Bezlotoxumab for prevention of Clostridium difficile infection recurrence: Distinguishing relapse from reinfection with whole genome sequencing.

BACKGROUND: Bezlotoxumab has been shown to prevent Clostridium difficile infection recurrence (rCDI) in high-risk patients. METHODS: We used whole genome sequencing to estimate the impact of bezlotoxumab on same-strain relapse or new-strain reinfection in MODIFY I/II trials. Reinfection with a new strain and relapse with the same strain were differentiated by the comparison of ribotype (RT) and pair-wise single-nucleotide whole genome sequencing (WGS) variations (PWSNV). Relapse was assigned if the baseline RT and the RT isolated during rCDI were the same, and if PWSNVs were ≤ 2. Reinfection was assigned if the baseline RT and the RT isolated during rCDI were different, or if the RT was the same but PWSNVs were > 10. Unknown status was assigned if the RT was the same but PWSNVs were 3-10. RESULTS: 259 rCDI events were evaluable (50 [19.3%] reinfection; 198 [76.4%] relapse). The proportion of relapses was higher for ribotype 027 (84.5%) compared with other ribotypes (74.1%). Cumulative incidence of relapse was significantly lower for bezlotoxumab versus no bezlotoxumab (p < 0.0001), with a non-significant trend towards reduction for reinfection (p = 0.14). CONCLUSION: Bezlotoxumab treatment significantly reduced the rate of CDI relapse versus a regimen without bezlotoxumab. (NCT01241552/NCT01513239).

Effect of Endogenous Clostridioides difficile Toxin Antibodies on Recurrence of C. difficile Infection.

BACKGROUND: Endogenous antibodies (eAbs) against Clostridioides (Clostridium) difficile toxins may protect against recurrence of C. difficile infection (rCDI). This hypothesis was tested using placebo group data from MODIFY (Monoclonal Antibodies for C. difficile Therapy) I and II (NCT01241552 and NCT01513239, respectively), global, randomized phase 3 trials that assessed the efficacy and safety of the antitoxin monoclonal antibodies bezlotoxumab and actoxumab in participants receiving antibiotic therapy for CDI. METHODS: A placebo infusion (normal saline) was administered on study day 1. Serum samples were collected on day 1, week 4, and week 12, and eAb-A and eAb-B titers were measured by 2 validated electrochemiluminescence immunoassays. Rates of initial clinical cure and rCDI were summarized by eAb titer category (low, medium, high) at each time point. RESULTS: Serum eAb titers were available from a total of 773 participants. The proportion of participants with high eAb-A and eAb-B titers increased over time. Rates of initial clinical cure were similar across eAb titer categories. There was no correlation between eAb-A titers and rCDI rate at any time point. However, there was a negative correlation between rCDI and eAb-B titer on day 1 and week 4. rCDI occurred in 22% of participants with high eAb-B titers at baseline compared with 35% with low or medium titers (P = .015). CONCLUSIONS: Higher eAb titers against toxin B, but not toxin A, were associated with protection against rCDI. These data are consistent with the observed efficacy of bezlotoxumab, and lack of efficacy of actoxumab, in the MODIFY trials. CLINICAL TRIALS REGISTRATION: NCT01241552 and NCT01513239.

Generation of Markerless Deletions in the Nosocomial Pathogen Clostridium difficile by Induction of DNA Double-Strand Breaks.

Clostridium difficile is an important nosocomial pathogen associated with potentially fatal disease induced by the use of antibiotics. Genetic characterization of such clinically important bacteria is often hampered by lack of availability of suitable tools. Here, we describe the use of I-SceI to induce DNA double-strand breaks, which increase the frequency of allelic exchange and enable the generation of markerless deletions in C. difficile The usefulness of the system is illustrated by the deletion of genes encoding putative AddAB homologues. The ΔaddAB mutants are sensitive to ultraviolet light and the antibiotic metronidazole, indicating a role in homologous recombination and the repair of DNA breaks. Despite the impairment in recombination, the mutants are still proficient for induction of the SOS response. In addition, deletion of the fliC gene, and subsequent complementation, reveals the importance of potential regulatory elements required for expression of a downstream gene encoding the flagellin glycosyltransferase.IMPORTANCE Most sequenced bacterial genomes contain genes encoding proteins of unknown or hypothetical function. To identify a phenotype for mutations in such genes, deletion is the preferred method for mutagenesis because it reduces the likelihood of polar effects, although it does not eliminate the possibility. Allelic exchange to produce deletions is dependent on the length of homologous regions used to generate merodiploids. Shorter regions of homology resolve at lower frequencies. The work presented here demonstrates the utility of inducing DNA double-strand breaks to increase the frequency of merodiploid resolution in Clostridium difficile Using this approach, we reveal the roles of two genes, encoding homologues of AddAB, in survival following DNA damage. The method is readily applicable to the production of deletions in C. difficile and expands the toolbox available for genetic analysis of this important anaerobic pathogen.

