed to be essentially the same [18]. In short, the vegetative cell undergoes asymmetric division with complete copies of the genome being segregated into each of the daughter cells. The smaller cell will become the endospore. The prespore (as the smaller cell is now known) is engulfed by the larger (mother cell), creating a double membrane around the prespore. Two layers of peptidoglycan are deposited between the inner and outer membranes; the larger of the two becoming the spore cortex. The final stage in the process is to lay down a protein coat around the exterior of the spore (Figure 2.2). Autolysis of the larger mother cell releases the spore into the environment [18].

Germination As with sporulation, the processes involved in germination of C. difficile spores are similarly poorly understood, relative to those in Bacilli. In Bacillus subtilis, germinants bind to specific receptors on the inner spore membrane leading to the release of its huge depot of dipicolinic acid and cations, and replacement of these components by water. These latter events trigger the hydrolysis of the spore’s peptidoglycan cortex by either Figure 2.2. The Clostridium difficile spore. A

B

Coat Inner membrane Cortex

Core

Germ cell wall

Outer membrane

(A) The structure of a bacterial spore (not drawn to scale) illustrating the various layers of the spore. The core contains the spore DNA, RNA and most enzymes and is enclosed within an inner membrane that has a low permeability to small molecules, protecting the core from DNA-damaging chemicals. (B) A single Clostridium difficile spore observed using transmission electron microscopy. Heeg D, Pers. Comm.

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Pathogenicity mechanisms of Clostridium difficile of two redundant lytic enzymes, which in turn allows for complete rehydration and resumption of enzyme activity, metabolism and vegetative cell growth [19].

In order to establish infection, C. difficile needs to adhere to the gut epithelium. This is achieved through a variety of adhesins linked to the cell surface.

S-layer & cell wall proteins Despite the undoubted importance of the toxins and the sporulation/ germination processes, other virulence factors and mechanisms play important roles in pathogenesis. The early stages that lead ultimately to disease are first the ingestion of spores and subsequently germination into vegetative cells. This is only possible if C.  difficile can overcome the colonization barrier put in place by the resident gut microflora and subsequently adhere to and colonize the gut epithelium. In order to adhere, the bacteria need to reach the cells, a maneuver facilitated by motility and the production of degradative enzymes that assist the penetration of the mucosal layer. The surface of the bacterial cell is composed of an S-layer that consists of two molecules. The high-molecular-weight component is anchored in the membrane while the light-molecular-weight domain is externally located and has been described as an immune variable, representing a possible means to evade the host immune defense [20–22]. A number of other adhesins have been described, most significantly a family of cell wall proteins (Cwps). Among these, Cwp84 binds noncovalently in its inactive form to the S-layer, while also being tightly associated with the cell wall. It then auto processes into an active protease, degrading proteins of the extracellular matrix. Cwp84 is thought to be the main protease of C. difficile and to be important in the early stages of colonization (Figure 2.3). However, it remains important throughout the infection process as the degradation of the extracellular matrix leads to nutrient availability. Another protease, Cwp13, is not present in all C. difficile strains and only plays a minor role [23,24]. Enzymes other than Cwp84 may also be involved; gelatinases, collagenases and others have been described but their role and importance is as yet unknown [22]. Further members of the large cell-wall protein family have been studied in detail (28 paralogs have been described in strain 630 [25]). Cwp66 and CwpV have both been confirmed as adhesins [22]. Interestingly, the latter is regulated through phase variation and has been implicated in the formation of aggregates in vitro and in biofilm formation in vivo [26]. Other members of this large family of proteins have not been studied in detail and their role in pathogenesis and colonization has not been fully elucidated.

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Collery, Govind, Minton & Kuehne Figure 2.3. The S-layer of Clostridium difficile.

S-layer

Peptidoglycan Cwp84 Cytoplasmic membrane

LMW

HMW

S-layer protein A

The S-layer of Clostridium difficile is built of proteins consisting of a LMW and a HMW unit, which are synthesized as one precursor, S-layer protein A. This precursor is then cleaved by the protease Cwp84 followed by autoassembly of the subunits into the mature S-layer proteins. HMW: High molecular weight; LMW: Low molecular weight.

When coming into contact with epithelial cells, remodeling of the C. difficile cell surface occurs, most likely to expose virulence factors or factors necessary for spread. Most of these factors are either phase dependent or regulated by SigH, an alternative sigma factor active in the postexponential phase of growth. A recent study has shown that SigH is a global regulator, controlling both directly and indirectly sporulation, motility, metabolism and virulence [9]. Analysis of the surface heat-shock protein, GroEL, has suggested a possible role in adherence, but further studies are necessary to clarify its precise function in vivo [22,27].

Flagella & virulence Many strains of C. difficile possess flagella (Figure 2.4). In some cases, their primary role may not be for motility [28,29], rather, they may play a significant role in adherence. Accordingly, nonflagellated strains have been shown to adhere less to mouse caeca [30]. However, recently, mutants in fliC (encoding the flagellin subunit) and fliD (encoding the flagella cap) showed increased adherence to Caco-2 cells [29]. These mutants were also more virulent than the wild-type in the hamster infection model. A transcriptomic ana­lysis using microarrays demonstrated that the flagella genes are downregulated

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Pathogenicity mechanisms of Clostridium difficile when the cells came into contact with Caco-2 Figure 2.4. Transmission electron micrograph cells [31]. It is hypothesized that flagella adapt showing a Clostridium difficile cell with according to their environment; within the flagella. lumen they are required for motility, but when in contact with epithelial cells they might not be needed for colonization. The most recent data show a modulation of toxin transcription in C. difficile via the flagellar operon. Early flagella genes appear to positively regulate toxin transcription, whereas late flagella genes have the opposite effect. It remains to be determined whether regulation via the flagella operon is part of a 2 µm wider, complex network of regulation in C.  difficile that controls colonization, Clostridium difficile PCR ribotype 012. Arrows point persistence and virulence [28]. to the flagella.

Other factors Certain strains of C. difficile produce pili in vivo, which may contribute to adherence and promote effective host colonization. Fibronectin-binding protein A has been shown to bind both fibronectin and fibrinogen, and a mutant strain in which the encoding gene had been inactivated proved less effective in colonizing mice caeca, suggesting a role in intestinal colonization [32]. Recently, biofilm formation on abiotic surfaces has been described for C. difficile. Interestingly, the two published studies revealed a link between sporulation and biofilm formation as a mutant in Spo0A, the master regulator of sporulation, led to significantly reduced biofilms [33,34]. In one study, virulence factors such as Cwp84, flagellin and the putative quorumsensing regulator, LuxS, were also shown to be required for maximal biofilm formation [34]. Biofilms may act as a reservoir for vegetative cells and spores, lead to enhanced resistance to antibiotics and provide a privileged site to evade the host immune system. A further mechanism that C.  difficile deploys to gain a competitive advantage in the gut environment is to produce para-cresol from tyrosine, which is bacteriostatic but well tolerated by C. difficile itself [35].

Regulation of virulence Aside from the specific regulators of toxin expression discussed earlier, TcdR and TcdC, few other regulators have been described. In keeping with its role in other bacteria, CodY has been shown to be a global regulator in C. difficile,

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Collery, Govind, Minton & Kuehne responding to nutrient availability. Through repression of tcdR, it is a potent toxin repressor when grown in rich medium. A link to cyclic-di-GMP has been suggested as well as an involvement in regulation of overall physiology [36]. In Bacillus, Spo0A is the master regulator of sporulation. Not unexpectedly, C. difficile spo0A mutants do not form any spores. The comparative ana­lysis of a spo0A mutant and its progenitor in in vivo models has produced compelling evidence that spores are crucial for disease transmission and persistence as well as recurrence [37]. However, to date, there is conflicting evidence as to the involvement of Spo0A in toxin regulation [37,38]. The catabolite control protein CcpA has recently been studied through transcriptional comparison of a wild-type and an isogenic mutant. As in Bacillus, it is a global regulator being involved in sugar uptake, fermentation, amino acid metabolism, sporulation and also toxin expression. The toxin genes tcdA and tcdB are direct targets of CcpA, as is the positive regulator gene tcdR. As CcpA controls the selective utilization of carbon sources it is involved in the glucose-dependent repression of toxin transcription and also binds directly to many of the numerous phosphotransferase systems of C. difficile, emphasizing an important link between pathogenicity and metabolism [39]. Clearly C. difficile has adapted to its unique niche as an anaerobe in the gut, exemplified by the many phosphotransferase systems that have been found in its genome. Furthermore, the organism possesses an extraordinary number of two component systems [25,40], the function of the majority of which remains unknown. Finally, the luxS gene, responsible for the production of an autoinducer molecule (AI-2), has been implicated in the regulation of virulence through the demonstration of a correlation between AI-2 production and tcdA and tcdB transcription [41,42]. Homologs of an accessory gene regulator (agr) system, analogous to that found in Staphylococcus aureus, are evident in C. difficile genomes [25]. To date, there have been no reports linking agr to virulence, although equivalent systems are known to regulate sporulation and toxin production in other pathogenic clostridial species [43,44].

Role of phage in C. difficile virulence All C. difficile phages characterized so far are lysogenic in nature [40,45–47]. Although several virulence-associated genes are located on phages in other members of the genus [48], no such association has been demonstrated in C.  difficile [40,45–47]. Nonetheless, PaLoc genes share homology with bacteriophage genes, most notably the tcdE gene with phage holins [1], tcdA with a Clostridium tetani phage fCT2 gene [49], and tcdC with ORF22

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Pathogenicity mechanisms of Clostridium difficile Many of the regulatory pathways of C. difficile of Lacto­b acillus casei phage A2 [50] . are still poorly understood and require further Moreover, TcdE has been shown to act as a research. bacterio­ p hage holin through the observation that it could complement a holin-defective lambda phage to lyse Escheria coli cells [1]. These observations may suggest that the PaLoc of C. difficile is a remnant of an early prophage.

Phages can contribute to bacterial virulence by regulating virulence genes that are not encoded by the phage [51]. C. difficile prophages fC2, fC6 and fC8 have been suggested to modify toxin production in their lysogens [50]. Similarly, C.  difficile strains lysogenized with a phage demonstrated increased toxin production [52]. While these observations are intriguing, molecular details for this increased toxin production are absent. A detailed study on the phage fCD119 reported a negative effect on toxin production upon phage lysogenization [53]. Phage fCD119 produces RepR, a repressorlike protein that maintains the lysogenic cycle within its C. difficile host. It was shown that RepR can downregulate the toxin gene expression by controlling the transcription of tcdR. Interestingly, the presence of a cyclicdi-GMP riboswitch within the lysis module of the fCD119 genome has been noted, which indicates that phage fCD119 has also evolved to monitor the physiological status of C. difficile. If a phage contributes to bacterial virulence, its production and release during bacterial infections in a human or animal host could contribute to pathogenesis. In vivo lysogenization of a phage during CDI in its host has been recently reported, where phage fCD119 was found to lysogenize C. difficile during its infection in hamsters [54]. In a recent study, C. difficile fMMP04 was shown to be induced by subinhibitory concentrations of antibiotics: ciprofloxacin, moxifloxacin and levofloxacin [55]. These antibiotics are known risk factors that induce CDI and suggest that phage induction is possible in vivo during CDIs. These experiments add to the increasing body of information that implicates bacteriophages in C. difficile physiology and pathogenesis.

Conclusion The main virulence factors associated with CDI unequivocally remain its toxins and the spore. They represent the principle agents of disease and transmission, respectively. Over and above these, many other factors are involved and important in C. difficile disease. CDI is clearly a multifactorial process and is governed by complex regulatory mechanisms, the nature of which is only now beginning to be understood. Further research into disease mechanism and the organism’s regulatory networks is a prerequisite for the future development of more effective diagnostics, therapies and counter measures.

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Collery, Govind, Minton & Kuehne Acknowledgement The authors would like to thank D Heeg for supplying Figure 2.2. Financial & competing interests disclosure The authors have no relevant affiliations or financial involvement with any organization or entity with a financial interest in or financial conflict with the subject matter or materials discussed in the manuscript. This includes employment, consultancies, honoraria, stock ownership or options, expert testimony, grants or patents received or pending, or royalties. No writing assistance was utilized in the production of this manuscript.

Summary. ƒƒ Clostridium difficile infection is characterized by antibiotic-associated diarrhea, which can lead to potentially fatal disease. ƒƒ C. difficile has two major toxins both independently capable of causing disease. ƒƒ A third of all strains carry a further toxin, binary toxin. ƒƒ Sporulation is the process of vegetative cells forming endospores to overcome adverse environmental conditions. ƒƒ Germination is the process of spores returning to a vegetative state. ƒƒ A variety of cell-surface components are linked to colonization by the organism. ƒƒ C. difficile has a plethora of regulatory pathways to control virulence. ƒƒ Certain phage genes have been shown to be involved in toxin regulation.

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and B. Front. Cell. Infect. Microbiol. 2, 28 (2012). 5

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chromosome reveals a lack of association between the tcdC genotype and toxin production. Appl. Environ. Microbiol. 78, 4683–4690 (2012).

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About the Authors Ciarán P Kelly Ciarán P Kelly is Professor of Medicine at Harvard Medical School (MA, USA) and Director of Gastroenterology Training at Beth Israel Deaconess Medical Center (MA, USA). He has been involved in patient care and research into Clostridium difficile infection (CDI) for more than 20 years. His main research interest is immunebased approaches to CDI prevention and treatment. He has authored numerous clinical and basic research book chapters, invited reviews and more than 100 peer-reviewed publications, including articles on CDI that have appeared in leading journals such as: Gastroenterology, Vaccine, Infection and Immunity, Journal of Clinical Investigation, Lancet and New England Journal of Medicine.

Saurabh Sethi Saurabh Sethi is a gastroenterology fellow at the Beth Israel Deaconess Medical Center. He is a part of Ciaran Kelly’s research team. His research is focused on developing clinical prediction tools for the severity of CDI.

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Chapter

3 Immunology and Clostridium difficile

Toxin structure & mechanisms of action

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Role of toxins A & B in CDI

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Importance of adaptive immunity in CDI 

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Immunization against CDI in animal models 

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Immunization against CDI in humans 

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Innate immune responses in CDI 42

doi:10.2217/EBO.13.221

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Ciarán P Kelly & Saurabh Sethi Clostridium difficile infection (CDI) usually occurs as a complication of antibiotic use, and is mediated by toxins A and B released by pathogenic strains of the bacterium. Adaptive immune responses to these toxins influence the outcomes of CDI. More robust antitoxin immune responses are found in symptomless carriers of toxinogenic C. difficile and those with a single episode of CDI without recurrence compared with those with the symptomatic and recurrent disease. Active vaccination and passive immunotherapy targeting C. difficile toxins have been developed as new approaches to CDI therapy and prevention. Innate immune responses to C. difficile and its toxins are also important in the pathophysiology of CDI. CDI is characterized by an acute intestinal inflammatory response with prominent neutrophil infiltration and associated tissue injury. Inhibiting this acute inflammatory response in animal models can result in protection against the intestinal injury resulting from C. difficile toxin exposure. In this chapter, the authors will review the adaptive and innate immune responses in CDI. The former has been a focus of recent advances in the understanding of the pathogenesis of

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Kelly & Sethi Adaptive immune response: adaptive (or acquired) immunity creates immunological memory after an initial response to a specific pathogen, leading to an enhanced response to subsequent encounters with that same pathogen. Innate immune response: defence response mediated by germline-encoded components that directly recognize components of potential pathogens.

the disease, and is an active area for new drug development. The role played by the innate immune response in CDI has drawn relatively little attention from researchers. However, it may prove to be an important target for adjuncts to antibiotics, especially in severe disease.

Toxin structure & mechanisms of action C. difficile is a noninvasive organism, which possesses multiple virulence factors that aid in colonization and may promote disease. These include various adherence factors such as flagellar proteins, surface layer proteins (SLPs) and a surface-exposed adhesin. In addition, all pathogenic strains of C. difficile express large exotoxins, either A and B or B alone. Figure 3.1 outlines the mechanism of action of these toxins [1–3]. Role of toxins A & B in CDI When administered orally to animals, the purified toxins are capable of inducing the full spectrum of disease manifestations typical of CDI. Purified toxin A possesses potent enterotoxic and proinflammatory activity, and it is also cytotoxic to cultured cells. Toxin B exhibits little or no independant enterotoxic activity in some animal models but it is a more potent cytotoxin than toxin A. Toxin A is fatal to mice and hamsters whereas toxin B is not when administered intragastrically. The toxins act in a synergistic fashion when coadministered intragastrically, which suggests that toxin A may initially affect epithelial integrity allowing entry of the more potent toxin B. More recently, investigators have shown that both toxins A and B contribute to disease pathogenesis in animal models by using isogenic tcdA and tcdB mutants of C. difficile [4]. Importantly, studies using the human colon have shown that toxin B is a more potent enterotoxin than toxin A in the human intestine [5]. Furthermore, strains of toxin A-negative and toxin B-positive C. difficile have emerged that have been associated with sporadic and epidemic CDI, confirming the pathogenicity of toxin B alone in the human disease [6]. Therefore, it appears that vaccines and passive antibody products that protect against and treat CDI in humans must target both toxins A and B.

It is important that vaccines and passive antibody products that protect against and treat Clostridium difficile infection (CDI) in humans target both toxins A and B.

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Importance of adaptive immunity in CDI Many healthy older children and adults have detectable serum IgG and IgA

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Immunology & Clostridium difficile Figure 3.1. The pathogenicity locus of Clostridium difficile; in vitro production and structure of toxins A and B; and toxin binding, internalization and intracellular actions. TcdR

A

RNA polymerase

tcdR

+

19.6 kb tcdE

tcdB

+

tcdA

tcdC

TcdE Toxin B Toxin A Permeability of cytoplasmic membrane

Upregulates transcription of toxins A and B

Inhibits toxin transcription

Toxin release B Toxin A: amino acid 2710 (308 kDa) 102-Trp 958 1130

1848

Binding domain (amino acid 861)

2710 C

N Glucosyltransferase enzymatic domain 102-Trp

Hydrophobic translocation domain

546

956 1128

Binding domain (amino acid 515) 1851

2366

N Toxin B: amino acid 2366 (270 kDa) C Hydrophobic domain

Toxin B-binding domain

C

Cell-surface receptor

DXD

UDP-Glc + Rho

Rho-Thr-Glc

(A) The 19.6-kb pathogenicity locus encodes toxin A (tcdA), toxin B (tcdB), a positive regulator of toxin transcription (tcdR) and a putative negative regulator of transcription (tcdC). The function of the tcdE gene product is uncertain but may include the facilitation of toxin release by bacterial membrane lysis [1]. (B) Toxins A and B of Clostridium difficile show considerable sequence and structural homology. Both have a C-terminal receptor-binding domain and a central hydrophobic domain that is believed to mediate the insertion of the toxin into the membrane of the endosome, thereby allowing the N-terminal glucosyltransferase enzymatic domain to enter the cytosol [2]. (C) The interaction of the toxin B-binding domain with cell-surface receptors induces receptor-mediated endocytosis. The acidic pH of the endosome triggers the first conformational change and results in pore formation of the hydrophobic translocation domain. Within the cytosol, a second conformational change activates intrinsic protease activity. Autocatalytic cleavage of toxin B releases the catalytic DXD glucosyltransferase domain into the cytosol. Glucosylation of the cytosolic target Rho GTPases at a conserved threonine residue leads to disaggregation of the cytoskeleton and cell death. DXD: Two aspartic acid residues separated by any one other amino acid; Glc: d-glucose; UDP: Uridine diphosphate [3]. Reproduced with permission from [1–3].

