Tanya Monaghan

Recent advances in Clostridium difficile-associated disease

The main purpose of this article is to review recent developments in the management of acute and recurrent Clostridium difficile-associated disease, with consideration of existing and new antibiotic and non-antibiotic agents for treatment. Details of the current developmental stage of new agents are provided and the role of surgery in the management of severe disease is discussed. Infection control measures considered comprise prudent use of antimicrobials, prevention of cross-infection and surveillance. Other topics that are covered include the recent emergence of an epidemic hypervirulent strain, pathogenesis, clinical presentation and approaches to rapid diagnosis and assessment of the colonic disease.The main purpose of this article is to review recent developments in the management of acute and recurrent Clostridium difficile-associated disease, with consideration of existing and new antibiotic and non-antibiotic agents for treatment. Details of the current developmental stage of new agents are provided and the role of surgery in the management of severe disease is discussed. Infection control measures considered comprise prudent use of antimicrobials, prevention of cross-infection and surveillance. Other topics that are covered include the recent emergence of an epidemic hypervirulent strain, pathogenesis, clinical presentation and approaches to rapid diagnosis and assessment of the colonic disease.

Circulating antibody and memory B-cell responses to C. difficile toxins A and B in patients with C. difficile- associated diarrhoea, inflammatory bowel disease and cystic fibrosis

C. difficile infection (CDI) is rarely reported in cystic fibrosis (CF) patients despite frequent hospitalisations and antibiotic usage. Conversely, the prevalence of CDI in inflammatory bowel disease (IBD) has received increased attention. We investigated components of the IgG-specific humoral immune response to C. difficile toxins A and B in patients with C. difficile-associated diarrhoea (CDAD), IBD patients with CDI, CF patients and healthy controls. Serum anti-toxin IgG was determined by ELISA. Circulating antigen-activated B-cells were investigated using Alexa Fluor 488-labelled toxin A and assessed by flow cytometry. Following induction of differentiation of memory B-cells, toxin A- and B-specific antibody secreting cells (ASCs) were quantified using ELISpot. We present the first data showing levels of serum anti-toxin A and B antibodies were significantly higher in patients with CF (without a history of CDI) than in CDAD patients and were stably maintained over time. Notably, the CDAD patients were significantly older than the CF patients. We also show that circulating toxin A-specific memory B-cells (IgD-negative) can be detected in CDAD patients [0.92 (0.09–1.78)%], and were prominent (5.64%, 1.14%) in two CF patients who were asymptomatic carriers of C. difficile. There was correlation between toxin A- and B-specific ASCs, with significantly higher proportions of the latter seen. In some with CDAD, high serum antibody levels were seen to only one of the two toxins. Mucosal secretion of toxin-specific IgG was detected in an additional group of IBD patients with no history of CDI. We conclude that enhanced and stable humoral immune responses to toxins A and B may protect CF and some IBD patients against CDI. The impaired ability to generate strong and/or sustained toxin-specific antibody and memory B-cell responses may increase susceptibility of older patients to CDI and highlight the need to investigate the role of immune senescence in future studies.

Oxford Handbook of Clinical Examination and Practical Skills

The Oxford Handbook of Clinical Examination and Practical Skills is the first truly comprehensive pocket guide to clinical examination and practical skills for medical students and junior doctors. Providing clear and user-friendly guidance on all aspects of history taking, physical examination, common practical procedures, data interpretation and communication skills, it gives realistic advice on coping with common situations. In line with current teaching methods, the book takes a systems-based approach to medicine.