Bezlotoxumab for Prevention of Recurrent Clostridium difficile Infection.

BACKGROUND: Clostridium difficile is the most common cause of infectious diarrhea in hospitalized patients. Recurrences are common after antibiotic therapy. Actoxumab and bezlotoxumab are human monoclonal antibodies against C. difficile toxins A and B, respectively. METHODS: We conducted two double-blind, randomized, placebo-controlled, phase 3 trials, MODIFY I and MODIFY II, involving 2655 adults receiving oral standard-of-care antibiotics for primary or recurrent C. difficile infection. Participants received an infusion of bezlotoxumab (10 mg per kilogram of body weight), actoxumab plus bezlotoxumab (10 mg per kilogram each), or placebo; actoxumab alone (10 mg per kilogram) was given in MODIFY I but discontinued after a planned interim analysis. The primary end point was recurrent infection (new episode after initial clinical cure) within 12 weeks after infusion in the modified intention-to-treat population. RESULTS: In both trials, the rate of recurrent C. difficile infection was significantly lower with bezlotoxumab alone than with placebo (MODIFY I: 17% [67 of 386] vs. 28% [109 of 395]; adjusted difference, -10.1 percentage points; 95% confidence interval [CI], -15.9 to -4.3; P<0.001; MODIFY II: 16% [62 of 395] vs. 26% [97 of 378]; adjusted difference, -9.9 percentage points; 95% CI, -15.5 to -4.3; P<0.001) and was significantly lower with actoxumab plus bezlotoxumab than with placebo (MODIFY I: 16% [61 of 383] vs. 28% [109 of 395]; adjusted difference, -11.6 percentage points; 95% CI, -17.4 to -5.9; P<0.001; MODIFY II: 15% [58 of 390] vs. 26% [97 of 378]; adjusted difference, -10.7 percentage points; 95% CI, -16.4 to -5.1; P<0.001). In prespecified subgroup analyses (combined data set), rates of recurrent infection were lower in both groups that received bezlotoxumab than in the placebo group in subpopulations at high risk for recurrent infection or for an adverse outcome. The rates of initial clinical cure were 80% with bezlotoxumab alone, 73% with actoxumab plus bezlotoxumab, and 80% with placebo; the rates of sustained cure (initial clinical cure without recurrent infection in 12 weeks) were 64%, 58%, and 54%, respectively. The rates of adverse events were similar among these groups; the most common events were diarrhea and nausea. CONCLUSIONS: Among participants receiving antibiotic treatment for primary or recurrent C. difficile infection, bezlotoxumab was associated with a substantially lower rate of recurrent infection than placebo and had a safety profile similar to that of placebo. The addition of actoxumab did not improve efficacy. (Funded by Merck; MODIFY I and MODIFY II ClinicalTrials.gov numbers, NCT01241552 and NCT01513239 .).

International Clostridium difficile animal strain collection and large diversity of animal associated strains.

BACKGROUND: Clostridium difficile is an important cause of intestinal infections in some animal species and animals might be a reservoir for community associated human infections. Here we describe a collection of animal associated C. difficile strains from 12 countries based on inclusion criteria of one strain (PCR ribotype) per animal species per laboratory. RESULTS: Altogether 112 isolates were collected and distributed into 38 PCR ribotypes with agarose based approach and 50 PCR ribotypes with sequencer based approach. Four PCR ribotypes were most prevalent in terms of number of isolates as well as in terms of number of different host species: 078 (14.3% of isolates; 4 hosts), 014/020 (11.6%; 8 hosts); 002 (5.4%; 4 hosts) and 012 (5.4%; 5 hosts). Two animal hosts were best represented; cattle with 31 isolates (20 PCR ribotypes; 7 countries) and pigs with 31 isolates (16 PCR ribotypes; 10 countries). CONCLUSIONS: This results show that although PCR ribotype 078 is often reported as the major animal C. difficile type, especially in pigs, the variability of strains in pigs and other animal hosts is substantial. Most common human PCR ribotypes (014/020 and 002) are also among most prevalent animal associated C. difficile strains worldwide. The widespread dissemination of toxigenic C. difficile and the considerable overlap in strain distribution between species furthers concerns about interspecies, including zoonotic, transmission of this critically important pathogen.