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Kelly & Sethi antibodies to C. difficile toxins A and B, even in the absence of C. difficile colonization or active infection [7]. It is likely that environmental exposure to C. difficile itself, or to other crossreacting antigens, stimulate antitoxin antibody production in infancy that is perpetuated through adult life. Early studies suggested that immune responses to C. difficile and its toxins could influence the disease presentation and course [8–10]. Kyne et al. subsequently tested this hypothesis in a prospective study of 271 hospitalized antibiotic recipients in whom they measured serum and fecal antibody responses to C. difficile toxins A and B, and nontoxin antigens over time [11]. They found that at the time of colonization, asymptomatic carriers of C. difficile had significantly higher serum IgG antibody levels to toxin A compared with colonized patients who developed diarrhea (Figure 3.2A). The effect of higher serum IgG antitoxin A levels in protecting against diarrhea was less marked in patients who were severely ill. This moderation of the effects of protective immunity in severely ill patients may have implications for future immunotherapy-based treatments, which may be less potent in very ill patients. In patients with CDI, IgM levels against toxins A and B, and nontoxin antigens were significantly higher in patients who had a single episode of diarrhea than in patients who later developed recurrent CDI (Figure 3.2B) [12]. IgG2 and IgG3 subclasses of antitoxin A IgG antibodies were abnormally low in patients with recurrent CDI [13]. In the past, many researchers examined SLPs to evaluate the role of the host immune response to C. difficile antigens other than toxins A and B. SLPs have been shown to have adhesive properties that play a role in bacterial adhesion. It was found that antibody levels to SLPs were similar in patients with CDI, asymptomatic carriers and controls [14]. However, compared with patients with a single episode of CDI, patients with recurrent CDI were not able to produce an IgM immune response to SLPs. In another study, IgG but not IgM levels to SLPs were similar in cases and carriers, but higher compared with controls [7].

Immunization against CDI in animal models A scientific rationale for the development of antibody products and vaccines against C. difficile was provided by the results of the studies presented above [11–14]. Researchers have studied the ability of active and passive immunity to C. difficile toxins to protect against CDI in animal models and humans. Vaccines against SLPs, aimed at preventing or reducing colonization, were developed and tested in animals.

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Immunology & Clostridium difficile Figure 3.2. Levels of serum IgG antibody against toxin A in hospitalized patients with active Clostridium difficile infection, symptomless carriers of C. difficile, and noncolonized control subjects and the relationship between levels of IgM against toxin A and recurrent Infection. Serum IgG antibody against toxin A (ELISA units)

A

Patients with recurrent Clostridium difficile diarrhea (%)

B

2.5 2.0

Carriers Patients with colonization Patients with diarrhea

1.5 1.0 0.5 0

p = 0.06

p = 0.002

p = 0.001

p = 0.005

Admission

Colonization or midpoint of hospital stay

3 days after colonization or midpoint

Discharge

100 75 50 25 0 90%, while cure rates with metronidazole have gradually declined from >90% to approximately 70%, probably as a result of the emergence of metronidazoleresistant C. difficile strains [21]. Although the clinical response rates are high, both antibiotics are suboptimal owing to recurrence rates of up to 30%. [5]. Administration of antibiotics appears to disrupt the delicate balance between host and commensal microbiota, which further reduces the microbial diversity in the gut. Results from several studies are consistent with this hypothesis and show marked decreases in the diversity of gut bacteria after antibiotic treatment (both given for CDI and administered in other settings), particularly with respect to members of the Bacteroides genus and Firmicutes [11,22]. There is also preliminary evidence that the gut microbiome, while resilient, eventually succumbs to persistent disruption and may adopt a new permanent dysregulated state [22]. This may explain the observation that patients undergoing CDI recurrences have increased likelihood of recurrence of the disease with each repeated episode.

Fidaxomicin is a novel narrow-spectrum macrocycle antibiotic approved in 2011 for treatment of CDI by the US FDA after the results of two parallel, randomized clinical trials showed similar clinical cure rates to vancomycin, but a rate of recurrence reduced by almost 50% [13]. One of the principal factors behind this difference may be the extremely narrow spectrum of activity of fidamoxicin (confined to Gram-positive anaerobic bacteria, e.g., C. difficile) [12], which may result in decreased impact on the microbiome in patients with CDI [23,24]. To explore the microbiota-sparing properties of fidamoxicin, Louie et al. compared the baseline microbiome between patients with CDI and healthy Treatment with fidamoxicin results in preservation of a more robust microbiome controls by conventional bacterial culture, than treatment with oral metronidazole or and differences in the microbiome between vancomycin, which may explain why there is a vancomycin- and fidamoxicin-treated decreased risk of recurrent disease.

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The microbiome & Clostridium difficile infection patients by quantitative real-time PCR of major bacterial taxonomic groups (taxa) [23]. As expected, patients with CDI had a severe perturbation of the microbiome at baseline, with a 2–3-log decrease in the major bacterial taxa. Patients treated with vancomycin had a further 2–4-log reduction in Bacteroides/Prevotella group organisms over at least 28 days, with shortterm suppression of Firmicutes, whereas treatment with fidamoxicin resulted in preservation of the depleted major bacterial taxa at baseline. This study supported the contention that highly specific and targeted therapy for C. difficile such as by fidamoxicin results in preservation of a more robust microbiome and a decreased risk of recurrent disease.

Probiotics & the C. difficile microbiome Probiotics are live microorganisms that are used to prevent or treat CDI on the basis of attempting to restore the colonic microbiome [25,26]. Very few studies, however, have examined the effects of probiotic consumption on the intestinal microbiota in the setting of CDI. These studies also have employed traditional but limited methods such as culture, cloning or 16S rRNA Sanger sequencing to describe the microbial community in the gut. Furthermore, as probiotics are usually considered dietary supplements, various formulations do not undergo the same rigorous testing as regulated pharmaceutical agents, so there remain significant questions regarding their purity, safety and efficacy [27]. Thus, the data regarding the effectiveness of probiotics for C.  difficile and the impact on the gut microbiome is understandably limited. A recent meta-analysis of probiotics and C. difficile studies to date have concluded that there is no substantial data to support probiotic use for treatment of CDI in adults [25]. As an example, probiotic treatments with Lactobacillus casei and Lactobacillus bulgaricus administered empirically for CDI have been shown to be inferior to vancomycin and metronidazole therapy [28]. With respect to CDI prophylaxis, probiotic mixtures of L. casei, L. bulgaricus and Streptococcus thermophilus were found to be effective in primary prevention of CDI in a randomized, double-blind, placebo-controlled trial of 115 patients [29]. Two additional randomized control trials support the use of probiotics in addition to antibiotics for prevention of recurrent CDI [30,31]. Nevertheless, systematic reviews suggest that the results are limited and inconclusive on the overall effectiveness of probiotics for either prevention or treatment of CDI [26,32]. A truly effective probiotic formulation for CDI will probably require a global, in-depth understanding of the dynamic changes of the gut microbiome in response of FT, which may only be revealed with ultra-deep analyses such as those Probiotics: microorganisms, such as live provided by deep sequencing (see below). bacteria, that are introduced into the body to treat a disease.

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Chiu & Pillai FT & the C. difficile microbiome While many studies have reported on the efficacy and clinical success of FT, only a few have attempted to address the mechanistic basis for this success by interrogation of the microbiome. In a study by Khoruts et al., the authors used terminal restriction fragment length polymorphism and 16S rRNA gene Sanger sequencing approaches to describe changes in the gut microbiome in a single patient undergoing FT for recurrent CDI [33]. Prior to therapy, the patient’s colonic microbiota was found to deficient in members of the bacterial Firmicutes and Bacteriodetes taxa. By 14 days post-FT, the fecal bacterial composition was found to be highly similar to the donor and was dominated by Bacteroides strains and another uncharacterized butyrate-producing bacterium. The detection of the latter butyrate-producing strain in this study is intriguing because short-chainlength fatty acids such as butyrate have been shown to be critical in maintaining the integrity of the intestinal mucosa and host immune responses to CDI [34]. Another study by van Nood et al. used microarrays to study the effect of FT on the microbiota [20], also demonstrating a relative increase in Firmicutes and Bacteriodetes taxa after a successful FT. Traditional methods such as culture, cloning or Sanger sequencing of fulllength 16S rRNA following PCR are limited in their capacity to fully elaborate the microbial community in the gut. Next-generation sequencing, otherwise referred to as ‘deep sequencing’, is an emerging approach that involves generation and analysis of millions of DNA sequence reads derived from clinical specimens [35]. The power of this strategy for studying the microbiome is the ability to perform a comprehensive investigation of the bacterial species that are present in both healthy and diseased states [36]. Deep sequencing provides superior depth and breadth of coverage of the microbiome, especially of as yet uncultured anaerobic microorganisms in the gut. The standard technique is ultra-deep amplicon sequencing of highly conserved 16S rRNA sequences encoded in the bacterial genome to study bacterial phylogeny and taxonomy in depth [37]. Either the entire approximately 1.5-kB gene can be analyzed, or selective variable regions of the rRNA gene useful for discriminating bacterial taxa at the genus/species level, Next-generation sequencing: otherwise known such as V4–V5, can be targeted. A previous as ‘deep’ sequencing, a new technology that study comparing a deep sequencing enables the generation of millions of sequence reads method, pyrosequencing, with conventional directly from clinical samples, which can then be analyzed to profile the gut microbiome. 16S rRNA cloning followed by Sanger sequencing for characterization of the gut 16S rRNA sequencing: sequencing of the bacterial 16S rRNA gene and/or its variable regions to elucidate the microbiome found that pyrosequencing types of microorganisms that comprise an individual’s provided a more precise estimate of relative microbiome, as well as their relative abundances.

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The microbiome & Clostridium difficile infection abundance and an improved confidence of detection compared with a conventional ‘clone-and-sequence’ strategy [22]. Importantly, the taxonomic information provided by these two different methods was concordant. An alternative strategy involves unbiased shotgun sequencing of all DNA extracted from clinical samples, and subsequent classification of the fraction of sequence reads corresponding to bacteria by computational analysis. This approach is particularly useful for pathogen discovery [38], but may not be ideal for in-depth characterization of the microbiome since the number and percentage of bacterial shotgun reads from a diarrheal stool sample amidst a predominant human host or environmental background can be too few or randomly scattered to permit an in-depth metagenomic analysis. A paper by Shahinas et al. reports the use of pyrosequencing to characterize the effect of FT on the CDI microbiome [39]. The V5–V6 region of bacterial 16S rRNA was pyrosequenced and the deep-sequencing reads were classified to reference sequence-based operational taxonomic units, defined at 97% sequence identity. Dramatic shifts in microbial diversity and composition were found to occur with a successful FT that were not seen with a rare failed transplant, resulting in restoration of bacterial diversity and richness (Figure 4.1). Specifically, the eradication of key species of Proteobacteria and a corresponding restoration of species in the Firmicutes and Bacteriodetes taxa appeared to indicate a successful FT, in agreement with the studies by Khoruts et  al. [33] and van Nood et  al.  [20]. This study also confirmed the potentially important role of butyrate-producing bacteria in the healthy gut; few taxa from butyrateproducing bacteria were found in CDI stools at baseline, while these species were on average increased after a successful FT. In summary, a number of studies have addressed the microbial changes that take place with FT, although different approaches were used for bacterial identification, including DNA microarray [20], terminal restriction fragment length polymorphism and 16S rRNA Sanger sequencing [33], and deep sequencing [39]. Both Shahinas et  al. [39] and van Nood et  al.  [20] observed an increased diversity of microbiota in recipients after FT using the Simpson index. However, van Nood et al. showed higher diversity for recipients post infusion than donors [20], whereas in the study by Shahinas et al., higher diversity was observed for donors [39]. Hallmarks of a successful FT in all three studies were a significant reduction of species belonging to Proteobacteria and restoration of Firmicutes and Bacteroidetes (Table 4.1). These microbiome studies reveal that successful FT results in a radical shift in the phylogenetic diversity of the GI microbiome, and that this change is

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Chiu & Pillai Figure 4.1. Measurements of bacterial species diversity in the gut microbiome for donor and recipient before and after fecal stool transplantation for recurrent Clostridium difficile infection. Chao index (richness)

Shannon index (diversity)

400

4.0

350

3.5

300

3.0

Simpson index (diversity) 0.7

†

250 200

0.6

† †

† †

0.5

2.5

† †

0.4

2.0

†

†

†

†

150

1.5

†

100

1.0

†

50

0.5

0.3 0.2 †

0

D

R pre R post

0.0

0.1

D

R pre R post

0.0

D

R pre R post

The observed Chao index (taxon richness), Shannon index (diversity) and Simpson index (diversity) for each sample are shown. Please note that the Simpson index takes on small values in data sets of high diversity and large values in data sets of low diversity. The line in the plot represents the median for each group. † Failed fecal transplantation (FT) samples. Note that pre-FT samples are characterized by fewer taxa and lower diversity than post-FT and donor samples. R pre: Recipient pre-fecal transplantation samples; R post: Recipient post-fecal transplantation samples; D: Donor samples. Reproduced with permission from [39] American Society for Microbiology (2012).

associated with clinical cure of CDI [20,39,40]. These have significant implications for the development of therapeutics for infections such as CDI, in which the microbial community plays a significant or even primary role. Among all infectious diseases, CDI is one of few, if not the only, infectious disease proven to be best treated by modifying or perturbing the microbiome by FT rather than administering antimicrobial therapy [20]. Thus, analyses of the microbiome of CDI present a unique opportunity to explore the immediate clinical impact of microbiome research on a global and debilitating infectious disease. A probiotic containing such key bacterial species constituting a healthy gut may be successful in outcompeting C. difficile and restoring normal gut homeostasis. Indeed, simple defined bacteriotherapy consisting of only six phylogenetically diverse intestinal bacteria was sufficient to completely clear C. difficile ribotype 27/NAP1/BI infection in mice [40].

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www.futuremedicine.com C. difficile presence Abundant proteobacteria

Yes

Not available

C. difficile dominance Abundant proteobacteria

Abundant Firmicutes and Bacteriodetes

Restoration of butyrateproducing bacteria post-FT

PCR amplification of 16s rRNA prior to analysis

Diversity

Pre-FT profile

Post-FT profile

Significant common finding

Kluyvera Raoultella Parasutterella Escherichia/Shigella

Restoration of butyrateproducing bacteria post-FT

Abundant Firmicutes and Bacteriodetes

Yes

Deep sequencing by 454 pyrosequencing

FT: Fecal transplantation; T-RFLP: Terminal restriction fragment length polymorphism.

Pre-FT dominant genera Veillonella (significantly decreased post-FT) Lactobacillus Streptococcus Lachnospira

Lowest pre-FT Increased post-FT Highest in donors

T-RFLP confirmed by cloning and rRNA 16S sequencing

Microbiome analysis method

Enema

Colonoscope

FT method

6

Shahinas et al. (2012) [39]

1

Khoruts et al. (2010) [33]

Study (year)

FT patients (n)

Study features

Enterobacter Klebsiella Proteus Vibrio Yersinia

Restoration of butyrateproducing bacteria post-FT

Abundant Firmicutes and Bacteriodetes

C. difficile presence Abundant proteobacteria

Lowest pre-FT Highest post-FT

Yes

Phylogenetic microarray

Nasoduodenal infusion

17

van Nood et al. (2013) [20]

Table 4.1. Summary of the three microbiome analysis studies of fecal transplantation in recurrent Clostridium difficile infection patients.

The microbiome & Clostridium difficile infection

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56

FT: Fecal transplantation; T-RFLP: Terminal restriction fragment length polymorphism.

van Nood et al. (2013) [20]

Bacteriodetes Parabacteroides Ruminococcus Anaerostipes Alistipes Prevotella Aerococcus Bacteriodetes Parabacteroides Ruminococcus Alistipes Lachnospira Roseburia Faecalibacterium Dorea

Shahinas et al. (2012) [39] Khoruts et al. (2010) [33]

Post-FT dominant genera Bacteriodetes (significantly increased post-FT) Ruminococcus Anaerostipes Uncharacterized butyrateproducing bacteria

Study (year) Study features

Table 4.1. Summary of the three microbiome analysis studies of fecal transplantation in recurrent Clostridium difficile infection patients.

Chiu & Pillai Another preliminary yet dramatic example of the potential impact of the microbiome in the treatment of CDI can be found in a proof-ofprinciple study in two patients by Petrof et al. using an artificial stool substitute mixture as an alternative approach to FT [41]. A stool substitute preparation was generated by mixing purified intestinal bacterial cultures derived from a single healthy donor, in total representing 33 isolates. Both patients had been infected with the hypervirulent C. difficile strain, ribotype 078 and had recurrent disease, having failed at least one course of antibiotics for CDI. Treatment with the stool substitute resolved in resolution of the diarrhea, and patients remained symptom-free during the following 6 months of observation. Analysis by deep sequencing of the 16S rRNA gene demonstrated that distinctive bacteria taxa found in the stool substitute were nearly undetectable in the pre-FT stool samples but constituted over 25% of the sequences up to 6  months after treatment. Similar to previous results of the gut microbiome post-FT, an expansion of taxonomic diversity was noted in the two patients.

Conclusion & future research Much more research needs to be done to fully assess the perturbations to the microbiome that accompany CDI and interventions to prevent or treat the disease. A large-scale metagenomic database of stool specimens from not only patients with acute or recurrent CDI but also from donors of FT, healthy controls, and patients infected with other infectious and noninfectious causes of diarrhea is urgently needed to supplement the existing human gut microbiome database [42]. It is also unclear whether the V5–V6 region of the 16S rRNA gene alone is sufficient to provide a true repre­sentation of the gut microbiome, and it is possible that supportive data from other variable

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The microbiome & Clostridium difficile infection regions of the 16S rRNA gene or even other gene markers may be required [43]. Given ongoing efforts to establish specialized clinics and referral networks for CDI care and FT, dedicated resources to assessing the microbiome of individual patients with C. difficile by comprehensive techniques such as massively parallel next-generation sequencing may be as productive as personalized tumor sequencing has been in the cancer field [44]. Global assessment of the gut microbiome in individual patients with CDI may also help guide clinicians regarding optimal length and duration of antibiotic treatment and indications for FT. Finally, insights from microbiome research on CDI may ultimately further the development of novel approaches for preventing and/or treating the disease, such as inhibition of spore formation [45], treatment with short-chain-length fatty acids [46] or microbiome replacement with a defined probiotic cocktail instead of FT [40,41], which can be a suitable replacement for, or adjunct to, existing therapies. Acknowledgments The authors would like to gratefully acknowledge D Shahinas for assistance in construction of Table 4.1. Financial & competing interests disclosure CY Chiu is a member of the Speaker’s Bureau for Optimer Pharmaceuticals (fidamoxicin). DR Pillai has no relevant financial interest to report. The authors have no other relevant affiliations or financial involvement with any organization or entity with a financial interest in or financial conflict with the subject matter or materials discussed in the manuscript apart from those disclosed. No writing assistance was utilized in the production of this manuscript.