Pathogenesis of Clostridium difficile Infection and Its Potential Role in Inflammatory Bowel Disease

Abstract:Colonization with toxigenic Clostridium difficile may be associated with a wide spectrum of clinical presentation ranging from asymptomatic carriage to mild diarrhea to life-threatening colitis. Over the last 15 years, there has been a marked increase in the incidence of C. difficile infection, which predominantly affects elderly patients on antibiotics. More recently, there has been significant interest in the association between inflammatory bowel disease (IBD) and C. difficile infection. This review article discusses in some detail current knowledge of the mechanisms by which C. difficile toxins may mediate mucosal inflammation, together with the role of cell wall components of the microorganism in disease pathogenesis. Innate and adaptive host responses to C. difficile toxins and other components are described and include consideration of the potential role of known mucosal changes in IBD that may lead to an enhanced inflammatory response in the presence of C. difficile infection. Recent studies, which have characterized resident microbiota that may mediate protection against colonization by C. difficile, including their mechanisms of action, are also discussed. This includes the role of bile acids and 7&agr;-dehydroxylase-expressing bacteria, such as Clostridium scindens. Recent studies suggest a higher carriage rate of C. difficile in patients with IBD. It is anticipated that future studies will determine the role of dysbiosis in IBD in predisposing to colonization with C. difficile.

Differential binding and internalization of Clostridium difficile toxin A by human peripheral blood monocytes, neutrophils and lymphocytes.

Colitis due to Clostridium difficile infection is mediated by secreted toxins A and B and is characterized by infiltration by cells from the systemic circulation. The aim of our study was to investigate interactions between fluorescently labelled toxin A and peripheral blood monocytes, neutrophils and lymphocytes. Purified toxin A was labelled with Alexa Fluor® 488 (toxin A488) and incubated with isolated human peripheral blood mononuclear cells or washed whole blood cells for varying time intervals at either 37 or 4 °C/ice. The ability of trypan blue to quench cell surface–associated (but not cytoplasmic) fluorescence was also investigated. At 37 °C, toxin A488‐associated fluorescence in monocytes peaked at 1 h (majority internalized), with subsequent loss associated with cell death. In contrast to monocytes, binding of toxin A488 in neutrophils was greater on ice than at 37 °C. Studies using trypan blue suggested that over 3 h at 37 °C, most of the toxin A488‐associated fluorescence in neutrophils remained at the cell surface. Over 48 h (37 °C and ice/4 °C), there was minimal toxin A488‐associated fluorescence in lymphocytes. These studies suggest major differences in interactions between toxin A and circulating cells that infiltrate the mucosa during colonic inflammation in C. difficile infection.

New Perspectives in Clostridium difficile Disease Pathogenesis

Clostridium difficile is associated with a spectrum of clinical manifestations ranging from asymptomatic carriage to severe life-threatening pseudomembranous colitis. Current perspectives indicate that C difficile pathogenesis is a multifactorial disease process dictated by pathogenic toxin production, gut microbial dysbiosis, and altered host inflammatory responses. This article summarizes recent findings underpinning the cellular and molecular mechanisms regulating bacterial virulence and sheds new light on the critical roles of the host immune response, intestinal microbiota, and metabolome in mediating disease pathogenesis.

Potential of lactoferrin to prevent antibiotic-induced Clostridium difficile infection

Objectives Clostridium difficile infection (CDI) is a global healthcare problem. Recent evidence suggests that the availability of iron may be important for C. difficile growth. This study evaluated the comparative effects of iron-depleted (1% Fe3+ saturated) bovine apo-lactoferrin (apo-bLf) and iron-saturated (85% Fe3+ saturated) bovine holo-lactoferrin (holo-bLf) in a human in vitro gut model that simulates CDI. Methods Two parallel triple-stage chemostat gut models were inoculated with pooled human faeces and spiked with C. difficile spores (strain 027 210, PCR ribotype 027). Holo- or apo-bLf was instilled (5 mg/mL, once daily) for 35 days. After 7 days, clindamycin was instilled (33.9 mg/L, four times daily) to induce simulated CDI. Indigenous microflora populations, C. difficile total counts and spores, cytotoxin titres, short chain fatty acid concentrations, biometal concentrations, lactoferrin concentration and iron content of lactoferrin were monitored daily. Results In the apo-bLf model, germination of C. difficile spores occurred 6 days post instillation of clindamycin, followed by rapid vegetative cell proliferation and detectable toxin production. By contrast, in the holo-bLf model, only a modest vegetative cell population was observed until 16 days post antibiotic administration. Notably, no toxin was detected in this model. In separate batch culture experiments, holo-bLf prevented C. difficile vegetative cell growth and toxin production, whereas apo-bLf and iron alone did not. Conclusions Holo-bLf, but not apo-bLf, delayed C. difficile growth and prevented toxin production in a human gut model of CDI. This inhibitory effect may be iron independent. These observations suggest that bLf in its iron-saturated state could be used as a novel preventative or treatment strategy for CDI.