A prospective study of community-associated Clostridium difficile infections: the role of antibiotics and co-infections.

OBJECTIVES: This prospective study was performed to determine the incidence, risk factors, severity and outcomes of community-associated Clostridium difficile infection (CA-CDI) in the SE of Scotland. METHODS: All patients (335) diagnosed with laboratory confirmed CDI in the city of Edinburgh, East Lothian and Midlothian regions of Scotland between August 2010 and July 2011 were followed up for one year after diagnosis. Clinical details and laboratory markers were recorded. Stool samples were tested for C. difficile, other bacterial pathogens and norovirus. Molecular epidemiology of C. difficile isolates was studied by PCR-ribotyping. RESULTS: Of the total 335 confirmed CDI cases, PCR-ribotype 001 was the commonest (14.1%), followed by PCR-ribotypes 078 (12.9%) and 015 (11.7%), respectively. CA-CDI represented 12.5% of the cases. In these, PCR-ribotype 078 was the commonest (19.0%), followed by PCR-ribotypes 014/020 (16.7%), PCR-ribotype 015 (14.3% and PCR-ribotype 001 (11.9%). A lower Charlson co-morbidity index and a lower age was observed in the CA-CDI group as was total number of different antibiotic classes whereas age >75 was more common in the HA-CDI group. On multivariable analysis presence of PCR-ribotype 078 was significantly associated with community acquisition (p = 0.006) whereas a greater proportion of immunosuppressed patients and those on antibiotics 8 weeks preceding diagnosis (p = 0.035 and p = 0.005 respectively) were found among HA-CDI cases. Charlson co-morbidity index, number of different antibiotics given in the eight weeks preceding onset, severity of infection and rural residence were not significantly different between the two groups. CONCLUSION: This study demonstrates that patients with CA-CDI may also present with severe infection, are less likely to receive antibiotics prior to CDI, more likely to be younger in age and have a greater proportion of PCR-ribotype 078 compared with CDI acquired in a hospital setting. Hence a high level of vigilance must be maintained to detect CDI cases which present in the community without the traditional predisposing factors.

Clostridium difficile infections in South East Scotland: mortality and recurrence in a region without PCR ribotype 027.

Three hundred and thirty-five patients with laboratory-confirmed Clostridium difficile infections (CDIs) were studied for epidemiological features, clinical presentation and laboratory markers. They were followed up for 1 year to determine recurrence and mortality. Four hundred and thirty-two episodes were recorded. One year mortality was 41.8 % of which CDI was listed on 20 % of the death certificates. One year recurrence rate was 22.9 %. PCR ribotype 001 was the commonest epidemiological type and ribotype 027 was not detected. High total leucocyte count and low albumin were significantly associated with mortality, as was the absence of a GI-invasive procedure in the 12 weeks preceding CDI diagnosis, probably due to patients being unfit for the procedure. No association with acid suppressants, deletion in the tdcC anti-sigma factor or vancomycin-resistant enterococcus/methicillin-resistant Staphylococcus aureus co-infection was detected. One year mortality was higher in patients who developed recurrent infections (P<0.001). Differences in ribotype were observed in 2.3 %, 11.11 %, 20 % and 32.4 % isolates with time intervals between sampling of 0-20, 21-40, 41-60 and >60 days, respectively, suggesting that the arbitrary cut-off of 28 days to call a repeat infection a reinfection may not be correct in some cases.

The changing faces of Clostridium difficile: a personal reflection of the past 34 years.

Late in 1978 my boss gave me a folder with "Clostridium difficile (diffikilé)" written on it. Inside were a few recent and now classic papers by Bartlett, Larson and co. It was suggested that this might be an interesting research topic. So began a continuing adventure which has resulted in at least 50 publications from my group. Over the years we have made several important contributions to the field. Beginning in 1982 we showed that C. difficile was a common cause of community-acquired infection! During the next few years we did extensive structural studies on the bacterium. This culminated in 1984 with a fingerprinting study (by immunoblotting surface antigens), on Swedish strains supplied by Carl-Erik Nord, which was probably the first study to demonstrate that C. difficile was really an infectious agent. This was later reinforced with strains sent from Amsterdam by Ed Kuijper. Later in the 1980s, in a study of recurrent disease, we showed that ca. 50% of recurrences were due to infection with a different strain. During my term as chair of the European Study Group for C. difficile, we began to define the status of C. difficile infection (CDI) in Europe and develop guidance for diagnosis and treatment. Recently we utilised our extensive culture collection, with isolates from the 1970s to the present, to observe how epidemiology has been driven largely by antibiotic usage. We have now come full circle: in the early years C. difficile infection was caused by many different strains. Then in the period beginning in the 1990s, characterised by often-large outbreaks and poor infection control, disease was caused by a few endemic strains highlighted by the 027/NAP1/BI pandemic. Now in a much-improved local situation, we are seeing again that the majority of cases (largely sporadic) is caused by multiple types. Current studies range from molecular studies on toxin and spore production, immune responses, novel observations on CDI in children, to what is the best way of decontaminating the anaerobe laboratory.