Summary. ƒƒ The gut microbiome refers to the totality of microorganisms that exist in the human gastrointestinal tract in healthy and diseased states. ƒƒ Perturbations in the gut microbiome, such as from recent antibiotic exposure, play a critical role in the pathogenesis of Clostridium difficile infection (CDI) and the likelihood of recurrence. ƒƒ Antibiotics that specifically target Gram-positive anaerobic bacteria, such as fidamoxicin, may be associated with a lower risk of recurrent disease due to decreased impact on the microbiome. ƒƒ Data on the efficacy of probiotics for the prevention or treatment of CDI to date are limited. ƒƒ Fecal transplantation, or the transplant of stool from a healthy individual to a patient, is vastly superior to conventional antibiotics for CDI, and appears to work by restoring a healthy, balanced microbiome. ƒƒ The emergence of new technologies for interrogating the microbiome such as next-generation sequencing, will probably guide the development of novel approaches for preventing and/or treating CDI in the future.

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About the Authors Samuel L Aitken Samuel L Aitken is an infectious diseases fellow at the University of Houston College of Pharmacy (TX, USA) and St Luke’s Episcopal Hospital (TX, USA). His research is focused on the clinical impact of Clostridium difficile and its treatment. He also maintains interest in the treatment of infections in the immunocompromised host.

Dhara N Shah Dhara N Shah is a Research Assistant Professor in Kevin Garey’s group. One of her research interest areas includes outcomes and translational research in the field of primary and recurrent C. difficile infection.

Kevin W Garey Kevin W Garey is an Associate Professor at the University of Houston College of Pharmacy and Chair of the Department of Clinical Sciences and Administration. He is an Adjunct Associate Professor at the University of Texas School of Public Health (TX, USA) and a Clinical Specialist and Researcher at St Luke’s Episcopal Hospital. He received a BSc in pharmacy from Dalhousie University (NS, Canada), a Doctor of Pharmacy from SUNY Buffalo (NY, USA) and a MS in biometry from the University of Texas School of Public Health. Postdoctoral training included a pharmacy practice residency at Bassett Healthcare (NY, USA) and infectious disease specialty residency and fellowship training at the University of Illinois (IL, USA). His research interests involve clinical and translational research involving healthcareassociated infections, including postsurgical infections, candidemia and C. difficile infection.

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Chapter

5 Economic burden of Clostridium difficile infection

Attributable costs

62

Total costs

64

Special populations

67

The cost of CDI recurrence70 Conclusion71

Samuel L Aitken, Dhara N Shah & Kevin W Garey Clostridium difficile infection (CDI) is an increasingly prevalent cause of morbidity and mortality worldwide. The increasing burden of CDI has led to substantial rises in the costs associated with the care of patients with CDI. Variations in the patient populations being studied lead to significant heterogeneity in assessments of the economic burden; however, it is clear that CDI contributes significantly to overall healthcare costs. Novel preventative strategies and new treatment options may reduce the economic burden of CDI.

doi:10.2217/EBO.13.185

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Aitken, Shah & Garey Clostridium difficile infection (CDI) is the leading cause of infectious diarrhea among hospitalized in-patients [1] , with an estimated 284,875  cases per year in the USA [2]. Between 1993 and 2005, the incidence of CDI rose threefold in the USA, from 25 to 76.9 cases per 10,000 hospital discharges with a 1–2.5% attributable mortality rate. During the same time frame, mortality due to C. difficile was nearly sixfold more common in Britain. The increased incidence and mortality rates have been attributed to the emergence of the epidemic BI/NAP1/027 strain in the early 2000s [3]. Adding to the growing initial burden of disease, recurrence occurs in roughly one quarter of patients, with recurrence causing a substantial increase in the risk of subsequent recurrences [4]. The economic impact of disease is significant, with the total cost to the US healthcare system placed between US$750 million and US$3.2 billion per year [5–7]. New treatment options, such as fidaxomicin and fecal transplantation, may lead to increased cure rates and decreased recurrences compared with current standard-of-care therapies; however, their role in the economics of CDI remains to be elucidated [8–10]. This chapter aims to summarize the current understanding of the economic impact of CDI from a global perspective.

Clostridium difficile infection (CDI) recurrence is a substantial clinical problem, occurring in approximately onequarter of all patients with an episode of CDI. The economic impact of a CDI recurrence has not been well studied, but may be assumed to be associated with a minimum cost of US$15,000.

Attributable costs Several single-center studies have attempted to determine the directly attributable costs of an initial episode of CDI to the total cost of hospitalization. It is clear that additional costs are primarily driven by an increased hospital length of stay and associated boarding fees, rather than additional laboratory or pharmacy costs [11–13]. See Figure 5.1 for an estimate of the attributable cost of CDI in different countries. Wilcox et al. conducted a prospective case–control study of patients with CDI during a 7-month span in England [11]. In addition to identifying over GB£4000 in CDI-attributable costs for each case, a more in-depth analysis found that boarding costs accounted for roughly 94% of these charges. Vonberg et al. performed a retrospective case–control analysis at a tertiarycare center in Germany to determine attributable costs of CDI [12]. In their analysis, a case of CDI was associated with an increase of €7147; however, daily hospital costs were similar between the cases and control groups, at €1110 and The directly attributable costs of CDI are largely €1034, respectively. The substantial due to increases in hospital length of stay, with increase in total hospital costs despite additional laboratory and pharmacy costs accounting for a small fraction of the total additional costs similar daily charges is explained by a incurred.

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Economic burden of Clostridium difficile infection Figure 5.1. Estimated attributable cost by study country. 12,000

Attributable cost (US$)

10,000 8000 6000 4000 2000 0 England

Germany

Northern Ireland

USA

All costs are converted to 2012 US$. Data adapted from [7,11–13].

median 8-day increase in length of stay. Al-Eidan et al. determined an increase in total costs of GB£2691 for patients with CDI in Northern Ireland, with pharmacy and laboratory costs accounting for only GB£177 of these costs [13]. These additional total costs probably represent an overestimate, as their analysis extrapolated costs from an increased length of stay of 13 days, and failed to adjust for clinical variables that may have contributed. In the largest single-center study of the economic impact of CDI to date, Dubberke et  al. evaluated the attributable costs of CDI among 24,691 hospitalized patients at a tertiary-care center in St Louis (MO, USA) [7]. During the index hospitalization, hospitalized patients with CDI were found to have an excess cost of US$3240 based on a propensity-matched case– control analysis and US$2454 with a linear-regression methodology. These increased costs were durable when extended to 180 days from the index case, with attributable costs due to in-patient care rising to US$7179 and US$5042 using case–control and linear-regression methodologies, respectively. When the specific cost was analyzed by center (e.g., pharmacy, laboratory or respiratory therapy), patients with CDI had significantly higher total cost in each center compared with patients without CDI. Unfortunately, the contribution of CDI to each of those cost centers was not provided. Kyne et al. performed a prospective cohort study in order to determine the attributable cost of CDI at a tertiary-care hospital in Boston (MA, USA),

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Aitken, Shah & Garey as well as to provide an estimate for total cost to the US healthcare system [6]. After adjusting for numerous potential demo­ graphic and clinical confounders, the authors estimated that a hospital course that was complicated by CDI was associated with an additional US$3669, with a mean total hospitalization cost of US$4657. By using nationwide hospital admission data, the attributable annual cost to the US healthcare system of CDI was estimated at over US$1.1 billion – it is important to note that the data used to generate this estimate do not include outpatient cost data or information or subsequent readmissions due to CDI, and is therefore probably a substantial underestimate of the true financial burden of CDI.

The total individual costs associated with CDI vary widely based on the specific study population and method of statistical analysis, but in general, are higher among patients who die in hospital and among those with specific comorbid conditions.

Total costs A number of studies have attempted to estimate the total costs of a CDI case using a number of different administrative databases. The cost estimates provided by these models are heterogeneous and are somewhat difficult to reconcile, as each study utilizes unique study populations and methods of case matching or adjustment for confounding variables. However, these studies do provide valuable insight into the economic burden of CDI and allow for rough estimation of the total cost of disease. Table 5.1 provides a summary of the estimated annual total cost of CDI in the USA. O’Brien et al. performed a retrospective analysis of acute-care hospitals in Massachusetts (USA) between 1993 and 2003 in order to estimate the Table 5.1. Estimated total cost of Clostridium difficile to the USA per year. Study (year)

Study population

Estimated cost (US$ billion)

Ref.

Dubberke et al. (2008) Single center, St Louis (MO, USA). In-patients only 0.897–1.3 (extended to 180-day total costs)

[7]

Ghantoji et al. (2010)

Systematic literature review. All peer-reviewed studies reporting economic data

0.433–0.797

[5]

Kyne et al. (2002)

Single center (MA, USA). In-patients only

1.1

[6]

Lipp et al. (2012)

Select acute-care hospitals (NY, USA). In-patients 0.792 only

[18]

McGlone et al. (2011)

Economic simulation. Estimates incorporate multiple economic perspectives

0.496–0.796

[32]

O’Brien et al. (2007)

Acute-care hospitals (MA, USA). In-patients only

3.2

[14]

64

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Economic burden of Clostridium difficile infection economic burden of CDI for an individual patient and the US healthcare system [14]. Cases of CDI were identified on the basis of the presence of the International Classification of Diseases, Ninth Edition (ICD-9) code corresponding with CDI and then classified as primary (i.e., CDI was the main discharge diagnosis for that admission) or secondary cases. CDI cases were then further stratified based on hospital-onset healthcare facility- or community-acquired status. Primary cases of CDI were associated with a mean total cost of US$10,212, with costs for patients who died in hospital (US$26,507) substantially higher than those who survived to discharge (US$9434). Secondary cases had a mean total cost of US$29,946, with an attributable cost of US$13,675, or 46% of the total in-patient costs. For this analysis, the authors attributed the additional costs of CDI to an average 2.9-day increase in the length of stay. By extrapolating costs to the US healthcare system, the annual economic burden of CDI was estimated at US$3.2 billion. To provide a national perspective on the burden of CDI, Stewart and Hollenbeak reviewed the Nationwide In-patient Sample (NIS) for the year 2007 [15]. The NIS provides discharge-level information on a representative 20% sample of in-patients in the USA, and is the largest publically available, all-payer database for this information. Using a propensity score casematched design, CDI cases were found to have a mean total hospital cost of US$23,344 compared with US$14,918 for those without CDI. CDI cases were again found to have a longer length of stay than those without, with a mean length of stay of 13 days for CDI cases versus 7.9 days for controls. Numerous comorbid conditions, including congestive heart failure, cardiopulmonary disease, valvular heart disease and coagulopathy, were found to further increase costs among patients with CDI. Interestingly, and somewhat counterintuitively, patients with chronic renal failure and diabetes and CDI experienced lower total hospitalization costs than matched cases without CDI. Wang and Stewart performed a similar analysis using data from the Pennsylvania Health Care Cost Containment Council database from 2005 to 2008 [16]. Cases were again identified using the ICD-9 code for CDI and matched using a propensity score case-matched design. CDI cases had a mean US$22,094 in total costs, compared with US$10,865 in the non-CDI cohort. The mean cost for a CDI case rose 9% between 2005 and 2008, while the costs for those without CDI remained roughly static. The nature of the database also allowed the authors to analyze costs based on the size, hospital teaching status and location of the hospital for each CDI case. These detailed analyses revealed that mean total costs for a CDI case were far lower in small, rural (US$24,465); large, rural (US$22,068); and urban,

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Aitken, Shah & Garey nonteaching hospitals (US$25,239) in comparison with urban teaching hospitals (US$37,100). The data utilized were insufficient to allow for a more in-depth analysis of this information to determine if differences in a patient’s clinical status between these hospital categories contributed to these large discrepancies. Pakyz et al. performed a retrospective, case–control study of 45 hospitals in the University Health Consortium database, using data from 2002 to 2007 [17]. Cases were identified as having an ICD-9 code for CDI as well as receiving either oral vancomycin or metronidazole for at least 4 days during the hospitalization. Control patients were matched by diagnosis-related group code, quarter and year of discharge, and age. Using a multivariate linear-regression analysis, mean costs of hospitalization for a patient with CDI were US$55,769 compared with US$28,069 for those without. This increase in costs corresponded with a near doubling in length of stay for cases compared with controls (21.1 vs 10.0 days, respectively). The authors also compared total costs for cases on the basis of anti-CDI antibiotics received, and found that mean total costs were highest for those treated with metronidazole following oral vancomycin (US$82,792), while those treated with oral vancomycin alone had the lowest total costs (US$29,260). Information on whether the more costly capsule formulation of oral vancomycin was utilized instead of the more common compounded liquid formulation was not provided. Lipp et  al. utilized data from the Statewide Planning and Research Cooperative System database in order to determine the costs associated with hospital-acquired CDI across New York state (USA) [18]. Cases were identified on the basis of ICD-9 code corresponding to CDI, as well as having a modifier indicating that the condition was not present on admission to the hospital; however, no specific time frame for the development of CDI during the hospitalization was available through the database. Through generalized linear regression modeling, CDI was associated with nearly US$29,000 in additional charges and an increase in length of stay of almost 12 days. Based on these estimates, the estimated total annual cost of CDI to New York state (USA) was roughly US$55  million, and in the USA, US$792 million. These additional costs were associated with an estimated additional 209,000 in-patient hospital days per year nationwide. A wide-ranging analysis of gastrointestinal disorders, utilizing multiple public and private databases, was conducted by Peery et al. in order to place the burden of CDI within the context of other gastroenterological complaints [19]. Based on the analysis of the NIS, CDI was found to be the tenth most frequent gastrointestinal-related principal discharge diagnosis, with data from the US

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Economic burden of Clostridium difficile infection Total costs to the US healthcare system have CDC’s National Vital Statistics System been estimated between US$750 million and showing CDI as the ninth leading cause of US$3.2  billion. These costs are probably an death among gastro­intestinal disease. The underestimate, as they do not include many of the impact of CDI on health-related quality of costs associated with outpatient care, long-term life was assessed using the 2010 US National follow-up costs or indirect nonmedical costs. Health and Wellness Survey, which provides information on health-related quality of life and work/activity impairment through the use of an annual, online, cross-sectional survey [19]. CDI was associated with the highest work-impairment score of all gastrointestinal disorders analyzed along with the third highest impairment of daily activities, ranking behind only chronic liver disease and chronic constipation. However, the economic impact of these lost wages has not been assessed. Using additional information from the NIS, total annual costs to the US healthcare system were estimated at roughly US$1.1 billion; however, this estimate includes only in-patient costs, and does not assess the contribution of decreased quality of life or outpatient burden of CDI.

In the only study to date that assessed both the in-patient and outpatient burden of CDI, Kuntz et  al. performed a retrospective cohort study of members of the Kaiser Permanente Colorado and Northwest healthcare plans between 2005 and 2008 [20]. Outpatient cases of CDI were identified on the basis of ICD-9 code or positive C. difficile toxin test along with a prescription for either metronidazole or vancomycin, while in-patient cases were identified on the basis of ICD-9 code alone. A total of 56% of all cases in this study were identified as outpatients. Mean total outpatient costs during the 180-day study period were US$859.40, and in contrast to in-patient costs, the cost of the anti-CDI drugs made up a substantial portion of these costs (US$424.30). This figure is inclusive of all costs associated with a patient’s care during the study time frame, including follow-up visits, laboratory testing, and telephone follow-up. Total in-patient costs were similar to those identified by other authors, at US$10,708.40. During the time period of analysis, only metronidazole and vancomycin were used as treatment for CDI. These data indicate that other estimates of the total annual national cost of CDI are probably significant underestimates, as these analyses have failed to identify the nearly 50% of cases that are identified and treated solely on an outpatient basis [20].

Special populations Other investigators have examined the impact of CDI in specific subpopulations, allowing for determination of whether the burden of disease falls disproportionately on specific groups of patients. See Figure 5.2 for a summary of the excess costs of CDI in select populations with CDI.

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Aitken, Shah & Garey

100,000

16

90,000

14

80,000 70,000

12

60,000

10

50,000

8

40,000

6

30,000

Mechanical ventilation

Irritable bowel disease

Trauma

0

Medical intensive care

10,000

0

Liver transplant

20,000

2 Surgical inpatients

4

Excess cost (2012 US$)

18

Overall

Excess length of stay (days)

Figure 5.2. Estimated excess cost and length of stay increase for select populations.

The pink line represents the excess length of stay and the bars represent the increase in costs attributable to Clostridium difficile infection. All values are converted to 2012 US$. Adapted from [15,22–27].

Two studies have used the NIS to examine the effects of CDI on in-patient costs for patients with solid organ transplants (SOTs). The first analysis, conducted by Pant et al., identified recipients of liver, kidney, heart or lung transplants as well as patients with CDI by utilizing ICD-9 codes [21]. Total charges, rather than cost, were assessed and compared between patients with SOTs, patients with CDI and patients with both. Median total charges were highest in the SOT group, with CDI at US$53,808, compared with US$37,917 in the CDI-alone group and US$31,488 in the SOT-alone group. It is not clear from the data what the reason for admission for the non-CDI SOT patients was; therefore, comparison of these charge estimates is difficult to decipher. Ali et al. conducted a similar analysis, analyzing the impact of CDI on patients with liver transplants [22]. In patients with both liver transplant and CDI, median overall hospital charges were US$143,000 compared with US$73,000 for those without. Charges were also further divided by patients who survived to discharge versus those who did not, with charges substantially higher in the CDI population in both the survivor group (US$129,000 vs 69,000) and nonsurvivor groups (US$393,000 vs 206,000). The dramatic differences in total charges accrued in these two

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Economic burden of Clostridium difficile infection analyses despite using the same data set raise questions about how the specific analyses were conducted, and the failure to convert charge to cost data prevents these data from being compared with those found in the general population. Two studies have evaluated the costs associated with CDI among patients in intensive care units (ICUs). The first, conducted by Lawrence et al., was a retrospective cohort review of patients in a 19-bed medical ICU over a 30-month span [23]. Patients with CDI were found to have significantly longer median ICU and total hospital stays versus those without (6.1 and 24.5 days compared with 3.0 and 10.1 days, respectively) as well as significantly higher median ICU and total hospital costs (US$11,353 and US$45,910 vs US$6,028 and US$18,620, respectively). A second study, performed by Zilberberg et al., examined the impact of CDI on a nationwide cohort of patients receiving prolonged, acute mechanical ventilation [24]. All patients with ICD-9 codes for either prolonged, acute mechanical ventilation or CDI were identified through the 2005 NIS, with a propensity score-based model used to adjust for confounding variables. After adjustment for comorbid conditions, demographics, and hospital and admission characteristics, CDI was found to result in a median hospital length-of-stay increase of 6.1 days and US$10,355 in additional costs. Ananthakrishnan et al. utilized the NIS data from 2003 to examine the impact of CDI in patients with coexisting inflammatory bowel disease (IBD) [25]. All patients in the data set with ICD-9 codes corresponding with CDI, and either Crohn’s disease or ulcerative colitis were identified; patients with Crohn’s disease or ulcerative colitis and no CDI served as a comparator group. Patients with CDI were found to have an overall length-of-stay increase of approximately 3 days, with a concomitant increase in hospital charges of US$13,652. This analysis was unable to adjust for severity of the underlying IBD, and patients with both CDI and IBD were more likely to have elevated Charlson comorbidity scores, introducing the possibility of a selection bias in the underlying estimates. Three studies have explored the economic impact of CDI in surgical patients. Zerey et al. reviewed the NIS from 1999 to 2003, using ICD-9 codes to identify patients who had undergone a wide variety of surgical procedures and were diagnosed with CDI [26]. After controlling for type of surgical procedure and a number of potential postoperative complications, patients with CDI were found to have a total length of stay that was 16 days longer with an additional US$77,483 in additional hospital charges. Of note, the median charges for patients with CDI varied widely between procedure types, ranging from US$23,349 for an appendectomy to US$100,845 for

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Aitken, Shah & Garey Heller myotomy, indicating that the overall cost estimate cannot be applied universally across all surgical types. Glance et al. also utilized the NIS to study the economic impact of CDI, but restricted their analysis to trauma patients from 2005 to 2006 [27]. Patients with CDI had a median 10-day difference in hospital length of stay and US$20,445 in costs, although these estimates were not adjusted for confounders. As in the general surgery population, patients with CDI had highly variable total costs, ranging from US$21,276 for a low fall to US$90,691 for a pedestrian trauma. Kim et al. utilized the NIS from 2001 through to 2008 to investigate the burden of CDI in patients undergoing a radical cystectomy for bladder cancer [28]. Using similar methods of adjustment as Zerey et al., CDI was associated with a 9-day increase in median length of stay and an additional US$8,340,806 in attributable costs for the total population studied [26].

