Humoral response to Clostridium difficile in inflammatory bowel disease, including correlation with immunomodulatory treatment

Background and Aim: An abnormal immune response to intestinal bacteria has been observed in Crohn’s disease (CD). Clostridium difficile infection incidence and severity are increased in CD, but reports on the humoral response have provided conflicting results. We aimed to shed light on the possible role of C. difficile in CD pathogenesis by paying attention to the influence of immunomodulatory treatment on the humoral response. Methods: A total of 71 consecutive outpatients with CD, 67 with ulcerative colitis (UC), and 121 healthy controls were analyzed for serum IgA and IgG to C. difficile toxins A and B. Results: IgA levels were similar in all study groups. IgG to toxin A was increased similarly in CD and UC (P = 0.02 for both). In contrast, IgG to toxin B was elevated only in CD patients not receiving disease-modifying anti-inflammatory bowel disease drugs (DMAID) (n = 16) (P = 0.0001), while the CD medication subgroup (n = 47) had a level similar to healthy controls. The UC results were not influenced by DMAID treatment. Conclusion: Our findings add support to the idea of a disturbed interaction between intestinal cells and the microbiota being part of the CD disease mechanism. An abnormal immune response to C. difficile toxin B may be a critical component of this interaction.publishedVersio

Lymphocyte and monocyte flow cytometry immunophenotyping as a diagnostic tool in uncharacteristic inflammatory disorders

<p>Abstract</p> <p>Background</p> <p>Patients with uncharacteristic inflammatory symptoms such as long-standing fatigue or pain, or a prolonged fever, constitute a diagnostic and therapeutic challenge. The aim of the present study was to determine if an extended immunophenotyping of lymphocytes and monocytes including activation markers can define disease-specific patterns, and thus provide valuable diagnostic information for these patients.</p> <p>Methods</p> <p>Whole blood from patients with gram-negative bacteraemia, neuroborreliosis, tuberculosis, acute mononucleosis, influenza or a mixed connective tissue disorders, as diagnosed by routine culture and serology techniques was analysed for lymphocyte and monocyte cell surface markers using a no-wash, no-lyse protocol for multi-colour flow cytometry method. The immunophenotyping included the activation markers HLA-DR and CD40. Plasma levels of soluble TNF alpha receptors were analysed by ELISA.</p> <p>Results</p> <p>An informative pattern was obtained by combining two of the analysed parameters: (i), the fractions of HLA-DR-expressing CD4+ T cells and CD8+ T cells, respectively, and (ii), the level of CD40 on CD14+ CD16- monocytes. Patients infected with gram-negative bacteria or EBV showed a marked increase in monocyte CD40, while this effect was less pronounced for tuberculosis, borrelia and influenza. The bacterial agents could be distinguished from the viral agents by the T cell result; CD4+ T cells reacting in bacterial infection, and the CD8+ T cells dominating for the viruses. Patients with mixed connective tissue disorders also showed increased activation, but with similar engagement of CD4+ and CD8+ T cells. Analysis of soluble TNF alpha receptors was less informative due to a large inter-individual variation.</p> <p>Conclusion</p> <p>Immunophenotyping including the combination of the fractions of HLA-DR expressing T cell subpopulations with the level of CD40 on monocytes produces an informative pattern, differentiating between infections of bacterial and viral origin. Furthermore, a quantitative analysis of these parameters revealed the novel finding of characteristic patterns indicating a subacute bacterial infection, such as borreliosis or tuberculosis, or a mixed connective tissue disorder. The employed flow cytometric method is suitable for clinical diagnostic laboratories, and may help in the assessment of patients with uncharacteristic inflammatory symptoms.</p

Clostridium difficile-associated diarrhoea - aspects of hospital epidemiology and treatment