Induction of cytokines in a macrophage cell line by proteins of Clostridium difficile.

Clostridium difficile is a major cause of nosocomial diarrhoea. The toxins produced by C. difficile are responsible for the characteristic pathology observed in C. difficile disease, but several surface-associated proteins of C. difficile are also recognized by the immune system and could modulate the immune response in infection. The aim of this study was to assess the induction of cytokines in a macrophage cell line in response to different antigens prepared from five C. difficile strains: the hypervirulent ribotype 027, ribotypes 001 and 106 and reference strains VPI 10463 and 630 (ribotype 012). PMA-activated THP-1 cells were challenged with surface-layer proteins, flagella, heat-shock proteins induced at 42 and 60 °C and culture supernatants of the five C. difficile strains. The production of the pro-inflammatory cytokines such as TNF-α, IL-1ß, IL-6, IL-8 and IL-12p70 was observed in response to the surface-associated proteins, and high levels of TNF-α, IL-1ß and IL-8 were detected in response to challenge with culture supernatants. The immune response triggered by the surface-associated proteins was independent of the strain from which the antigens were derived, suggesting that these proteins might not be related to the varying virulence of the hypervirulent ribotype 027 or ribotypes 001 and 106. There was no interstrain difference observed in response to the culture supernatants of the tested C. difficile strains, but this was perhaps due to toxicity induced in the macrophages by large amounts of toxin A and toxin B.

Efficacy of decontaminants and disinfectants against Clostridium difficile.

Clostridium difficile is a common nosocomial pathogen transmitted mainly via its spores. These spores can remain viable on contaminated surfaces for several months and are resistant to most commonly used cleaning agents. Thus, effective decontamination of the environment is essential in preventing the transmission of C. difficile in health-care establishments. However, this emphasis on decontamination must also be extended to laboratories due to risk of exposure of staff to potentially virulent strains. Though few cases of laboratory-acquired infection have been reported, the threat of infection by C. difficile in the laboratory is real. Our aim was to test the efficacy of four disinfectants, Actichlor, MicroSol 3+, TriGene Advance and Virkon, and one laboratory decontaminant, Decon 90, against vegetative cells and spores of C. difficile. Five strains were selected for the study: the three most commonly encountered epidemic strains in Scotland, PCR ribotypes 106, 001 and 027, and control strains 630 and VPI 10463. MICs were determined by agar dilution and broth microdilution. All the agents tested inhibited the growth of vegetative cells of the selected strains at concentrations below the recommended working concentrations. Additionally, their effect on spores was determined by exposing the spores of these strains to different concentrations of the agents for different periods of time. For some of the agents, an exposure of 10 min was required for sporicidal activity. Further, only Actichlor was able to bring about a 3 log(10) reduction in spore numbers under clean and dirty conditions. It was also the only agent that decontaminated different hard, non-porous surfaces artificially contaminated with C. difficile spores. However, this too required an exposure time of more than 2 min and up to 10 min. In conclusion, only the chlorine-releasing agent Actichlor was found to be suitable for the elimination of C. difficile spores from the environment, making it the agent of choice for the decontamination of laboratory surfaces.

Comparison of toxin and spore production in clinically relevant strains of Clostridium difficile.