The cost of CDI recurrence Although many of the previously mentioned studies, particularly those studying large, administrative databases, have potentially included recurrent CDI in their cost estimates, the economic impact of recurrent CDI has been explicitly evaluated in only two studies. McFarland et al. utilized data obtained as part of a USA-based prospective, placebocontrolled, randomized trial that evaluated treatment options for recurrent CDI [29]. Direct costs of the medical care provided, including outpatient items (e.g., antibiotics, clinic costs and laboratory testing) as well as in-patient costs (e.g., daily charges, physician fees and procedural costs) were evaluated. Indirect medical costs, such as patient travel time or lost work days, were not included in the analysis. The total costs of lifetime medical care for the 209 patients enrolled in the trial were calculated at US$2,292,856, or an average of US$10,970 per patient. When evaluated by treatment episode (i.e., initial or subsequent episodes), the cost of recurrent disease was found to be substantially higher for recurrences (US$3103) compared with the initial episode (US$1914). The authors attributed this difference to the shorter duration of antibiotic therapy utilized in the treatment of an initial episode of CDI. Miller et  al. conducted a prospective, multicenter study to assess the outcomes associated with nosocomial CDI in Canadian hospitals in 1997 [30]. Patients were enrolled if a laboratory test was positive for C. difficile toxin and met the case definition of CDI on the basis of the Society for Healthcare Epidemiology of America’s definition of nosocomial acquisition. A total of 269 patients met the criteria for inclusion in the study, of whom 19 (7%) were readmitted within 30 days for management of CDI. The mean length of stay associated with these readmissions was 13.6 days per case.

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Economic burden of Clostridium difficile infection The authors calculated a total cost of CAN$128,200 to each hospital per year, assuming a 7% admission rate for CDI and incorporating estimated minimum daily bed rates and the cost of antibiotic therapy. Despite the paucity of data evaluating costs for recurrent CDI, inference can be made on the basis of surveillance data that estimates the rate of recurrence as well as the frequency of rehospitalization for recurrences. O’Brien et al. noted a 13% admission rate within 1 year following their index admission, of which 32% again received a primary diagnosis of CDI [14]. These readmissions were associated with a mean 15-day hospital length of stay. These data are similar to that of Miller et  al., both in terms of total rehospitalizations for CDI and length of stay, allowing for a reasonable estimate of a range to aid in making a gross cost estimate of recurrent CDI [30]. By comparison, Kuntz et al. noted a 21.6% readmission rate for CDI within 180 days of their index case, substantially higher than what had previously been reported [20]. These readmissions were associated with a 15-day length of stay, remarkably similar to the results of both O’Brien et al. and Miller et al. Based on these similarities, an approximation of the cost of a readmission for CDI in the USA can be made by using average daily cost information provided by the US Census Bureau [101]. Conservatively, assuming a daily cost of US$1853 and a total hospital stay of 15 days, the estimated minimum cost of a readmission for CDI is US$27,795. It is important to note that this estimate does not account for the likelihood of increased severity of disease with a recurrence [31], nor the no-longernegligible cost of drugs such as fidaxomicin, which may be more likely to be used in the setting of a disease recurrence [10].

Conclusion Comparison of the costs of CDI is difficult due to the substantial heterogeneity of the studies on the topic, with in-study population and methods of cost calculation leading to an enormous range of estimates of total cost. Despite these limitations, two key conclusions can be drawn from these studies: first, that C. difficile dramatically increases total cost of hospitalization and second, that this cost is directly related to an increased length of stay. Regardless of the population studied, data sources utilized, or the country in which the study was performed, these conclusions hold true. Further study is needed to determine the exact economic burden of CDI on outpatients, as well as the impact of recurrent CDI and its relation to the total cost of CDI. Table 5.2 provides a summary of the characteristics of the studies that have evaluated the economics of CDI. When viewed from any perspective, the total cost of C. difficile is substantial. For an individual patient with CDI, overall hospital costs may range from

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71

72

Retrospective, USA, 193,174 inhospital nationwide patients with liver transplant­ ation, with or without CDI

Ali et al. (2012)

52.97

US$5042

US$2454

US$11,000

NR

NR

Attributable or incremental costs 13

Attrib­ utable LOS (days)

NA

7 vs 4

NA

3

17.8 vs 7.7 NR

16.9 vs 3.9

Total LOS versus controls (days)

[7]

[25]

[22]

[13]

Ref.

CDI: Clostridium difficile infection; HMO: Health-maintenance organization; IBD: Irritable bowel disease; ICU: Intensive care unit; LOS: Length of stay; NA: Not applicable; NR: Not reported; SOT: Solid organ transplant.

24,691 hospital Primary nonsurgical admission: in-patients US$8394 vs 5940 In-patient costs over 180 days: US$14,560 vs 9518

41.31

MO, USA

Dubberke et al. (2008)

Retrospective cohort, hospital

45.11

In-patients 95.89 with or without CDI: US$143,000 vs 73,000

GB£3675.3

Change in costs versus controls (%)

Ananthakrishnan Retrospective, USA, 44,400 CDI IBD patients et al. (2008) hospital nationwide patients with with or or without IBD without CDI: US$19,548 vs 13,471

87 hospital in-patients

Retrospective, Northern hospital Ireland

Al-Eidan et al. (2000)

Sample Total costs characteristic, (vs controls setting where applicable)

Study design, Location economic perspective

Study (year)

Table 5.2. Characteristics of the studies that have evaluated the economics of Clostridium difficile.

Aitken, Shah & Garey

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NA

77.29

159.12

Change in costs versus controls (%)

US$3,669

NR

NR

Attributable or incremental costs

10.2 vs 6.6

NR

17 vs 8

16 vs 6

Total LOS versus controls (days)

3.6

NR

NR

Attrib­ utable LOS (days)

[6]

[20]

[28]

[27]

Ref.

CDI: Clostridium difficile infection; HMO: Health-maintenance organization; IBD: Irritable bowel disease; ICU: Intensive care unit; LOS: Length of stay; NA: Not applicable; NR: Not reported; SOT: Solid organ transplant.

271 hospital in-patients

In-patients with or without CDI: US$10,489 vs 6820

Prospective, hospital

Kyne et al. (2002)

MA, USA

In-patients with CDI: US$10,708.40 Outpatients with CDI: US$859.40

Retrospective, CO and payer Northwest (USA)

Kuntz et al. (2012) 3067 HMO members

In-patients with or without CDI: US$51,919 vs 29,285

In-patients with or without CDI: US$33,294 vs 12,849

Kim et al. (2012) Retrospective, USA, 10,856 inhospital nationwide patients undergoing radical cystectomy with or without CDI

Retrospective, USA, 155,891 hospital nationwide trauma patients with or without CDI

Glance et al. (2011)

Sample Total costs characteristic, (vs controls setting where applicable)

Study design, Location economic perspective

Study (year)

Table 5.2. Characteristics of the studies that have evaluated the economics of Clostridium difficile.

Economic burden of Clostridium difficile infection

73

74

Retrospective cohort, hospital

Lawrence et al. (2007)

4,853,800 inpatients

1835 ICU patients

Change in costs versus controls (%)

NR

NR

NR

8.8

NR

24.5 vs 10.1

US$27,290

US$29,000

6.1 vs 3

Total LOS versus controls (days)

US$5325

Attributable or incremental costs

NA

12

14.4

3.1

Attrib­ utable LOS (days)

[29]

[18]

[23]

Ref.

CDI: Clostridium difficile infection; HMO: Health-maintenance organization; IBD: Irritable bowel disease; ICU: Intensive care unit; LOS: Length of stay; NA: Not applicable; NR: Not reported; SOT: Solid organ transplant.

NA

NA

CDI patients vs 88.34 non-CDI patients ICU stay only: US$11,353 vs 6028 CDI patients vs 146.56 non-CDI patients entire hospitalization US$45,910 vs 18,620

Sample Total costs characteristic, (vs controls setting where applicable)

McFarland et al. Prospective USA, 209 patients Primary (1999) cohort, patient nationwide with recurrent episode: CDI US$1914 Recurrent episodes: US$3103 Primary plus recurrent: US$10,970

Lipp et al. (2012) Retrospective, NY, USA hospital

USA

Study design, Location economic perspective

Study (year)

Table 5.2. Characteristics of the studies that have evaluated the economics of Clostridium difficile.

Aitken, Shah & Garey

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Retrospective cohort, payer

Retrospective, USA, 30,071 inhospital nationwide patients

Pakyz et al. (2011)

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In-patients with or without CDI: US$55,769 vs 28,609

Peery et al. (2012)

NA

70.88

94.94

NR

5

9 vs 4

21.1 vs 10.0

15.7

US$13,675

NA

6.4

13.6

Total LOS versus controls (days)

NA

NA

Attributable or incremental costs

NR

NA

2.9

NA

NA

Attrib­ utable LOS (days)

[19]

[21]

[17]

[14]

[30]

Ref.

CDI: Clostridium difficile infection; HMO: Health-maintenance organization; IBD: Irritable bowel disease; ICU: Intensive care unit; LOS: Length of stay; NA: Not applicable; NR: Not reported; SOT: Solid organ transplant.

Retrospective, USA, hospital nationwide

NA

Change in costs versus controls (%)

Primary NA admission: US$10,212 CDI-secondary diagnosis: US$29,946

In-patients with or without CDI: US$53,808 vs 31,488

3692 patients in-patient data with recurrence evaluation

CDI primary diagnosis: CDN$12,456

Pant et al. (2012) Retrospective, USA, 49,198 inhospital nationwide patients with SOT with or without CDI

MA, USA

2062 hospital in-patients

O’Brien et al. (2007)

Canada

Laboratorybased prevalence, hospital

Miller et al. (2002)

Sample Total costs characteristic, (vs controls setting where applicable)

Study design, Location economic perspective

Study (year)

Table 5.2. Characteristics of the studies that have evaluated the economics of Clostridium difficile.

Economic burden of Clostridium difficile infection

75

76

Prospective case–control, hospital

UK

142 geriatric in-patients

78,273 inpatients

NR

In-patients with or without CDI: US$10,931 vs 6485

In-patients with or without CDI: €33,840 vs 18,981

In-patients with or without CDI: US$23,344 vs 14,918

NA

68.56

78.28

56.48

Change in costs versus controls (%)

£4107

€7147

NA

Attributable or incremental costs

46.5 vs 25.2

NA

27 vs 20

13 vs 7.9

Total LOS versus controls (days)

21.3

NA

7

NA

Attrib­ utable LOS (days)

[11]

[16]

[12]

[15]

Ref.

CDI: Clostridium difficile infection; HMO: Health-maintenance organization; IBD: Irritable bowel disease; ICU: Intensive care unit; LOS: Length of stay; NA: Not applicable; NR: Not reported; SOT: Solid organ transplant.

Wilcox et al. (1996)

Retrospective, PA, USA hospital

Wang and Stewart (2011)

45 in-patients

Prospective case–control, hospital

Vonberg et al. (2008)

Germany

Retrospective, USA, 82,414 inhospital nationwide patients

Stewart et al. (2011)

Sample Total costs characteristic, (vs controls setting where applicable)

Study design, Location economic perspective

Study (year)

Table 5.2. Characteristics of the studies that have evaluated the economics of Clostridium difficile.

Aitken, Shah & Garey

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Retrospective, USA, 64,910 inhospital nationwide patients undergoing prolonged acute mechanical ventilation with or without CDI

Zilberberg et al. (2009) In-patients with or without CDI: US$57,607 vs 42,785

Surgical inpatients with or without CDI: US$63,184 vs 17,232 34.64

266.67

Change in costs versus controls (%)

US$10,355

US$77,483 (total attributable charges)

Attributable or incremental costs

25 vs 17

18 vs 4

Total LOS versus controls (days)

6.1

16

Attrib­ utable LOS (days)

[24]

[26]

Ref.

CDI: Clostridium difficile infection; HMO: Health-maintenance organization; IBD: Irritable bowel disease; ICU: Intensive care unit; LOS: Length of stay; NA: Not applicable; NR: Not reported; SOT: Solid organ transplant.

Retrospective, USA, 8113 surgical hospital nationwide in-patients

Zerey et al. (2007)

Sample Total costs characteristic, (vs controls setting where applicable)

Study design, Location economic perspective

Study (year)

Table 5.2. Characteristics of the studies that have evaluated the economics of Clostridium difficile.

Economic burden of Clostridium difficile infection

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Aitken, Shah & Garey approximately US$10,000 to over US$50,000 – of which, US$2500 to nearly US$5000 may be directly attributable to the disease. For certain specific populations, such as surgical in-patients with CDI, cost estimates may increase to over US$100,000 per episode. Placed in the context of the overall healthcare system the costs are staggering. In the USA, reasonable estimates place the total cost of CDI at US$3.2  billion per year. Novel therapeutic strategies and preventive measures will hopefully decrease this substantial burden. Financial & competing interests disclosure KW Garey and DN Shah have ongoing research support from Merck Inc. The authors have no other relevant affiliations or financial involvement with any organization or entity with a financial interest in or financial conflict with the subject matter or materials discussed in the manuscript apart from those disclosed. No writing assistance was utilized in the production of this manuscript.

Summary. ƒƒ The economic impact of disease is significant, with the total cost to the US healthcare system placed between US$750 million and 3.2 billion per year. ƒƒ Additional costs are primarily driven by an increased hospital length of stay and associated boarding fees, rather than additional laboratory or pharmacy costs. ƒƒ Hospitalized patients with Clostridium difficile infection (CDI) have excess costs of at least US$2500–3000 compared with patients without CDI. ƒƒ CDI is associated with an estimated additional 209,000 in-patient hospital days per year in the USA. ƒƒ CDI is the tenth most frequent gastrointestinal-related discharge diagnosis, and is the ninth leading cause of death among gastrointestinal disorders.

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McGinley EL, Binion DG. Excess hospitalisation burden associated with Clostridium difficile in patients with inflammatory bowel disease. Gut 57(2), 205–210 (2008).

26 Zerey M, Paton BL, Lincourt AE

et al. The burden of Clostridium difficile in surgical patients in the United States. Surg. Infect. (Larchmt) 8(6), 557–566 (2007).

27 Glance LG, Stone PW,

Mukamel DB et al. Increases in mortality, length of stay, and cost associated with hospitalacquired infections in trauma patients. Arch. Surg. 146(7), 794–801 (2011).

28 Kim SP, Shah ND, Karnes RJ

et al. The implications of hospital acquired adverse events on mortality, length of stay and costs for patients undergoing radical cystectomy for bladder cancer. J. Urol. 187(6), 2011–2017 (2012).

29 McFarland LV, Surawicz CM,

Rubin M et al. Recurrent Clostridium difficile disease: epidemiology and clinical characteristics. Infect. Control Hosp. Epidemiol. 20(1), 43–50 (1999).

30 Miller MA, Hyland M, Ofner-

Agostini M et al. Morbidity, mortality, and healthcare burden of nosocomial Clostridium difficileassociated diarrhea in

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Aitken, Shah & Garey Canadian hospitals. Infect. Control Hosp. Epidemiol. 23(3), 137–140 (2002). 31 Pepin J, Routhier S, Gagnon S

et al. Management and outcomes of a first recurrence of Clostridium difficileassociated disease in Quebec,

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Canada. Clin. Infect. Dis. 42(6), 758–764 (2006). 32 McGlone SM, Bailey RR,

Zimmer SM et al. The economic burden of Clostridium difficile. Clin. Microbiol. Infect. 18(3), 282–289 (2011).

Website 101 Average Cost to Community

Hospitals per Patient: 1990–2009. US Census Bureau; 2012. www.census.gov/compendia/ statab/2012/tables/12s0173. pdf

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About the Author Peter H Gilligan Peter H Gilligan has participated in the evolution of Clostridium difficile diagnostic tests in the clinical laboratory for the past 35 years. He currently advocates the use of algorithmic approaches for the diagnosis of C. difficile infections as a sensitive, specific, cost-effective solution to this difficult clinical problem.

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Chapter

6 Laboratory diagnosis of Clostridium difficile infection Peter H Gilligan Since the advent of medical microbiology in the late 19th century, culture of bacteria and later fungi and viruses was the standard method used to diagnose infectious etiologies. However, the initial human case of Clostridium  difficile pseudomembranous colitis was detected by placing stool filtrates from a child onto tissue culture cells in the hopes of detecting a viral cause of the illness [1]. What was subsequently understood is that the cytopathic effect observed was due to a specific protein cytotoxin produced by an anaerobic bacteria called Clostridium  difficile [2]. Later, it was determined that C.  difficile produced two proteins, toxins A and B, and that they both had cytotoxic activity, although the cytotoxic activity of toxin B was logs greater than that of toxin A [3]. C. difficile is now recognized to be a leading cause of healthcare-associated infection.

doi:10.2217/EBO.13.210

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Gilligan Unlike most bacterial and fungal infections, Clostridium difficile is one of a small number of agents, including enteroxigenic and shiga-toxin producing Escherichia coli and Clostridium botulinum, which is diagnosed by demonstrating the presence of toxin or toxigenic organism in feces [4]. C. difficile toxin can be detected either directly in fecal specimens by tissue culture cytotoxin neutralization assay (CTN) or enzyme immunosorbent assays (EIAs), or indirectly by the detection of toxin genes or toxigenic organisms [4].