Clostridium difficile is a spore-forming bacterium that causes antibiotic-associated diarrhoea and is responsible for hospital outbreaks of diarrhoea. In the first study, 173 consecutive isolates of C. difficile collected from 147 patients at Malmö University Hospital during 1995 were typed by AP-PCR to determine whether cross-transmission could explain the increased incidence of Clostridium difficile-associated diarrhoea (CDAD) at our hospital. Overall DNA analysis of the combined AP-PCR data yielded 140 types, of which 130 were unique types, whereas 10 common types occurred repeatedly in 33 patients. In eight of these 33 patients epidemiological data confirmed a high probability of nosocomial transmission. Thus, our study suggested a low frequency of nosocomial transmission of CDAD during the study period. In a follow-up study the same C. difficile isolates were examined by PCR-ribotyping, and results were compared with those generated by AP-PCR. PCR-ribotyping demonstrated a somewhat higher rate of nosocomial transmission of C. difficile than shown by AP-PCR. Hospital environments are considered to be one of the most important reservoirs of C. difficile spores. C. difficile spores are resistant to most commonly used disinfecting chemicals. Spores of four strains of C. difficile were tested against isopropanol 70%, glutaraldehyde 2%, peracetyl ions 1.6% and acidified nitrite 0.1M using a dilution-neutralisation method. Peracetyl ions and acidified nitrite showed highly sporicidal activity independently of organic load conditions. Both agents appear to be suitable for the inactivation of C. difficile spores in hospital environments. There are currently few options for the treatment of CDAD. In an investigator-initiated, randomised controlled, double-blind trial 114 patients with an initial episode of CDAD received either fusidic acid or metronidazole. Of the patients in the fusidic acid group 83% were clinically cured in comparison to 93% in the metronidazole group (P=0.116). Bacteriological cure did not differ between the two groups. Clinical and bacteriological recurrences were noted in 27% and 13% respectively of the patients receiving fusidic acid and in 29% and 10% of those given metronidazole. The results indicate that fusidic acid seems to be equally effective to metronidazole in curing an initial episode of CDAD. It is poorly understood why some individuals in particular suffer multiple recurrences of CDAD. The disturbed colonic microflora in these patients is considered as a main cause for recurrent episodes. Biotherapy aims to restore the commensal gut flora and hence prevent colonisation of C. difficile. A double-blind, placebo-controlled trial was initiated by the author to analyse the ability of Lactobacillus plantarum 299v to prevent further recurrent episodes of CDAD. Recurrence of clinical symptoms was seen in four of eleven patients who received metronidazole in combination with L. plantarum 299v and in six of nine treated with metronidazole in combination with placebo. Although the small sample size does not allow any conclusion to be drawn concerning the efficacy of L. plantarum in patients with RCDAD, the results of this trial may contribute to the ongoing discussion about the benefits of probiotics in patients with RCDAD and encourage the performance of larger multicenter studies

Pharmacodynamic studies of vancomycin, metronidazole and fusidic acid against Clostridium difficile

Background: Pharmacodynamic studies of antibiotics have attracted great interest in recent years. However, studies on the pharmacodynamics of different antibiotics against Clostridium difficile are scarce. Methods: The postantibiotic effects (PAE) and the postantibiotic sub-minimum inhibitory concentration (MIC) effects (PA SME) of vancomycin, metronidazole and fusidic acid were investigated by viable counts against three different strains of C. difficile. The killing rate and extent of the three antibiotics against the same strains were also studied by adding 2, 4, 8, 16 and 32 ! MIC of the three antibiotics, respectively. Results: Metronidazole exerted a very rapid bactericidal effect at concentrations of 8 ! MIC and above against all three strains investigated. Vancomycin gave overall less kill in comparison to metronidazole and was bacteriostatic against two of the three strains. Fusidic acid exerted a concentration-dependent killing against two of the strains. Vancomycin exerted short PAEs and PA SMEs against all three strains. Significantly longer PAEs and PA SMEs were noted for fusidic acid. Metronidazole gave similar short PAEs like vancomycin but longer PA SMEs were noted against two of the investigated strains. Conclusion: Metronidazole exerted the most prominent bactericidal effect greater than fusidic acid and greater than vancomycin. Fusidic acid gave overall the longest PAEs and PA SMEs greater than metronidazole and greater than vancomycin. Copyright (C) 2007 S. Karger AG, Basel

IgG Antibody Response to Toxins A and B in Patients with Clostridium difficile Infection.

IgG antibodies against Clostridium difficile toxins A and B were followed in controls and patients with an initial C. difficile infection (CDI). Of the 50 CDI patients, 38 were cured and 12 developed recurrence. Compared to controls, patients had significantly lower anti-toxin A and B IgGs at inclusion, but the subsequent levels rose slightly regardless of clinical outcome. The results imply that the general serum reactivity against toxins A and B in the population reduces the risk of CDI, which suggests implications for vaccine strategies