Clostridium difficile is a major cause of nosocomial diarrhoea. The toxins that it produces (TcdA and TcdB) are responsible for the characteristic pathology of C. difficile infection (CDI), while its spores persist in the environment, causing its widespread transmission. Many different strains of C. difficile exist worldwide and the epidemiology of the strains is ever-changing: in Scotland, PCR ribotype 012 was once prevalent, but currently ribotypes 106, 001 and 027 are endemic. This study aimed to identify the differences among these ribotypes with respect to their growth, and toxin and spore production in vitro. It was observed that the hypervirulent ribotype 027 produces significantly more toxin than the other ribotypes in the exponential and stationary phases of growth. Further, the endemic strains produce significantly more toxins and spores than ribotype 012. Of note was the observation that tcdC expression did not decrease into the stationary phase of growth, implying that it may have a modulatory rather than repressive effect on toxin production. Further, the increased expression of tcdE in ribotype 027 suggests its importance in the release of the toxins. It can thus be concluded that several genotypic and phenotypic traits might synergistically contribute to the hypervirulence of ribotype 027. These observations might suggest a changing trend towards increased virulence in the strains currently responsible for CDI.

A proposed nomenclature for cell wall proteins of Clostridium difficile.

Strains of Clostridium difficile produce a number of surface-localized proteins, including the S-layer proteins (SLPs) and other proteins that have suspected roles in pathogenesis. During the Third International C. difficile Symposium (Bled, Slovenia, September 2010) discussions were held on standardization of nomenclature. Gene designations were proposed for the large family of cell wall proteins that are paralogues of the SLP and contain putative cell wall binding motifs. This paper summarizes the agreed nomenclature, which we hope will be used by research groups currently active in the field.

Fidaxomicin: a new macrocyclic, RNA polymerase-inhibiting antibiotic for the treatment of Clostridium difficile infections.

Clostridium difficile infection is now a major concern throughout the developed world and its occurrence is a consequence of broad-spectrum antibiotic therapy primarily in the elderly in-patient population and high spore loads in hospitals in these regions. With the emergence of a hypervirulent, endemic strain, more severe disease is being recognized and is occurring in previously considered unusual patient groups. Vancomycin and metronidazole are the current mainstays for therapy of severe and nonsevere disease, respectively. Relapse is a major concern, with treatment options for these cases often difficult. Any new drug must be better than vancomycin for severe disease with fewer relapses. Fidaxomicin, a macrocyclic RNA polymerase inhibitor, has a narrow spectrum of activity, which is almost C. difficile specific. This drug appears to have a higher clinical cure rate than vancomycin, and fewer patients relapse following initial treatment. From the results of a recent Phase III trial, fidaxomicin appears to be an extremely promising drug for treating C. difficile infection and preventing relapses.

Changes in antibiotic susceptibility and ribotypes in Clostridium difficile isolates from southern Scotland, 1979-2004.

An increase in the incidence of clinical cases of Clostridium difficile infection has been reported in recent years, but few studies have examined changes in molecular epidemiology and antibiotic resistance over a long period of time. A collection of 179 isolates of C. difficile obtained from symptomatic adult patients in southern Scotland between 1979 and 2004 was used to determine changes in the prevalence of epidemiological types and antibiotic susceptibilities to common antibiotics. PCR ribotyping and MIC determination were performed on all isolates. A total of 56 different ribotypes were identified, among which ribotype 002 was the commonest type overall (14 .0%), followed by ribotypes 014 (7.3 %), 012 (5 .0%), 015 (5.0 %), 020 (5 .0%) and 001 (4.5 %). Ribotype 078 was also identified. The 10 commonest ribotypes comprised 55 % of the total isolates. Ribotype 001 increased in prevalence from 1.5 to 12.2 % over the study years, whereas the prevalence of ribotype 012 decreased from 8.7 to 2 .0%. Resistance to clindamycin, erythromycin and ceftriaxone was found in 95.5, 14.0 and 13.4 % of isolates, respectively. Resistance to vancomycin or metronidazole was not detected. Thirty-two (17.9 %) and 14 (7.8 %) isolates were resistant to two and three or more antibiotics, respectively. Ribotype 001 displayed maximum resistance, with 50 % of isolates resistant to erythromycin, moxifloxacin and ceftriaxone, and 100 % resistant to clindamycin. Over the 26 years of the study, antibiotic resistance and ribotype prevalence have changed, and antibiotic pressures may have been the major driver of this change.

Potential use of a liposome-encapsulated mixture of lipopolysaccharide core types (R1, R2, R3 and R4) of Escherichia coli in controlling colisepticaemia in chickens.