Cytotoxin neutralization assay: reference test in which Clostridium difficile toxin A and B specific antibodies neutralize cytotoxic effect of these toxins on tissue culture monolayers.

The evolution of C. difficile diagnostic tests over the past five decades can be seen in Table 6.1. The initial test, the CTN, was developed as an outgrowth of the observation of Larson. This assay was dependent on the development of antibodies specific for C. difficile toxins. In the CTN, stool filtrates are applied to a monolayer of tissue culture cells typically grown in 96-well flat-bottom microtiter trays. Fibroblast cell lines are most widely used because the cytopathic effect is most clearly seen with this cell type. If toxin is present, the cells demonstrate actinomorphic changes after as little as 4 h. These apoptotic changes are characterized by rounded cells Table 6.1. Diagnostic testing for toxigenic Clostridium difficile. Test

Sensitivity/ specificity

Availability First used Expense† Utilization clinically (US$)

CTN

Sensitivity: moderate Limited Specificity: high

1979

15–25

Reference method Limited diagnostic use Epidemiology No diagnostic use

Toxigenic culture

Sensitivity: high Limited Specificity: moderate

1979

10–30

Reference method Epidemiology No diagnostic use

Toxin EIA tests

Sensitivity: low Specificity: high

Widely

1991

5–15

Must detect toxin A and B; inferior sensitivity

GDH

Sensitivity: high Specificity: low

Widely

2001

5–15

Diagnostically as a screening test; must be confirmed

NAATs

Sensitivity: high Widely Specificity: moderate

2006

20–50

Use only in acute disease; false positives of concern

† Cost of goods; does not reflect laboratory charges. CTN: Cytotoxin neutralization assay; EIA: Enzyme immunosorbent assay; GDH: Glutamate dehydrogenase; NAAT: Nucleic acid amplification test. Adapted with permission from [38].

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Laboratory diagnosis of Clostridium difficile infection with thin, ray-like projections [5]. To prove that this effect is due to the C. difficile toxin, a combination of cytopathic-producing stool filtrate and antitoxin specific for C. difficile toxins is applied to a separate monolayer [6]. If no cytopathic effect is observed with this filtrate-antitoxin combination, the toxins are ‘neutralized’, indicating the presence of the C. difficile toxins. During the late 1970s to the mid-1990s, this assay was widely used diagnostically. Today, it is only infrequently used diagnostically but continues to be an important reference method in research studies. C. difficile got its name in part because it was difficult to recover from feces on culture [7]. However, the development of a selective medium, cycloserine–cefoxitin fructose agar (CCFA), in 1979 greatly simplified the recovery of this organism from fecal specimens [8]. This medium has undergone several modifications but the basic idea of using antimicrobials to select for this organism is still the basis for the isolation of this organism [9]. The spore-forming nature of this organism is also used to enhance recovery. Stools are either treated with alcohol or are heated to kill all vegetative cells. Spores survive this treatment and treated stools are then placed on C. difficile-selective agar for isolation [9]. This approach obviously is very labor intensive and takes several days for recovery and identification of the organism. In addition, three other problems were quickly discovered concerning the utility of culture as a diagnostic test. First, it was recognized that patients who received antimicrobials but who did not have diarrhea had a carriage rate of C. difficile as high as 20%, calling into question the value of culture diagnostically [10]. Second, it was recognized that some strains of C. difficile did not produce toxin and those strains are not associated with disease [11]. As a result, for culture to be used diagnostically, the ability of an isolated strain to produce toxin needed to be determined, further lengthening the turnaround time of culture. Third, it was noted that a segment of patients who were treated for C. difficile infection continued to carry the organism despite the resolution of symptoms [12]. Currently, toxigenic culture is used primarily for research and epidemiologic purposes and rarely if at all for diagnostic ones. When it is used as a reference method in research studies, it must be demonstrated that the patient has clinical disease usually on the basis of a strict definition of diarrhea indicated by three or more bowel move­ ment in a 24-h period where the stool is liquid or takes the form of the cup [13].

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The pathophysiology of C. difficile disease is mediated by two protein exotoxins, toxins A and B produced by most but not all strains. Diagnosis of C. difficile infection is dependent on the detection of toxin or toxin producing organisms in patient feces. Only stools specimens from patients with diarrheal disease should be tested for C. difficile. The reason for this is high rates of asymptomatic carriage have been observed in specific patient populations such as those receiving antimicrobial agents.

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Gilligan Because toxigenic culture and CTN had turnaround times of at least 24  h and in most incidences much longer, alternative methods were sought. Two of which were Toxins A and B: two large protein exotoxins produced tried but soon abandoned were based on by C. difficile that induce cell rounding, apoptosis, and the detection of toxin directly in stools by cytokine-induced inflammation resulting in the immunologic methods. One was counter­ pathophysiologic effects caused by this organism. immuno­electrophoresis, the other latex agglutination. Both assays had poor sensitivity and specificity compared with CTN, the commonly used reference method in the early 1980s. The reason for poor specificity was the result of the finding that the major antigen being detected by these two assays was not toxin A as originally thought but a cell wall protein, glutamate dehydrogenase (GDH), produced in abundance by both toxigenic but also nontoxigenic strains of C. difficile [14].

Glutamate dehydrogenase: cell wall protein made in large quantities found in both toxigenic and nontoxigenic C. difficile strains; target for diagnostic screening test.

The quest for rapid diagnostic tests for the detection C. difficile continued resulting in the development of solid-phase enzyme immunoassays (EIAs) for toxin A by a variety of commercial manufacturers in the early 1990s [15,16]. The assays were developed to detect only toxin A rather than toxins A and B because in animal models of C. difficile disease, toxin A was believed to be the toxin more responsible for clinical disease [17]. These tests were an improvement over the failed immunologic methods, in part because of the improvement in the antibody preparations used in the assay and increased sensitivity compared with latex agglutination assays. These assays had sensitivities of 80–90% and specificities of >95% when compared with CTN and had turnaround times of less than 2 h [15,16]. Subsequently, it was determined the strains of C. difficile that produced only toxin B could cause disease and further animals studies suggested that toxin B made important contribution to pathogenicity [18–20]. As a result, toxin A/B EIA replaced toxin A EIAs for the detection of C. difficile by the late 1990s [21,22]. During the early 2000s, an approach that detected C. difficile was revisited. Using a novel immunochromatographic approach with an assay time of 15 min, a test that detected both toxin A and GDH was developed. The GDH portion of the test had excellent sensitivity and negative predictive value for C. difficile, in part because the organism produces a large amount of this antigen compared with the amount of toxin A produced [14,23]. Because the toxin A portion of the assay was insensitive compared with CTN, the test was found to be nonspecific and have a low positive predictive value. Test with characteristics of high sensitivity and negative predictive values are viewed as excellent screening tests but at the time, the poor specificity and positive predictive value resulted in this test not finding favor in the clinical microbiology community.

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Laboratory diagnosis of Clostridium difficile infection This all changed due to two factors. One was a seminal 2006 paper by Ticehurst and colleagues in which they reported that the sensitivity of a toxin A/B EIA test was only 38% compared with a 100% sensitivity for a solid-phase EIA GDH assay [24]. Because of a poor positive predictive value for the GDH assay, this test needed to be confirmed by CTN. Subsequent studies using other toxin A/B EIAs and immunochromatographic GDH test confirmed these findings [25]. Further commercial development resulted in a new generation of a combination GDH–toxin A/B immunochroma­to­ graphic EIA test [26,27]. When compared with CTN, the toxin A/B portion of this test has high specificity but low sensitivity meaning that GDHpositive/toxin A/B-negative specimen need to be assayed by a confirmatory test but that GDH and toxin A/B negative could be reported as negative and GDH and toxin A/B positive could be reported as positive. Metaanalysis of GDH tests demonstrate high sensitivity and negative predictive value making this an ideal screening approach [28]. Using this testing approach, approximately 85% of specimens could be reported within 15 min, while 15% required further testing by CTN. The second factor that has changed the C. difficile testing landscape has been the development of nucleic acid amplification tests (NAATs) for the detection of C. difficile toxin genes. These tests have been found to be more sensitive than the GDH assays and have excellent specificity [29–31]. They also are considerably more expensive. There are currently four commercially available PCR tests, and two commercial isothermal amplification tests, one using loop-mediated isothermal/DNA amplification (LAMP) technology, and the other that uses Helicase technology [13,30–36]. All have excellent sensitivity and specificity with none showing clear superiority. All have assays times of less than 2 h with varying amounts of complexity and hands on time. Meta-analysis indicates that NAAT is highly sensitive and specific [30,31]. Currently there are three diagnostic approaches that are widely used for detection of C. difficile infection, NAAT as a standalone test, GDH as a screening test with NAAT or occasionally CTN as a confirmatory test for GDH-positive specimens, or toxin A/B EIAs [29,37]. The NAAT-alone approach has found favor in many large tertiary centers. NAAT is rapid and sensitive but requires care in how the test is used to insure its specificity. It is well established that toxigenic C.  difficile organism can still be detected in patients who have resolved their infections and in patients who do not have clinical Nucleic acid amplification test (NAAT): include disease but have received antimicrobials [38]. PCR, loop-mediated isothermal/DNA ampli­ In addition, NAAT detects DNA and can fication, or helicase amplification for the detection of detect both live as well as dead organisms. C. difficile toxin genes.

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Gilligan Because of this, NAAT testing needs to be limited to specific clinical situations. In fact, it is fair to say all C. difficile testing needs to be limited to these situations: n Only stools from patients with diarr­ heal disease should be tested. For the laboratory, that has traditionally meant testing only fecal specimens that take the form of the specimen container. However at least two factors may confound this approach. First, patients with the most severe manifestation of C. difficile disease, toxic megacolon, may not have diarrhea. In those patients and those patients only should formed stools be tested [38]. Second, a study from Washington University (MI, USA) showed that up to 20% of patients received laxatives in a 48-h period prior to testing which may challenge the diagnosis of diarrheal disease in those patients [39];

Two approaches to diagnose C.  difficile infection are widely used. One is an algorithmic approach where stools are screened for a C. difficile: cell wall protein, glutamate dehydrogenase. If positive, the specimen is then screened for the presence of C. difficile toxin or toxin genes by nucleic acid amplification test. Alternatively, NAAT can be used as a stand-alone test being more sensitive than this algorithm but less specific.

Test of cure for C. difficile should not be performed. Because C. difficile can persist for at least 30 days in as many as 20% of patients who are treated for C. difficile and resolve their symptoms, the meaning of a positive test in this patient population is difficult to determine [12,38];

n

Only a single stool specimen should be tested. NAAT for C. difficile is a highly sensitive test with repeat testing being positive less than 2 weeks. One of the studies that was included in the above analysis showed that nine out of 19 (47%) patients who were C. difficile-negative at admission and subsequently acquired toxigenic C. difficile developed CDI within 1 week of their first positive screen [4]. Based on this small number of patients, the authors concluded that progression to CDI after spore acquisition occurs either early (52

Time of onset (weeks after discharge) Bar is shortened, n = 118 cases. CA: Community-associated; CDI: Clostridium difficile infection; CO-HCFA: Community-onset, healthcare facility-associated. Adapted from [5] with permission from University of Chicago Press.

†

discharge, becoming more sporadic subsequently [5]. To enhance the specificity of the epidemiologic definition of hospital-acquired CDI, the US CDC has defined healthcare facility-acquired, hospital-onset CDI as disease with symptom onset more than 48 h following admission through discharge [6]. Healthcare-associated, community-onset disease is defined as CDI occurring at any time up to 4 weeks from the last hospital discharge, with cases occurring more than 4 but less than 12 weeks from discharge defined as ‘indeterminate’ with respect to exposure category. CA-CDI is defined as occurring more than 12 weeks from any in-patient stay at a healthcare facility [6]. These interim definitions, while useful as a tool for measuring the effectiveness of interventions to control CDI within healthcare facilities, have several limitations when applied at the level of individual patients. In part due to the unknown incubation period for CDI, the definitions do not account for the large number of patients who have been in two or more healthcare facilities prior to discharge other than to assume that the facility

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Curry at discharge was the source of the community-onset CDI. Given that CDI may be a two-hit process requiring oral ingestion of C. difficile spores, and administration of antimicrobials, these definitions cannot account for instances where different healthcare facilities both contribute to a given patient’s CDI. Prolonged incubation periods may also cause misattribution of CDI to the facility at discharge rather than to a preceding facility. A series of laboratory-based surveillance studies has suggested that CA-CDI is an emerging problem. Several studies have reported that up to 25% of all CDI is unassociated with hospital care and that cases arising in the community account for a disease burden of 5–20 cases per 100,000 personyears [7–11]. A recent prospective study of 1091 outpatients with diarrheal illness, however, showed that only three out of 39  individuals testing positive for C. difficile toxin had no recent hospital exposures or coinfections with pathogens such as norovirus or Clostridium perfringens [12]. This study highlights that laboratory-based surveillance studies of CA-CDI suffer from substantial diagnostic testing bias, as clinicians do not have ready access to testing for common outpatient diarrheal illnesses such as norovirus. Even if current estimates of laboratory-based studies of CA-CDI are accurate, the incidence of CDI in most hospitals is expressed in cases per 10,000 patient-days compared with cases per 100,000 person-years for CA-CDI, reflecting that the incidence of CDI is approximately 1000–5000-times greater within hospital populations than within the communities that surround them.

Microbiology of C. difficile infection control The endospore formed by C. difficile is the key means of its survival in hospital environments, as the vegetative organism is aero-intolerant and easily killed by quaternary ammonium-based disinfectants used in hospitals. C.  difficile spores, however, have been shown to be nearly indefinitely viable. Using purified spores stored at room temperature on stainless steel disks, Perez et al. observed only a 1.5-log10 decrease in recoverable spores over 16 months [13]. A previous study in a nursing home showed that asymptomatic carriers and CDI patients have log 10 3.6 ± 1.3 CFU/g and log10 5.6 ± 1.4 CFU/g stool, respectively, and in the same study 59% of carriers and 78% CDI patients had contaminated environmental surfaces in their rooms [14]. Beyond environmental contamination, C. difficile also contaminates the skin surfaces of patients both during and after CDI therapy. In a prospective study of 27 patients with active CDI, Bobulsky et al. showed that 25 out of 27 (93%) had skin contamination for at least one of five sites (hand, forearm, chest, abdomen and groin), and contact with a subset of ten of

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Infection control issues in Clostridium difficile these patients resulted in acquisition of C.  difficile colonies on sterile gloved hands for seven out of ten groin contacts and three to five out of ten hand, forearm, chest, and abdomen contacts (Figure 8.2) [15]. Of ten patients in this study that remained hospitalized on days 9–14 of treatment (the end of a typical CDI treatment course), six (60%) had chest and/or abdominal skin sites positive for C. difficile. In a larger study of 52 CDI patients by Sethi et  al., the percentage of positive stool, skin, and environmental cultures gradually decreased throughout treatment but rebounded substantially after treatment completion, with approximately 50% of stool, skin and environmental samples positive for C. difficile in these patients up to 3–4 weeks after treatment (Figure 8.3) [16]. Together, Figure 8.2. Contamination of skin sites and examiners’ hands with Clostridium difficile. A 70 Positive (%)

60 50 40 30 20 10 0

Hand

Forearm

Chest Skin site

Abdomen

Groin

Hand

Forearm

Chest Skin site

Abdomen

Groin

Positive (%)

B 80 70 60 50 40 30 20 10 0

(A) Frequency of Clostridium difficile contamination of skin sites of 27 patients with C. difficile infection and (B) frequency of acquisition on sterile gloves after contact with skin sites of a subset of ten patients. Adapted from [15] with permission from University of Oxford Press.

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Curry

Cultures positive for C. difficile (%)

Figure 8.3. Percentage of stool, skin (chest and abdomen), and environmental (bed rail, bedside table, call button and toilet seat) cultures positive for Clostridium difficile among 52 patients with C. difficile infection. 100 Stool

90

Skin

Environment

80 70 60 50 40 30 20 10 0 Prior to treatment

Day 3 of Resolution treatment of diarrhea

End of 1–2 weeks 3–4 weeks 5–6 weeks treatment after after after treatment treatment treatment

The numbers of patients who had samples cultured at each time point were 52 before treatment, 48 on day 3 of treatment, 43 after resolution of diarrhea, 28 at the end of treatment, 22 at 1–2 weeks after treatment, 15 at 3–4 weeks after treatment, and 8 at 5–6 weeks after treatment. Adapted from [16] with permission from University of Chicago Press.

these data suggest that the transmission of CDI from symptomatic patients can occur long after the resolution of diarrhea, and many hospitals have implemented a strategy of contact isolation precautions for CDI patients until hospital discharge rather than for the standard duration recommended by the CDC for enteric bacterial diseases – that is, for the duration of symptoms. The optimum duration of contact precautions is unknown, however, in facilities in which discharge is infrequent, such as long-term acute care facilities, psychiatric facilities, nursing homes and rehabilitation units, settings in which mixing of patients in common areas is more common than in acute care. Extending contact precautions based on arbitrary time frames following resolution of diarrhea can also have disadvantages; as can be seen in Figure 8.2 this unnecessarily commits an increasing fraction of patients (50–90%) to contact isolation that may include placement of the culture-negative former CDI patient in a room with an active CDI patient.

Modifying CDI risk factors: antimicrobial stewardship A large body of observational literature on the risk of CDI after various antimicrobials can be summarized as concluding that all antibiotics – including

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Infection control issues in Clostridium difficile For prevention of CDI, antimicrobial those such as oral vancomycin that are used stewardship focuses on restriction/reduction to treat CDI – raise the risk of subsequent CDI. of use of clindamycin, fluoroquinolones and This is thought to result from the collateral cephalosporins. effect of antibiotics on the nonpathogenic, obligate anaerobic stool microflora that are thought to be one of the principal defenses against C. difficile colonization and CDI (i.e., colonization resistance). However, the risk of CDI following exposure to various antibiotics has not been observed to be equal. For example, clindamycin confers a unique CDI risk presumably due to its wide spectrum and prolonged activity against anaerobic flora for up to 12 weeks following its administration. Fluoroquinolones and cephalosporins have also been observationally linked to large C. difficile epidemics [17]. Cephalosporins are some of the most widely used antibiotics in hospitals due to their status as recommended agents for community-acquired pneumonia and surgical infection prophylaxis, but C. difficile is intrinsically resistant to them, and the sheer frequency of use for these antibiotics make them responsible for more CDIs than for agents such as clindamycin and fluoroquinolones that have greater odds ratios for CDI in some studies [17]. Fluoroquinolones have received much attention as potential drivers of the large post-2000 CDI epidemics in hospitals [18–20]; first introduced into widespread clinical use in the 1980s, these wide spectrum agents have been implicated in the emergence of the epidemic strain (BI/NAP1/027) emergence 20 years later, as these strains have evolved resistance to this antibiotic class.