Protective antibodies against Clostridium difficile are present in intravenous immunoglobulin and are retained in humans following its administration

The prevalence of serum antibodies against Clostridium difficile (CD) toxins A and B in healthy populations have prompted interest in evaluating the therapeutic activity of intravenous immunoglobulin (IVIg) in individuals experiencing severe or recurrent C. difficile infection (CDI). Despite some promising case reports, a definitive clinical role for IVIg in CDI remains unclear. Contradictory results may be attributed to a lack of consensus regarding optimal dose, timing of administration and patient selection as well as variability in specific antibody content between commercial preparations. The purpose of this study was to investigate retrospectively the efficacy of three commercial preparations of IVIg for treating severe or recurrent CDI. In subsequent mechanistic studies using protein microarray and toxin neutralization assays, all IVIg preparations were analysed for specific binding and neutralizing antibodies (NAb) to CD antigens in vitro and the presence of anti‐toxin NAbs in vivo following IVIg infusion. A therapeutic response to IVIg was observed in 41% (10 of 17) of the CDI patients. Significant variability in multi‐isotype specific antibodies to a 7‐plex panel of CD antigens and toxin neutralization efficacies were observed between IVIg preparations and also in patient sera before and after IVIg administration. These results extend our current understanding of population immunity to CD and support the inclusion of surface layer proteins and binary toxin antigens in CD vaccines. Future strategies could enhance IVIg treatment response rates by using protein microarray to preselect donor plasma/serum with the highest levels of anti‐CD antibodies and/or anti‐toxin neutralizing capacities prior to fractionation.

Profiling Humoral Immune Responses to Clostridium difficile-Specific Antigens by Protein Microarray Analysis.

ABSTRACT Clostridium difficile is an anaerobic, Gram-positive, and spore-forming bacterium that is the leading worldwide infective cause of hospital-acquired and antibiotic-associated diarrhea. Several studies have reported associations between humoral immunity and the clinical course of C. difficile infection (CDI). Host humoral immune responses are determined using conventional enzyme-linked immunosorbent assay (ELISA) techniques. Herein, we report the first use of a novel protein microarray assay to determine systemic IgG antibody responses against a panel of highly purified C. difficile-specific antigens, including native toxins A and B (TcdA and TcdB, respectively), recombinant fragments of toxins A and B (TxA4 and TxB4, respectively), ribotype-specific surface layer proteins (SLPs; 001, 002, 027), and control proteins (tetanus toxoid and Candida albicans). Microarrays were probed with sera from a total of 327 individuals with CDI, cystic fibrosis without diarrhea, and healthy controls. For all antigens, precision profiles demonstrated <10% coefficient of variation (CV). Significant correlation was observed between microarray and ELISA in the quantification of antitoxin A and antitoxin B IgG. These results indicate that microarray is a suitable assay for defining humoral immune responses to C. difficile protein antigens and may have potential advantages in throughput, convenience, and cost.

Varicella pneumonia in an immunocompromised inflammatory bowel disease patient.