Infections caused by Escherichia coli have an economically significant impact on the poultry industry and a non-serotype-specific vaccine appears to be the most logical method of controlling them. The core oligosaccharide-lipid A region of bacterial lipopolysaccharide (LPS) is well conserved and highly immunogenic but toxic. This study determined the possible use of a liposome-encapsulated mixture of rough LPSs of core types R1, R2, R3 and R4 in controlling infections caused by E. coli in chickens. The liposome which encapsulated the LPS consisted of egg phosphatidylcholine, bovine brain phosphatidylserine and cholesterol. As determined by Limulus amoebocyte lysate assay, incorporation of LPS into the liposome reduced the endotoxicity of LPS to 0.7 % of its initial value. When tested on a chicken macrophage cell line (HD11), liposome-incorporated LPS produced a significantly lower amount of nitric oxide (<5 microM) than that produced by free LPS (22 microM). Transcription of the genes for interleukin-1beta and inducible nitric oxide synthase was lower in cells treated with liposome-incorporated LPS than in cells treated with free LPS. When chickens were immunized with 0.2 microg, 1 microg and 5 microg liposome-encapsulated mixture of LPS core types, the antibody response increased with increasing dose. When challenged with the virulent E. coli O78 strain, the birds which received 1 microg liposome-encapsulated LPS and 5 microg LPS had significantly lower lesions scores (P <0.05) and high body weight when compared with the birds in the control group as well as with the birds immunized with a suboptimal dose (0.2 microg) of liposome-encapsulated LPS.

Bacteroides fragilis signals through Toll-like receptor (TLR) 2 and not through TLR4.

Although it is desirable to identify the interactions between endotoxin/LPS and the innate immune mechanism, it is often not possible to isolate these interactions from other cell wall-related structures of protein or polysaccharide origin. There is no universally accepted method to extract different LPSs from different bacteria, and their natural state will be influenced by their interactions with the associated molecules in the bacterial outer membrane. It is now believed that Toll-like receptor (TLR) 4 is the main signal transducer of classical LPS (i.e. Escherichia coli LPS), while TLR2 is used by certain non-classical LPSs. There are contradictory reports as to whether Bacteroides fragilis LPS, a non-classical LPS, signals primarily through TLR2 or TLR4. This study was designed to address this problem. Different non-purified and purified B. fragilis LPSs extracted by different methods together with different heat-killed, whole-cell populations of B. fragilis were used to elucidate the TLR specificity. All of these B. fragilis preparations showed a significant signalling specificity for TLR2 but not for TLR4. This indicates that changing the extraction methods, with or without applying a repurification procedure, and varying the cell populations do not alter the TLR specificity of B. fragilis LPS.

Systemic antibody response to Clostridium difficile in colonized patients with and without symptoms and matched controls.

It has been proposed that patients who develop Clostridium difficile-associated disease (CDAD) do so because they are unable to mount an adequate immune response. Serum was collected from three groups of elderly in-patients: (i) cases (n=21) of CDAD, being toxin A/B-positive; (ii) carriers (n=21) asymptomatic for CDAD (no diarrhoea) but at least toxin or culture positive; and (iii) controls (n=26) asymptomatic for CDAD and negative for both C. difficile toxin and culture. The age and gender of each group were compared, and the colonizing strains were ribotyped and toxinotyped. Serum antibodies (IgG and IgM) were measured by ELISA using different antigen preparations: EDTA extract (containing cell-surface proteins and carbohydrates), guanidine hydrochloride extract (surface-layer proteins), aqueous phenol-extracted lipocarbohydrate (LC); crude toxin (dialysis culture supernatant) and purified toxin A. LPS from Escherichia coli was used as a control antigen. Antibodies were also tested for toxin neutralization on tissue monolayers and for binding to EDTA-extracted antigens by Western blotting. IgG antibody measurements to cytomegalovirus (CMV) were included as an indicator of potential immunosenescence. Results showed that the patient groups were well matched by age and gender, and the colonizing strains were similar in cases and carriers, being predominantly ribotype 001 and toxinotype 0. By ELISA, IgG levels to most of the antigens were highest in the cases and lowest in the controls, with the exception of antibodies to the LC, which were higher in the controls than the cases. Levels in the carriers tended to be of intermediate level or similar to the controls. For all antigens, the levels of IgM were not significantly different among cases, carriers and controls. Serum from all groups was able to neutralize the cytotoxic action of toxin on both Vero and Caco2 cells, and all to a similar extent. Western blots showed an overall higher level of IgG antibodies to the EDTA-extracted antigens in the cases. The results of the CMV ELISA showed that specific IgG was detected in more cases (78%) than carriers and controls (both 65%), but this difference in seropositivity was not significant. The conclusion is that, during symptomatic infection, patients respond to protein antigens of C. difficile in a manner typical of a secondary antibody response, with no evidence that an inability to respond predisposes to the appearance of symptoms.