Owing to the apparently unequal risk conferred by different antibiotic classes, antimicrobial stewardship for control of CDI has focused principally on restriction of those antimicrobials associated with the highest risk of CDI. The specific agents most associated with CDI vary by center, but fluoroquinolones, clindamycin, and third-generation cephalosporins are consistently associated with CDI in multiple studies and are the principal targets of restriction. The logistics of antimicrobial stewardship programs vary, but most programs use a system of prior authorization for high-risk CDI agents requiring staffing by clinical pharmacists or infectious diseases physicians. Most programs also include retrospective review of empiric antimicrobial prescriptions, streamlining or discontinuing therapy as indicated. Although antimicrobial stewardship programs involve investments in personnel and information technology resources, many of these programs result in substantial pharmacy cost savings resulting from reductions in usage of high-cost antimicrobials – particularly in antifungals and antivirals – as a collateral benefit to reductions in hospital-acquired CDI (HA-CDI), the cost savings for which are more difficult to enumerate. In a large study of HA-CDI at two hospitals in Quebec (Canada), Valiquette

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Curry et al. observed that antimicrobial stewardship (without prior authorization or restriction) was successful in reducing HA-CDI rates whereas enhanced infection control measures were not (Figure 8.4) [21]. Of note, the rate of HA-CDI observed in this study at the end of this effective intervention was still approximately ten cases per 10,000 patient-days, a rate that has been considered epidemic in other studies [22].

The logistics of enhanced infection control precautions for CDI In contrast to the findings of Valiquette et al. [21], the University of Pittsburgh (PA, USA) observed a substantial drop in its HA-CDI rate after adoption of a bundle of nine interventions for control of CDI, only two of which involved antimicrobial restriction [22]. Most of the decrease in the HA-CDI rate occurred before the full implementation of antimicrobial restriction (Figure 8.5), but since the interventions were made over a short time frame, it is impossible to attribute the control of the epidemic to any particular intervention. The key distinction between the approach of Valiquette and

3.5

Implementation of infection control measures

250

Abx optimization intervention

CDI Targeted Abx 200

3.0 2.5

150

2.0 100

1.5 1.0

50

0.5 0.0

Sep 2003

Incidence of CDI/1000 patient-days

4.0

1 Jan 2003 1 Apr 2003

0 1 Apr 2004

1 Apr 2005

Patient-days of antibiotic use/1000 patient-days

Figure 8.4. Hospital-acquired C. difficile infection rates (per 1000 patient-days) in a tertiary-care hospital in Quebec (Canada).

1 Apr 2006

Dates shown after implementation of enhanced infection control measures (red arrow) and establishment of an antimicrobial stewardship program (green shaded area). Abx: Antibiotic; CDI: Clostridium difficile infection. Adapted from [21] with permission from University of Oxford Press.

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Infection control issues in Clostridium difficile Figure 8.5. Hospital-acquired Clostridium difficile infection rates and intervention time line at a tertiary-care hospital in Pittsburgh (PA, USA).

1999

2000

2001

2002

2003

2004

2005

2006

2.7

7.2

5.6

5.0

5.2

4.6

5.5

3.0

12

10

10

8

8

6

6

4

4

2

1

2 3 4 5

6

2

78 9

0

Monthly HA-CDI

Sep 06 Jan 07

Jan 06

May 06

Sep 05

May 05

Sep 04 Jan 05

May 04

Sep 03 Jan 04

May 03

Sep 02 Jan 03

Jan 02

May 02

Sep 01

May 01

Sep 00 Jan 01

Jan 00

May 00

Sep 99

Jan 99

0

Yearly HA-CDI rate no. of infections per 1000 hospital discharges

12

May 99

Monthly HA-CDI rate no. of infections per 1000 hospital discharges

Year

Yearly HA-CDI

Monthly HA-CDI rates are reported forthe period January 1999–January 2007 on the primary X and Y axes, and yearly HA-CDI rates (for the period 1999–2006) are shown on the secondary X and Y axes. Intervention points 6 and 8 mark the implementation of antimicrobial stewardship. HA-CDI: Hospital-acquired Clostridium difficile infection. Adapted from [22] with permission from University of Oxford Press.

Muto et al., however, was that the latter achieved a substantially lower HA-CDI rate and has maintained an annual rate less than seven cases per 10,000 patient-days since publication [Muto C, Pers. Comm.]. The actual infection control interventions used in the study were: enhanced case finding through email alerts to physicians identifying highrisk patients for CDI and through allowing nurses to send C. difficile stool toxin tests, even if formed; real time unit notification from the microbiology laboratory upon diagnosis of CDI; antimicrobial restriction of high-risk antimicrobials; extending contact isolation for CDI patients for the length of stay; switching to 500 ppm then later 5000 ppm sodium hypochlorite for terminal and daily disinfection of CDI patient rooms; mandating use of hand hygiene with soap and water (not alcohol sanitizers) for CDI Hand hygiene (washing with soap and water) patients; and regular audits of compliance alone cannot completely remove the number of C. difficile spores acquired by contact with a CDI with hand hygiene and CDI contact patient.

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Curry isolation precautions by unidentified observers. These measures have remained in effect since the publication of the study, and since its publication the hospital has switched to universal use of 5000  ppm sodium hypochlorite for daily and terminal disinfection of all patient rooms. The association of markedly similar infection control interventions with reduced rates of HA-CDI was confirmed at Maisonneuve-Rosemont Hospital in Quebec, Canada, where a 61% reduction in annual rates was observed between 2002 and 2007 despite no significant change in antibiotic consumption patterns at this institution [23]. While it is impossible to state which of the infection control interventions described by Muto and Weiss et al. was vital to the control of its CDI epidemic, the necessity of contact precautions with gloves was established in a 6-month ward-based prospective trial of gloves versus enhanced education about hand hygiene. In this study, HA-CDI rates fell from 7.7 to 1.5 cases per 1000 discharges on units using gloves, while two control units showed no significant changes [24]. These results are hardly surprising in light of data in volunteers using nontoxigenic C. difficile spores that show hand washing with varying soap and water combinations capable of achieving only approximately 2-log10 reduction in viable spore counts from hand surfaces [25]. As can be seen in Figure 8.6, contact with CDI patients’ skin surface easily results in acquisition of Figure 8.6. Hand-print culture showing acquisition of Clostridium difficile on sterile gloves more spores than hand hygiene alone can eliminate (i.e., >100 spores). Experimental after contact with a C. difficile-infected data using the new mouse model of CDI have patient’s groin. shown that the inoculum necessary to infect 50% of mice (ID50) is only five C.  difficile spores/cm2 [26]. Thus, hand hygiene alone is probably insufficient for prevention of transmission of infectious doses of C. difficile to new patients. The use of disposable gowns to supplement the barrier of glove use has not been tested in clinical trials, but their use has been recommended as a precautionary addition at most centers due to the widespread environmental contamination in the rooms of CDI patients. The larger yellow colonies outlining the fingers are C. difficile. Of note, the patient had showered 1 h before collection of the culture specimen. Adapted from [15] with permission from University of Oxford Press.

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Terminal disinfection for C. difficile control: challenges & questions Cleaning of in-patient hospital rooms upon in-patient discharge (terminal cleaning) has

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Infection control issues in Clostridium difficile been traditionally carried out with quaternary ammonium, phenolic or alcohol-based disinfectants that can be combined with surfactant (detergent) solutions to create general-purpose cleaning solutions capable of rendering most pathogens noninfectious after relatively short contact times (30 s to 5 min). These solutions have the advantage of being able to disinfect hospital surfaces while also serving as general-purpose cleaners capable of removing organic soil burdens (feces, blood and other excreta), while not damaging electronic equipment or plastic surfaces. Unfortunately, none of these hospital disinfectants has any sporicidal activity against C. difficile. Environmental transmission of C.  difficile has long been theoretically important in the transmission of C. difficile to new in-patients, but a large retrospective cohort study of intensive-care unit (ICU) patients in a large academic medical center in Michigan confirmed that occupancy of a hospital room previously occupied by a CDI patient is an independent risk factor for development of CDI after ICU admission [27]. This study confirmed the findings of multiple prior environmental culture studies establishing that both CDI patients and carriers contaminate a substantial number of room surfaces with C. difficile spores [2,14,16]. In a study of nine rooms contaminated with C. difficile spores, seven rooms remained positive on one or more sites after disinfection by housekeeping staff using 10% bleach solution (5000 ppm sodium hypochlorite) despite prior education on extra steps needed in C. difficile disinfection practice, and awareness that the room to be cleaned was previously occupied by a C. difficile patient [28]. In this same study, bleach disinfection by research personnel was effective for eight out of nine rooms, with a single site (toilet) positive in the remaining room. This study highlights several practical difficulties in establishing terminal disinfection procedures that are effective against C.  difficilecontaminated rooms. Even after using an effective sporicide, nonblinded housekeeping personnel were unable to disinfect hospital rooms reliably. Such personnel are frequently poorly paid and trained, and their job performance is most often related to their speed in turning over rooms, never on the efficacy of their disinfection effort. Recently, there has been considerable attention on disinfection technologies that are less operator-dependent, such as hydrogen peroxide vapor (HPV) and UV light disinfection, both of which can be used for terminal room disinfection in unoccupied patient rooms. A recent quasi-interventional study of HPV use showed that it was able to reduce acquisition of multidrug-resistant organisms in patients occupying a room with a previous occupant with multidrug-resistant organisms by 64% (p 700 clinical trials in patients and healthy volunteers have been conducted using probiotics, and one may think that all options have been examined. However, the effects of probiotics are dose- and strain-specific, making the task of comparison between studies almost impossible. However, the nature of C. difficile pathogenesis means that a multistrain cocktail could address the various issues posed by C. difficile. Hell et al. studied a product, Ecologic® AAD, that is an assembly of equal ratios of ten bacterial strains with a total dose of 5 g per sachet and 109 CFU/g [13]. The ten strains consisted of six Lactobacillus species, three Bifidobacteria and one Enterococcus species. The authors studied almost 1000 CDI patients over the period 2009–2011. Of this total, almost 10% died with a causative and contributive role to mortality assigned to 4–6.9% of patients, depending on age. From this cohort, ten patients were selected to receive vancomycin 125 mg qid for 10 days plus two sachets of Ecologic AAD. Five patients had recurrent CDI. Complete clinical resolution occurred in nine out of ten patients and one subject died within 3-month follow up from pneumonia. No adverse events were reported. This impact of normal flora has been confirmed in both murine and human studies. Lawley et  al. evaluated a simple, defined bacteriotherapy in relapsing CDI in mice [14]. They studied mice infected with 027 C. difficile and observed the ‘supershedder’ phenomenon and the impact on the normal flora. They examined the bowel flora of these and other mice using whole-genome molecular studies. They used species identified as being favorable to establishing a normal flora in 027 CDI: Anaerostipes spp. nov., Staphylococcus warneri, Lactobacillus reuteri, Enterococcus hirae, Enterorhabdus spp. nov., and Bacteroidetes spp. nov. Several species were also identified as being more frequent in supershedder mice feces: Blautia producta, Enterococcus faecium, E.  faecalis, Parabacterioides distasonis, Klebsiella pneumoniae, Citrobacter rodentum, Escherichia coli and Proteus mirabilis.

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Future & alternative approaches to managing C. difficile infection The authors concluded that targeting a dysbiotic microbota with a defined mix of phylogenetically diverse bacteria can lead to major shifts in the microbial community structure, which displaces C. difficile and consequently resolves disease and contagiousness. A randomized, placebo-controlled prophylactic study of a probiotic containing two strains of Lactobacillus, L. acidophilus CL1285 and L. casei LBC80R was conducted by Gao et al. [15] to determine whether enhancing the gut flora had an effect on the development and course of CDI and antibiotic-associated diarrhea (AAD). The probiotic was dosed in a sachet containing 50 billion cfu of live strains of both Lactobacilli. Three cohorts received either two sachets per day, one sachet or placebo starting within 36 h of initial antibiotic administration and continued for 5 days after the last antibiotic dose. Patients were followed for a further 21 days. The twosachet-per-day group had a 15.5% incidence of AAD compared with 28.2% in the one-sachet-per-day group. Both groups were lower than placebo at 44.1%. Moreover, the two-dose group had a lower incidence of CDI than the once-daily dosing group (1.2 vs 9.4%) and each treatment group had a shorter symptom duration than placebo. The authors concluded that the 100 billion cfu dosing yielded superior outcomes in hospitalized patients with CDI and AAD than did those given 50 billion cfu. It is important to note that this was a single study and confirmation of these effects in CDI would be essential. Furthermore, probiotics are not governed by the same strict US FDA guidelines as are antibiotics, and thus further studies are required to ensure the quality and safety of this group of potential CDI therapies. Perhaps one of the most fascinating options to manage recurrent CDI is FMT. This process has been used in patients in whom virtually all other treatments have failed and this has been the last option. In essence, FMT is the replacement of the normal fecal flora of a patient. Previously, the fecal transplant came from a partner, but current studies employ a pooled donor approach. Kassam et al. recently conducted a review and meta-analysis of 11 studies that recruited a total of 273 CDI patients, each of whom was treated with FMT [16]. No randomized clinical trials were identified. In total, 245 out of 273 patients experienced a clinical resolution (unweighted pooled resolution rate [UPR]: 89.7%; weighted pooled resolution rate [WPR]: 89.1% [95%CI: 84–93%]). An a priori subgroup analysis suggested that lower GI FMT delivery led to a trend towards higher clinical resolution rates compared with the upper GI route. No difference in clinical outcomes was detected between anonymous versus patient-selected donors. There were no reported adverse events associated with FMT, and follow-up varied from weeks to years. The authors concluded that FMT holds considerable promise

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Tillotson & Weiss as a therapy for recurrent CDI but further randomized, controlled trials and long-term safety registries are still required. This conclusion has to some extent been answered by a study conducted by van Nood et al., in which subjects with recurrent CDI were randomized to receive either oral vancomycin (500 mg q.i.d. for 4 days) followed by bowel lavage and subsequent infusion of a solution of donor feces through a naso–duodenal tube, or a standard oral vancomycin regimen (500 mg q.i.d. for 14 days), or a standard vancomycin regimen with bowel lavage [17]. The primary end point was resolution of diarrhea associated with CDI without relapse after 10 weeks. The study was stopped early after an interim analysis. Out of 16 patients in the FMT infusion group, 13 (81%) had resolution of CDI after the first infusion, and the remaining three  patients had a second infusion from a different donor and two resolved. Patients receiving vancomycin alone and vancomcyin plus bowel lavage had response rates of 31 and 23%, respectively (p 65 years [21]. In both healthy adults and the elderly, vaccination resulted in an increase in antibody titers. As anticipated, maximal IgG antibody responses occurred after the third dose. Overall, the response to toxin B was lower than that seen with toxin A. For both toxins, there was a dose response seen as the antibodies increased with escalating dosing (2, 10 or 50 µg). The optimal dose was seen with the 50-µg dose. No vaccine-related serious adverse events were reported in the Phase 21 studies. Any reactions that occurred were self-limiting and similar to those seen with other aluminum-adjuvanted vaccines [21]. Using the 50-µg dose, there are two Phase  II studies underway – one is a therapeutic trial while the other is a novel evaluation of the use of the vaccine as a prophylactic agent. Both studies will enroll 650 patients but the study in already-infected subjects is posing recruitment challenges. Owing to this vaccine meeting an unmet clinical need, ACAM-DIFF is being fast-tracked by the FDA.

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Recombinant fusion protein consisting of truncated toxins A and B

Combination of toxoid A and B

Four-component vaccine comprising TcdA, TcdB and CDTa and CDTb

IC84 (Intercell)

Pfizer

Merck Research Laboratories

Antitoxin A and B fullyhuman monoclonal antibodies targeting the receptor-binding domain of the toxins

Development stage 1 × 36 healthy volunteers and 2 × 48 healthy volunteers. Latter study had two age cohorts, 18–55 years and >65 years 650 subjects without CDI (aged 40–57 years) 650 subjects with first event of CDI (aged >65 years)

Patient groups

NR

NR

4 × 400 (1600) subjects with symptomatic CDI and on antibiotic therapy

Phase II: completed 200 patients with symptomatic CDI

Preclinical

Phase I

Phase I (open-label 160 healthy patients, dose-escalation one cohort includes study) subjects >65 years

Phase II: prophylactic use Phase II: therapeutic use

MK-3415 (antitoxin A), MK 6072 Phase III: (antitoxin B) or MK 3145A (combination in progress of both antibodies) at a single dose of 10 mg/kg with antibiotic therapy

CDA1 (antitoxin A) plus CDB1 (antitoxin B) once at 10 mg/kg in parallel with standard antibiotics

NR

NR

IC84 (20–75–200 mg) ± aluminum adjuvant given i.m. days 0, 7 and 21

ACAM-CDIFF ± adjuvant, 0.5 ml given at days 0, 7 and 28 ACAM-CDIFF ± adjuvant, 0.5 ml given at days 0, 7 and 30

ACAM-CDIFF ± alum Phase I Dose escalation study. Administration Three studies at 0, 4 and 8 weeks performed

Formulation

CDI: Clostridium difficile infection; i.m.: Intramuscular; NR: Not reported. Adapted from [28].

Bristol-Myers Squibb–Merck

Passive vaccination

Formalin-inactivated toxins A and B from strain VPI 10463 (nonhypervirulent strain expressing high toxin levels)

ACAM-CDIFF™ (Sanofi-Aventis)

Active vaccination

Sponsor/ Therapeutic agent candidate agent

Table 9.2. Status of immune-based strategies in development in Clostridium difficile infection.