To the Editor: We present the case of a 65year-old man who developed lifethreatening varicella zoster pneumonia after starting azathioprine for Crohn’s disease (CD). Initial diagnosis of distal ileal CD was made 7 months previously on established clinical and radiological criteria. The patient’s disease was steroid-dependent and he was begun on azathioprine 25 mg 3 times each day 1 week prior to presentation. Other medications included prednisolone 35 mg once daily and Pentasa 2 g twice daily. On presentation the patient reported a 4-day history of fever, headache, shortness of breath, nausea, vomiting, and abdominal discomfort after most meals. On examination he was pyrexial, tachycardic, hypotensive, and tachypnoeic. There was evidence of a pruritic vesiculopustular rash on his forehead and trunk. All other systems examinations were unremarkable. Laboratory investigations showed a normochromic normocytic anemia, mild renal impairment, deranged liver enzymes, and an elevated C-reactive protein of 306 mg/L. Admission chest and abdominal radiographs were both normal. Arterial blood gas analysis revealed a respiratory alkalosis and elevated lactate. The provisional clinical diagnosis was varicella zoster sepsis likely secondary to azathioprineinduced immunosuppression. Azathioprine therapy was immediately discontinued. The patient was transferred to the intensive care unit where he required inotropic support for septic shock. Intravenous broad spectrum antibiotics, antifungals, and antivirals (acyclovir, at a dose of 10 mg/ kg every 8 hours) were also started, along with activated protein C for the systemic inflammatory response syndrome and hydrocortisone. Blood cultures were negative and an echocardiogram was reported as normal. Serum anti-varicella IgG antibodies were detected on day 3 of admission. His condition further deteriorated as a result of respiratory embarrassment secondary to varicella pneumonia and he required intubation and invasive ventilation. The patient’s condition improved with aggressive supportive management and he was stepped down to a medical ward on day 8 of admission. In this case the diagnosis of varicella was based on the presence of a rash with a typical distribution affecting mainly the trunk and face and the detection of varicella antibodies in the serum. Azathioprine-induced varicella infection was presumed to be a probable reactivation, since the patient had reported experiencing chickenpox as a child. Fulminant infection caused by varicella zoster has rarely been reported in patients with CD undergoing treatment with azathioprine and 6-mercaptopurine and the true incidence in this group is not known. Chickenpox, or varicella, is a well-recognized systemic infection caused by the herpes virus, varicella zoster (VZV). It is commonly perceived to be a mild, self-limiting infection of childhood. However, in adults, pregnant women, and immunocompromised hosts varicella infection is associated with significant morbidity and mortality. Varicella pneumonia is the most frequent complication in adults and often develops insidiously, usually a few days after the onset of the rash, and can progress to respiratory failure and acute respiratory distress syndrome (ARDS). Mortality is 10%–30% in the general population, 40% in pregnancy, and greater than 50% in immunocompromised adults. Smoking and preexisting lung disease are 2 other well-known risk factors linked to the development of varicella pneumonia in adults. Other complications include encephalitis, hepatitis, and secondary skin and soft tissue infections. The incubation period is 10–21 days for a primary infection. Infectivity is at its highest 2 days prior to onset of the rash, until all vesicles become crusted. Immunity in contacts can be assumed if there is a clinical history of chickenpox or shingles in the past. If varicella infection or reactivation occurs, prompt diagnosis and treatment with antiviral therapy and discontinuation of azathioprine or other immunosuppressive therapy should be initiated without delay to limit viremia and avoid potentially fatal complications. This case highlights the importance of having a high index of clinical suspicion of varicella infection in the context of a patient with inflammatory bowel disease (IBD) and a new rash, particularly in those who are receiving concomitant immunosuppressives. In patients without a clear history of varicella infection and in those testing negative for the presence of antibodies to VZV, consideration should be given to administering varicella vaccine before starting treatment with immunosuppressive agents. The best time for immunization may be at initial diagnosis of IBD and attention to this should perhaps become a routine part of the initial workup.