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Future & alternative approaches to managing C. difficile infection In parallel, Intercell (Vienna, Austria) is testing the safety, tolerability and immunogenicity of IC84 in a Phase I study in healthy volunteers; in this study, one cohort includes subjects aged >65 years. This vaccine is based on a recombinant fusion protein containing part of the receptor-binding domain of both toxins A and B, adjuvanted with aluminum and given thrice daily. Pfizer Vaccines Research (NY, USA) is developing a toxoid-based vaccine that has proven to be 100% effective in the hamster model. Nonhuman primates immunized with adjuvanted toxoids generated robust antitoxin A and B neutralizing antibodies, which were maintained for 1 year. Clinical trials are planned [22]. Merck (NJ, USA) have developed a vaccine based on detoxified TcdA and TcdB but it was insufficient to protect animals; however, they followed up by developing a four-component vaccine consisting of CDTa and CDTb as well as the toxoids. This vaccine is immunogenic in various animal species, and antisera raised following immunization are capable of neutralizing TcdA, TcdB and binary toxin in Vero cells. Clinical trials are warranted [23]. Passive vaccination via oral administration of colostrums from cows immunized with C.  difficile was shown to protect antibiotic-treated hamsters from a lethal challenge with C.  difficile spores. These data prompted the use of intravenous immunoglobulin (IVIG) with 15 trials on a small group of volunteer patients and showed efficacy in severe CDI as well as refractory disease. Abougergi et al. treated 21 of 1230 CDI patients with IVIG. The cohort had severe disease and had an Acute Physiology and Chronic Health Evaluation II score of 25 on day 1 of IVIG infusion [24]. All patients had pancolitis or ileus. Nine patients (43%) survived their hospitalization while 12 (57%) died. Symptoms resolved after an average of 10 days for survivors. The authors concluded that IVIG, while benefiting some patients, was clearly not ideal, and the outcomes appear to benefit from this therapy. A lack of randomized clinical trials precludes the widespread use of this modality. Fully-human monoclonal antibodies have been developed and tested in the treatment of CDI. A combination of monoclonal antibodies raised towards the receptor-binding regions of toxins A and B was demonstrated to protect antibiotic-treated hamsters. These data led Medarex (NJ, USA) and Bristol-Myers Squibb (NY, USA) to conduct a placebo-controlled Phase II study in 200 subjects receiving metronidazole or vancomycin alone or in combination with antibodies to both toxins A and B. Although there was no significant difference in the reduction of diarrhea, there was a statistical reduction of recurrence

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Tillotson & Weiss in the group receiving the monoclonal antibody compared with the placebo group, thus showing proof of concept [25]. Merck have acquired the rights to this antibody and are running a Phase III study of 1600 subjects (Table 9.2). In spite of this encouraging news, the development of monoclonal antibodies to the two toxins creates a significant challenge, as so far no epitope-specific antibody that crossreacts with both toxins has been identified, thus resulting in the need for two separate antibodies. While monoclonal antibody therapies have been successful in treating cancer and autoimmune inflammatory disorders, their high costs limit their utility in large populations, and, with CDI increasing markedly each year, the widespread use of such a therapy is going to be limited to patients who are likely to become severely ill. This approach can provide an immediate immunological barrier to CDI, whereas vaccination takes several weeks to mount a clinically meaningful response. Current therapeutic strategies focus on the vegetative cells and established infection whereas Abel-Santos et al. have chosen to focus on preventing C. difficile from sporulating by the use of a novel agent, a cholate metabenzene sulfonic derivative, CamSA, which is a strong inhibitor of C. difficile germination [26]. They demonstrated that CamSA when given orally to antibiotic-treated mice completely protected the mice from CDI. A dosedependent response was observed with 50 mg/kg when administered at the same time as infection, whereas the same dose given 24 h earlier was only partially effective. Of particular interest is the lack of relapse in the treated mice, suggesting that the C. difficile and its spores were eliminated from the mouse bowel; however, Cam SA failed to protect mice from vegetative C.  difficile bacteria. In a recent editorial, Armstrong et  al. commented that this molecule may represent another paradigm shift in the management of CDI, one that involves a prophylactic approach by using the antigermination effect of taurocholate-based drugs such as CamSA [27]. CDI has presented an increasingly complex disease over the past 15 years, but in parallel our understanding and appreciation of the interplay of toxins, spores, surface layer proteins, pili and flagella is enabling us to confront the disease with a little more confidence. It is also clear that a multipronged approach to not only treating the active infection but also the spore form is essential. These prongs could include vaccination, antibiotics, antibodies or other as yet undiscovered therapies. Perhaps more importantly, it is the maintenance of the normal bowel flora that avoids this disease is the underlying secret to winning the battle against C. difficile.

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Future & alternative approaches to managing C. difficile infection Financial & competing interests disclosure GS Tillotson has served as Consultant for Summit PLC, Basilea and Astellas EU, and is an employee of TranScrip Partners US. K Weiss has received research grants from Optimer Pharma, Merck, Cubist and Novartis, and served as Speaker for Optimer Pharma and Merck. The authors have no other relevant affiliations or financial involvement with any organization or entity with a financial interest in or financial conflict with the subject matter or materials discussed in the manuscript apart from those disclosed. No writing assistance was utilized in the production of this manuscript.

Summary. ƒƒ As scientific advances occur with Clostridium difficile, it is clear that we are developing more targeted therapies. ƒƒ Much narrower spectrum antibiotics such as CB183,315 and SMT 19969 hold promise in having a lower dysbiotic effect, which should enable the normal bowel flora to be re-established sooner. ƒƒ Four vaccines are in development, each using different approaches, but the singular larger issue is who should receive these vaccines and when in order to establish adequate immunity, especially among the ‘at-risk’ subpopulations. ƒƒ Monoclonal antibodies have found significant success in fields such as oncology or rheumatology, but mindsets will have to change if we are to see wider use of these costly agents in infectious diseases. ƒƒ Perhaps the most intriguing area for managing C. difficile is the replacement of bowel flora or the use of competitive strains in patients with recurrent C. difficile infection. After the issues with unrecognized pathogens in plasma-based therapies in the 1980s, we face the uncertainty of potentially transferring a communicable condition in these transplanted microbes, whereas, with accurately defined populations of organisms, M3 C. difficile or a proven ‘probiotic’/bacteriotherapy, these risks are much lower. ƒƒ Finally, one characteristic of the C. difficile epidemic has been the emergence of the ‘supershedding’ sporulating strains (e.g., 027 or 001). The novel approach to treating C. difficile infection by stopping germination and hence enhanced sporulation deserves further investigation.

References 1

Patino H, Stevens C, Louie T et al. Efficacy and safety of the lipopetide CB183,315 for the treatment of Clostridum difficile infection. Presented at: the Interscience Conference on Antimicrobial Agents. IL, USA, 17–20 September 2011.

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Cannon K, Byrne B, Happe J, Louie T. Enteric microbiome profiles during a Phase 2

clinical trial of CB 183.315 or vancomycin for the treatment of Clostridium difficile infection. Presented at: the 22nd European Congress of Clinical Microbiology and Infectious Diseases. London, UK, 31 March–3 April 2003. 3

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Louie T, BuitragoM,Cornely O et al. Multicentre, double blind, randomised, Phase 2 study evaluating the novel

antibiotic, cadazolid, in subjects with Clostridium difficileassociated diarrhea. Presented at: the 23rd European Congress of Clinical Microbiology and Infectious Diseases. Berlin, Germany, 27–30 April 2013. 4

Debast SB, Bauer MP, Sanders IMJG et al. Antimicrobial activity of LFF571 and three

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McKenney D, Williams A, LaManche M et al. Efficacy comparison between LFF571 and fidaxomicin in the hamster model of C. difficile infection. Presented at: the 52nd Interscience Conference on Antimicrobial Agents. San Francisco, CA, USA, September 9–12 (2012). Vickers R, Tillotson G, Echols R. Clostridium difficile: a pathogen still in need of new therapeutic options including SMT 19969. Presented at: British Society for Antimicrobial Chemotherapy Spring meeting. London, UK, 14 March 2013. Weiss WJ, Vickers R, Pulse M et al. Efficacy of SMT 19969 and SMT 21829 in a hamster model of Clostridium difficile associated disease (CDAD). Presented at: the Interscience Conference on Antimicrobial Agents. Chicago, IL, USA, 17–20 Septemer 2011. Baines SD, Freeman J, Huscroft GS et al. Efficacy of novel antimicrobial agent SMT-19969 (SMT) against simulated Clostridium difficile (CD) infection in an in vitro human gut model. Presented at: the Interscience Conference on Antimicrobial Agents. Chicago, IL, USA, 17–20 Septemer 2011. Citron DM, Warren YA, Tyrrell KL et al. Comparative in vitro activity of REP 3123 against Clostridium difficile

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et al. Fecal microbiota transplantation for Clostridium difficile infection: systematic review and metaanalysis. Am. J. Gastroenterol. 108(4), 500–508 (2013).

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O’Leary AL et al. Inhibitory effect of REP 3123 on toxin and spore formation in Clostridium difficile, and in vivo efficacy in a hamster gastrointestinal infection model. J. Antimicrob. Chemother. 63, 964–971 (2009).

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Nieuwdorp M et al. Duodenal infusion of donor feces for recurrent Clostridium difficile. N. Engl. J. Med. 368(5), 407–415 (2013).

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Burkhadt O et al. Intravenous tigecycline as adjunctive or alternative therapy for severe refractory Clostridium difficile infection. Clin. Infect. Dis. 48, 1732–1735 (2009).

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Johnson S et al. New approach to the management of Clostridium difficile infection: colonization with non-toxigenic C. difficile during daily ampicillin or ceftriaxone administration. Int. J. Antimicrob. Agents 33(Suppl. 1), s46–s50 (2009).

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Tatarowic W et al. Evaluation of an oral suspension of VP20261, spores of nontoxigenic Clostridium difficile strain M3, in health subjects. Antimicrob. Agents Chemother. 56, 5224–5229 (2012).

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Walker AER et al. Target restoration of the intestinal microbiota with a simple, defined bacteriotherapy resolves relapsing Clostridium difficile in mice. PLOS Pathog. 8, e1002995 (2012).

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casei LBC80R for antibioticassociated diarrhea and Clostridium difficileassociated diarrhea prophylaxis in adult patients. Am. J. Gastroenterol. 105(7), 1636–1641 (2010).

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Roopchand VS et al. A novel approach to a C. difficile toxoid vaccine: immunogenicity and preclinical efficacy. Presented at: 4th International Clostridium difficile Symposium. Bled, Slovenia, 20–22 September 2012.

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About the Authors Joni Tillotson Joni Tillotson graduated from Immaculata University in Biological sciences after an internship studying the impact of methicillinresistant Staphylococcus aureus in human infections and the environment at John A Burns Medical Center in Hawaii (USA). She is currently working at Aardvark Animal Hospital (PA, USA). She has demonstrated a keen interest in zoonotic and bacterial infections culminating in several publications including papers on Clostridium difficile.

Glenn S Tillotson Glenn S Tillotson has almost 30 years of pharmaceutical experience, including clinical research, commercialization, medical affairs, strategic drug development, life-cycle management and global launch programs. While at Bayer (Leverkusen, Germany), he was instrumental in the development of ciprofloxacin and moxifloxacin. He has held various medical affairs leadership roles in the past decade. He has published over 140 peer-reviewed manuscripts and is on several journal editorial advisory boards.

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Epilogue Recent advances with Clostridium difficile Joni Tillotson & Glenn S Tillotson The field of Clostridium difficile is changing on a daily basis with the number of new publications increasing annually, even since the completion of the chapters in this book and the final release of the publication each topic has seen further advances. In an effort to give readers a feel for the recent publications we have captured the abstracts of a select number of manuscripts. These we have put into broad categories such as epidemiology and infection control, clinical outcomes, in vitro or diagnostics and therapeutics.

Epidemiology & infection control Following the emergence of the hyperepidemic strain, type 027, in Canadian hospitals almost a decade ago it is timely that Simor et al. [1] investigated the prevalence of both C. difficile and methicillin-resistant Staphylococcus aureus (MRSA) in a selection of moderate-sized Canadian hospitals. Twothirds (176) of those eligible hospitals participated. The median prevalence rate of C. difficile colonization or infection was 0.9%. C. difficile infection (CDI) was thought to have been healthcare associated in 84% of cases. Higher bed occupancy rates were associated with higher rates of CDI (probability distribution: 1.02 [95% CI: 1.01–1.03]).These data provide the first national prevalence estimates for CDI in Canadian hospitals. Certain infection prevention and control policies were found to be associated with prevalence and deserve further investigation. doi:10.2217/EBO.13.461

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Tillotson & Tillotson The burden and source of illness associated with C. difficile varies between healthcare systems, so it is of interest that researchers in Rhode Island, USA, have evaluated the Rhode Island hospital discharge database for 2010, which included present on admission (POA) indicators. This database provided the opportunity to distinguish cases of hospital-onset CDI from cases of community-onset CDI and to assess the burden of hospital-onset CDI in patients discharged from Rhode Island hospitals during 2010 and 2011 [2]. Patients 18 years of age or older discharged from one of Rhode Island’s 11 acute-care hospitals between 1 January 2010, and 31 December 2011 were evaluated using the newly available POA indicators in the Rhode Island 2010 and 2011 hospital discharge database. Researchers identified patients with hospital-onset CDI and without CDI. Adjusting for patient demographic and clinical characteristics using propensity score matching, between-group differences in mortality, length of stay, and cost for patients with hospital-onset CDI and without CDI were measured. Over the period 2010–2011, the 11 acute-care hospitals in Rhode Island had 225,999 discharges. Of 4531 discharged patients with CDI (2.0% of all discharges), 1211 (26.7%) had hospital-onset CDI. After adjusting for patient demographic and clinical characteristics, discharged patients with hospital-onset CDI were found to have higher mortality rates, longer lengths of stay, and higher costs than those without CDI. The epidemiology of C. difficile has proven to be particularly complex with various patients groups being shown to be at higher risk of disease acquisition or disease recurrence. A growing population, diabetics, was the subject of an investigation in terms of diabetes as a risk factor for CDI. This prospective cross-sectional study involved 159 patients with established Type 2 diabetes mellitus admitted into acute medical wards who developed a hospital-acquired C. difficile infection [3]. Stools were tested for C. difficile toxins using a toxin A/B kit and a toxin A kit. Clinical features, laboratory findings, types of antibiotics, and use of a proton pump inhibitor were examined for their association with the infection. In total, 13 subjects were positive for toxin A and one for toxin B. Using univariate analysis, the authors found that patients with Type 2 diabetes mellitus and hospital-acquired C. difficile infection were younger (mean 53.8 years; p = 0.02), had diarrhea and abdominal pain (p = 0.001) but no fever. Sepsis (p = 0.02) and use of a proton pump inhibitor (p = 0.01) were more commonly implicated as the cause of the infection. Of the various types of antibiotics prescribed, carbapenem (28.6 vs 4.1%; p = 0.01) and metronidazole (42.9 vs 19.3%; p = 0.04) were significantly associated with hospital-acquired C. difficile infection. This team reported that patients with Type 2 diabetes mellitus admitted into acute medical

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Recent advances with Clostridium difficile wards and who developed hospital-acquired C. difficile infection have distinct characteristics.

Clinical outcomes Although C. difficile is not often associated directly with mortality it is often a ‘secondary’ condition to another cause of death, especially in those with comorbidities. Recent guidelines have recommended that treatment of CDI be divided based on the presenting signs and symptoms, mild–moderate or moderate–severe, such that metronidazole is the first-line agent in the first group. Venugopal et al. studied the 30-day all-cause mortality in those patients treated appropriately with metronidazole [4]. They retrospectively evaluated 285 patients who were initially treated with metronidazole and stratified them by severity of illness using the guideline criteria. We compared the outcomes in the two groups including the need to change therapy, recurrences, and 30-day all-cause mortality. There were no differences in recurrence rates based on severity of disease. From the multivariate analysis, severe CDI was predictive of 30-day all-cause mortality (odds ratio [OR]: 1.98; 95% CI: 1.07–3.67; p = 0.03), after controlling for intensive care unit (ICU) stay prior to diagnosis (OR: 2.94; 95% CI: 1.60–5.41; p = 0.001), age (OR: 1.02; 95% CI: 1.004–1.05; p = 0.02), and the modified Charlson score (OR: 1.31; 95% CI: 1.14–1.49; p < 0.0001). Thus the severity of presenting disease and use of metronidazole negatively impacts the 30-day all-cause mortality. CDI is significantly associated with subsequent all-cause mortality. Although a number of studies have investigated mortality associated with CDI, few have compared all-cause mortality between ribotypes. Inns et al. estimated all-cause mortality following CDI and to investigate the relationship between mortality, ribotype and other available variables [5]. A retrospective cohort study of all patients with toxin-positive CDI in North East England between July 2009 and June 2011 were matched to death registration data. Differences in all-cause 30-day case fatality were explored using Poisson regression with robust error variances. For survival analysis, an accelerated failure time model with generalized gamma distribution was chosen. A total of 1426 patients were included analysis showed all-cause case fatality was 10.2, 16.4, 25.7 and 38.1% at 7, 14, 30 and 90 days respectively. In a multivariate analysis, ribotype 027 (risk ratio: 1.34; 95% CI: 1.02–1.75) and ribotype 015 (0.46; 0.26–0.82) were significantly associated with higher and lower all-cause 30-day case fatality rates, respectively. In survival analysis, only ribotype 015 had significantly lower predicted mortality (p = 0.008). Patients whose infection was hospital-acquired had significantly higher predicted mortality (p < 0.001).This is the first population-based study of comparative mortality

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Tillotson & Tillotson between multiple ribotypes. The study identified a high rate of all-cause mortality following CDI. The authors found evidence of variability in mortality between ribotypes in this cohort with mortality significantly higher for ribotype 027 at 30 days following diagnosis and significantly lower for ribotype 015. CDI can cause life-threatening complications including shock, sepsis, ileus, megacolon, and colon perforation. Shivashankar et al. [6] created a model to identify clinical factors associated with severe complicated CDI. They analyzed data from 1446 inpatient cases of CDI (48.6% female, median age 62.5 years, range 0.1–103.7 years) over the period June 28, 2007 through June 25, 2010. Patients with severe complicated CDI (n = 487) were identified as those who required admission to the ICU or colectomy, or died, within 30 days of CDI diagnosis. Logistic regression models identified variables that were independently associated with the occurrence of severe complicated CDI in two cohorts. One cohort comprised all hospitalized patients; the other comprised a subset of these in-patients who were residents of Olmsted County (MN, USA). The linear combinations of variables identified using logistic regression models provided scores to predict the risk of developing severe-complicated CDI. In a multivariable model that included all in-patients, increasing age, leukocyte count >15 × 109/l, increase in serum level of creatinine >1.5-fold from baseline, and use of proton pump inhibitors or narcotic medications were independently associated with severe complicated CDI. Older age, high numbers of leukocytes in blood samples, an increased serum level of creatinine, gastric acid suppression, and use of narcotic medications were independently associated with development of severe complicated CDI in hospitalized patients.

Therapeutics Until the middle of 2011, the two mainstay drugs used in the treatment of CDI were vancomycin and metronidazole. Both agents were prescribed frequently in the management of CDI occurring in oncology patients. However these patients are at higher risk of clinical failure and recurrence due to the plethora of comorbid conditions and antimicrobial chemotherapy regimen. Cornely et al. used the Phase III database of fidaxomicin, approved in May 2011 to investigate these patients and their course of CDI when randomized to either vancomycin or fidaxomicin [7]. Two double-blind trials randomly allocated 1105 patients with CDI to fidaxomicin or vancomycin treatment (modified intent-to-treat [mITT]), and 183 of these had cancer. Univariate and multivariate post hoc analyses compared effects of treatment and patient characteristics on cure, recurrence, and sustained response after 4 weeks. Patients with cancer had a lower cure rate and

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Recent advances with Clostridium difficile longer time to resolution of diarrhea than patients without cancer. Recurrence rates were similar. Cure was more likely with fidaxomicin than vancomycin (OR: 2.0; p = 0.065), recurrence was less likely (OR: 0.37; p = 0.018), and sustained response more frequent (OR: 2.56; p = 0.003). In the full mITT population, age, hypoalbuminemia and cancer were inversely associated with clinical cure by multivariate analysis. Study treatment with vancomycin was a significant predictor of recurrence (p < 0.001). Within the cancer population, low albumin was negatively and fidaxomicin was positively associated with improved cure. For patients with cancer, fidaxomicin treatment was superior to vancomycin, resulting in higher cure and sustained response rates, and fewer recurrences. Despite the impressive results shown in oncology patients the cost of fidaxomicin is causing prescribers to carefully consider the overall health economic analysis before more widely using this drug. Bartsch et al. [8] developed a decision analytic simulation model to determine the economic value of fidaxomicin for CDI treatment from the third party payer perspective. The authors looked at CDI treatment in these three cases: no fidaxomicin; only fidaxomicin; and fidaxomicin based on strain typing results. They found that the incremental cost-effectiveness ratio for fidaxomicin based on screening given current conditions was over US$43.7 million/quality-adjusted life-year (QALY) and using only fidaxomicin was dominated (i.e., more costly and less effective) by the other two treatment strategies explored. The fidaxomicin strategy tended to remain dominated, even at lower costs. With approximately 50% of CDI due to the NAP1/BI/027 strain, a course of fidaxomicin would need to cost no more than US$150 to be cost effective in the treatment of all CDI cases and between US$160 and $400 to be cost-effective for those with a nonNAP1/BI/027 strain (i.e., treatment based on strain typing). The authors proposed that given the current cost and NAP1/BI/027 accounting for approximately 50% of isolates, using fidaxomicin as a first-line treatment for CDI is not cost effective. However, typing and treatment with fidaxomicin based on strain may be more promising depending on the costs of fidaxomicin. Probiotics are live organisms, generally bacteria, which are believed bring to balance to the gastrointestinal and other physical niches. There has been much debate as to the efficacy of these living organisms in C. difficile, often in recurrent disease this substantive meta-analysis provides moderate evidence that probiotics may be of some clinical value in patients who are not immunocompromised or seriously ill where perturbation of the bowel may allow ingress of the probiotic organisms that may lead to systemic infection by this strain.

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Tillotson & Tillotson Goldenberg et al. [9] conducted a large meta-analysis in which they evaluated two primary objectives, the efficacy and safety of probiotics for preventing C. difficile-associated diarrhea (CDAD) or CDI in adults and children. Two authors independently and duplicatively extracted data and assessed risk of bias. Any disagreements were resolved by a third adjudicator. For articles published in abstract form only, further information was sought by contacting principal authors. The primary outcome was the incidence of CDAD. Secondary outcomes included the incidence of CDI, adverse events, antibiotic-associated diarrhea (AAD) and length of hospital stay. Sensitivity analyses were conducted to explore the impact of missing data on efficacy and safety outcomes. The overall quality of the evidence supporting each outcome was assessed using the GRADE criteria. A total of 1871 studies were identified with 31 (4492 participants) meeting eligibility requirements for the evaluation. A complete case analysis (i.e., subjects who completed the study) of those trials investigating CDAD (23 trials, 4213 participants) suggests that probiotics significantly reduce this risk by 64%. The incidence of CDAD was 2.0% in the probiotic group compared with 5.5% in the placebo or no treatment control group (relative risk [RR]: 0.36; 95% CI: 0.26–0.51). Sixteen of 23 trials had missing CDAD data ranging from 5 to 45%. These results proved robust to sensitivity analyses of plausible and worst-plausible assumptions regarding missing outcome data and were similar whether considering trials in adults versus children, lower versus higher doses, different probiotic species, or higher versus lower risk of bias. The authors concluded that the overall evidence warrants moderate confidence in this large relative risk reduction. With respect to the incidence of CDI, a secondary outcome, pooled complete case results from 13 trials (961 participants) did not show a statistically significant reduction. The incidence of CDI was 12.6% in the probiotics group compared to 12.7% in the placebo or no treatment control group (RR: 0.89; 95% CI: 0.64–1.24). Adverse events were assessed in 26 studies (3964 participants) and the pooled complete case analysis indicates probiotics reduce the risk of adverse events by 20% (RR: 0.80; 95% CI: 0.68–0.95). In both treatment and control groups the most common adverse events included abdominal cramping, nausea, fever, soft stools, flatulence, and taste disturbance. For the short-term use of probiotics in patients that are not immunocompromised or severely debilitated, the authors consider the strength of this evidence to be moderate.

In vitro & diagnostics The impact of antibiotics on spore germination has been hypothesized to have a major bearing on disease progression and spread. Chilton et al.

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Recent advances with Clostridium difficile evaluated the effects of oritavancin exposure on C. difficile spore germination, outgrowth and recovery [10]. Germination and outgrowth of C. difficile spores exposed to different concentrations of oritavancin, vancomycin, or metronidazole (0.1–10 mg/l) were monitored at 0, 2, 4, 6, 24 and 48 h using phase-contrast microscopy. Recovery of antimicrobial-exposed spores was determined by viable counting on Brazier’s modified CCEYL agar. Persistence of oritavancin activity on spores after washing was determined by measuring activity against a S. aureus lawn. Oritavancin, vancomycin and metronidazole exposure did not prevent germination of phase-bright spores to phase-dark spores, but did inhibit further outgrowth into vegetative cells. The inhibitory effect of oritavancin persisted after washing, whereas the inhibitory effects of vancomycin and metronidazole did not. Oritavancin exposure affected spore recovery; fewer spores were recovered after washing following oritavancin exposure than vancomycin exposure. The extent of this effect was dependent on PCR ribotype, with recovery of ribotype 078 spores completely prevented, but recovery of ribotype 001 spores only slightly affected. Spores exposed to oritavancin, but not vancomycin, retained antimicrobial activity after washing, indicating adherence of oritavancin, but not vancomycin, to the spore surface The authors propose that oritavancin may adhere to spores, potentially causing early inhibition of germinated cells, preventing subsequent vegetative outgrowth and spore recovery, which may prevent some recurrences of symptomatic CDI. Recurrence of CDI ranges from 15–40% depending on the primary antibiotic prescribed however this repeated disease state has not been associated with antibiotic resistance, Wasels et al. have reported on the negative impact of acquisition of a resistance determinant gene in C. difficile [11]. In C. difficile, resistance to the macrolide-lincosamide-streptogramin B group of antibiotics generally relies on erm(B) genes. The authors investigated elements with a genetic organisation different from Tn5398, the mobilizable non-conjugative element identified in C. difficile strain 630. These results suggest that the elements most frequently found in strains isolated during the European Surveillance 2005 were related to Tn6194, the conjugative transposon recently detected in different C. difficile types, including PCRribotype 027. Wasels et al. characterized a Tn6194-like and a novel element rarely found in clinical isolates. A burden on the in vitro fitness of C. difficile was observed after the acquisition of these elements as well as of Tn5398. The Syrian Hamster model is the most predictive animal model of CDI in humans Douce’s group characterized three clinical strains of C. difficile, all differing in toxinotype; CD1342 (PaLoc negative), M68 (toxinotype VIII) and

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Tillotson & Tillotson BI-7 (toxinotype III) [12]. The naturally occurring nontoxic strain colonized all hamsters within 1 day postchallenge with high-levels of spores being shed in the feces of animals that appeared well throughout the entire experiment. C. difficile toxins (TcdA, TcdB and CDT) are absent from the genome. By contrast, hamsters challenged with strain M68 resulted in a 45% mortality rate, with those that survived challenge remaining highly colonized. It is unclear why some hamsters survive infection, as bacterial and toxin levels and histology scores were similar to those culled at a similar time point. Hamsters challenged with strain BI-7 resulted in a rapidly fatal infection in 100% of the hamsters approximately 26 h postchallenge. These data describe the infection kinetics and disease outcomes of three clinical C. difficile isolates differing in toxin carriage and provides additional insights to the role of each toxin in disease progression. Financial & competing interests disclosure GS Tillotson has served as Consultant for Summit PLC, Basilea, Astellas EU, and is an Employee of TranScrip Partners US. J Tillotson is an employee of Aardvark Animal Hospital (PA, USA). The authors have no other relevant affiliations or financial involvement with any organization or entity with a financial interest in or financial conflict with the subject matter or materials discussed in the manuscript apart from those disclosed. No writing assistance was utilized in the production of this manuscript.

References 1

2

Simor AE, Williams V, McGeer A et al. Prevalence of colonization and infection with methicillin-resistant Staphylococcus aureus and vancomycin-resistant enterococcus and of Clostridium difficile infection in Canadian hospitals. Infect. Control. Hosp. Epidemiol. 34(7), 687–693 (2013). Jiang Y, Viner-Brown S, Baier R. Burden of hospital-onset Clostridium difficile infection in patients discharged from Rhode Island hospitals, 2010–2011: application of present on admission indicators. Infect. Control. Hosp. Epidemiol. 34(7), 700–708 (2013).

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4

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Hassan SA, Rahman RA, Huda N, Wan Bebakar WM, Lee YY. Hospital-acquired Clostridium difficile infection among patients with Type 2 diabetes mellitus in acute medical wards. J. R. Coll. Physicians Edinb. 43(2), 103–107 (2013). Venugopal AA, Szpunar S, Sanchez K, Sessions R, Johnson LB. Assessment of 30-day all-cause mortality in metronidazole-treated patients with Clostridium difficile infection. Scand. J. Infect. Dis. (2013) (Epub ahead of print). Inns T, Gorton R, Berrington A et al. Effect of ribotype on all-cause mortality following Clostridium difficile infection.

J. Hosp. Infect. 84(3), 235–241 (2013). 6

Shivashankar R, Khanna S, Kammer PP et al. Clinical factors associated with development of severecomplicated Clostridium difficile infection. Clin. Gastroenterol. Hepatol. doi:10.1016/j. cgh.2013.04.050 (2013) (Epub ahead of print).

7

Cornely OA, Miller MA, Fantin B, Mullane K, Kean Y, Gorbach S. Resolution of Clostridium difficileassociated diarrhea in patients with cancer treated with fidaxomicin or vancomycin. J. Clin. Oncol. 31(19), 2493–2499 (2013).

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Bartsch SM, Umscheid CA, Fishman N, Lee BY. Is fidaxomicin worth the cost? An economic analysis. Clin. Infect. Dis. 57(4), 555–561 (2013).

9

Goldenberg JZ, Ma SS, Saxton JD et al. Probiotics for the prevention of Clostridium difficile-associated diarrhea in adults and children. Cochrane Database Syst. Rev. 5, CD006095 (2013).

10 Chilton CH, Freeman J, Baines

SD, Crowther GS, Nicholson S, Wilcox MH. Evaluation of the effect of oritavancin on Clostridium difficile spore germination, outgrowth and recovery. J. Antimicrob. Chemother. 68(9), 2078–2082 (2013).

11 Wasels F, Spigaglia P,

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Barbanti F, Mastrantonio P. Clostridium difficile

erm(B)-containing elements and the burden on the in vitro fitness. J. Med. Microbiol. 62(Pt 9), 1461–1467 (2013). 12 Buckley AM, Spencer J,

Maclellan LM, Candlish D, Irvine JJ, Douce GR. Susceptibility of hamsters to Clostridium difficile isolates of differing toxinotype. PLoS ONE 8(5), e64121 (2013).

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Index Symbols

16S rRNA sequencing, 52

A

active vaccination, 43, 133 Aitken, Samuel L, 61 algorithm, 88, 89, 90, 92 anaerobic Gram-positive bacteria, 99 antibiotic-associated diarrhea, 30, 59, 131, 138, 146 antimicrobial stewardship, 15, 111, 116, 117, 118, 119 asymptomatic carrier, 112

B

bacterial infection, 29, 140 BI/NAP1, 9, 10, 11, 13, 14, 16, 30, 31, 58, 62, 117 BI/NAP1/027, 9, 10, 11, 13, 14, 16, 30, 31, 58, 62, 117 binary toxin, 9, 23, 30, 42, 135 bleach, 121, 123

C

Chiu, Charles Y, 47 Chopra, Teena, 7 Clostridium difficile-associated disease, 15, 16, 17, 18, 58, 78, 79, 80, 107, 123, 124 Clostridium difficile infection, 5, 6, 7, 8, 15, 16, 17, 18, 21, 22, 30, 31, 33, 34, 44, 45, 47, 48, 54, 55, 57, 58, 59, 61, 62, 68, 72, 78, 79, 95, 96, 98, 100, 104, 105, 106, 107, 108, 112, 113, 118, 119, 123, 124, 127, 128, 134, 148, 150 colectomy, 48, 102, 108, 144 Collery, Mark M, 21 colonization, 9, 25, 27, 30, 31, 32, 36, 38, 39, 42, 79, 93, 99, 100, 104, 112, 117, 123, 132, 138, 141, 148 community-acquired Clostridium difficile infection, 18 community-associated Clostridium difficile, 18, 123 confirmatory test, 87, 88, 90 contact precaution, 12 Curry, Scott, 111 cytotoxin, 17, 36, 41, 83, 84, 90, 92, 129

D

deep sequencing, 51, 52, 53, 56, 59 diarrhea, 14, 15, 16, 17, 22, 23, 30, 38, 39, 40, 45, 48, 49, 56, 58, 59, 62, 78, 79, 85, 88, 91, 96, 105, 106, 107, 116, 123, 124, 131, 132, 135, 137, 138, 142, 145, 146, 148, 149 disinfection, 111, 119, 120, 121, 122, 123, 124

E

environmental contamination, 42, 114, 120

150

F

fecal bacteriotherapy, 49, 58, 79 fecal microbiota transplantation, 58, 105, 130 fecal transplantation, 47, 49, 50, 54, 55, 58, 62, 103, 104 fidamoxicin, 49, 50, 51, 57 flagellar protein, 31, 36

G

Garey, Kevin W, 61 Gilligan, Peter H, 83 GI tract, 48 global trend, 150 glove, 115, 120, 150 glutamate dedhyrogenase, 150 Govind, Revathi, 21 gut, 23, 25, 27, 28, 31, 47, 48, 50, 51, 52, 53, 54, 56, 57, 58, 59, 99, 100, 103, 106, 129, 131, 138, 150 gut commensal, 150 gut homeostasis, 54, 150

H

hand hygiene, 12, 119, 120, 150 healthcare-associated infection, 8, 16, 83, 90, 111, 150 helicase amplification, 87, 150 hospital-acquired infection, 7, 150 host inflammatory response, 43, 150 housekeeping, 121, 122, 150 hypervirulent strain, 8, 11, 13, 14, 15, 150

I

immunochromatographic device, 150 incidence, 7, 8, 9, 10, 11, 12, 13, 14, 15, 43, 48, 62, 114, 123, 127, 131, 146, 150 infection control, 3, 7, 12, 14, 15, 17, 111, 112, 114, 118, 119, 120, 122, 141, 150 infectious diarrhea, 48, 62, 150 infectious inoculum, 150 innate immune response, 36, 43, 150 intravenous immunoglobulin, 41, 105, 135, 150

K

Kelly, Ciarán P, 35 Kuehne, Sarah A, 21

L

loop-mediated isothermal/DNA amplification, 87, 150

M

Martin, Jessica, 95 megacolon, 88, 102, 144, 150 metagenomic, 53, 56, 59, 150

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metronidazole, 41, 44, 49, 50, 51, 66, 67, 91, 95, 97, 98, 99, 101, 102, 103, 105, 106, 107, 129, 135, 142, 143, 144, 147, 148, 150 microbiome, 5, 3, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 107, 137, 150 microbiota, 13, 18, 46, 48, 49, 50, 51, 52, 53, 58, 59, 79, 93, 103, 104, 105, 107, 108, 129, 130, 132, 138, 150 microbiota transplantation, 13, 49, 58, 79, 103, 104, 105, 108, 130, 138, 150 Minton, Nigel P, 21 multicomponent vaccine, 150

N

new strains, 8, 102, 150 next-generation sequencing, 46, 47, 57, 150 nontoxin antigen, 38, 150 North American pulsotype, 150 nucleic acid amplification test, 88, 89, 150

O

opportunistic infection, 150 oral vancomycin, 13, 49, 50, 66, 99, 102, 107, 117, 128, 132, 150 outbreak, 3, 8, 9, 12, 14, 16

P

passive immunotherapy, 35, 40, 41, 43, 150 PCR, 9, 10, 12, 16, 17, 23, 27, 51, 52, 55, 87, 92, 106, 125, 147, 150 peripartum Clostridium difficile infection, 150 Pillai, Dylan R, 47 polymorphism, 16, 52, 53, 55, 150 probiotics, 13, 47, 51, 57, 59, 96, 97, 130, 131, 145, 146, 150 pseudomembranous colitis, 83, 91, 106, 150 pyrosequencing, 52, 53, 55, 150

Q

quantitative real-time PCR, 51

R

recurrence, 7, 13, 28, 35, 40, 41, 47, 48, 49, 50, 57, 59, 61, 62, 70, 71, 75, 80, 89, 95, 98, 100, 101, 102, 103, 105, 107, 108, 128, 129, 133, 135, 142, 143, 144, 145 ribosomal RNA, 9 ribotype, 9, 11, 17, 23, 27, 48, 54, 56, 97, 100, 101, 102, 105, 129, 143, 144, 147, 148

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ribotype 027, 97, 129, 143, 144, 147 rifaximin, 108

S

screening test, 84, 86, 87, 88, 90 Sethi, Saurabh, 35 Shah, Dhara N, 61 short-chain-length fatty acid, 52, 57 skin contamination, 114, 124 spore, 21, 23, 24, 29, 31, 57, 85, 112, 120, 129, 133, 136, 138, 146, 147, 149 Suramaethakul, Nuttanun, 7 surface-exposed adhesin, 36 surface-layer protein, 31 surveillance, 8, 9, 11, 14, 15, 18, 71, 93, 101, 111, 114, 122, 123, 125

T

terminal restriction fragment length, 52, 53 Tillotson, Glenn S, 3, 127, 141 Tillotson, Joni, 141 toxigenic culture, 12, 85, 86, 88, 90 toxin, 9, 11, 12, 16, 17, 21, 22, 23, 27, 28, 29, 30, 31, 32, 33, 35, 36, 37, 38, 39, 40, 42, 43, 44, 45, 58, 59, 67, 70, 78, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 98, 106, 107, 114, 119, 124, 129, 133, 134, 135, 138, 139, 142, 143, 148 toxin A, 36, 37, 39, 40, 42, 44, 45, 83, 84, 86, 87, 88, 91, 92, 142 toxin B, 31, 36, 37, 43, 44, 83, 86, 91, 142 toxoid vaccine, 41, 42, 45, 139 transmission, 14, 15, 23, 24, 28, 29, 32, 94, 111, 112, 116, 120, 121, 122, 123, 124, 125 treatment, 7, 11, 12, 13, 14, 15, 34, 41, 43, 47, 49, 50, 51, 56, 57, 58, 59, 60, 61, 62, 67, 70, 79, 85, 91, 92, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 115, 116, 124, 131, 133, 135, 137, 138, 139, 143, 144, 145, 146

V

vancomycin, 13, 41, 49, 50, 51, 58, 59, 66, 67, 78, 91, 95, 96, 97, 98, 99, 100, 101, 102, 103, 105, 106, 107, 117, 124, 128, 129, 130, 132, 135, 137, 144, 145, 147, 148, 150

W

Weiss, Karl, 3, 127 Wilcox, Mark, 95

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