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Summary 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.

Background Clostridium difficile is a nosocomial pathogen prevalent in hospitals worldwide and increasingly common in the community. Sequence differences have been shown to be present in the Surface Layer Proteins (SLPs) from different C. difficile ribotypes (RT) however whether these differences influence severity of infection is still not clear.ResultsWe used a molecular evolutionary approach to analyse SLPs from twenty-six C. difficile RTs representing different slpA sequences. We demonstrate that SLPs from RT 027 and 078 exhibit evidence of positive selection (PS). We compared the effect of these SLPs to those purified from RT 001 and 014, which did not exhibit PS, and demonstrate that the presence of sites under positive selection correlates with ability to activate macrophages. SLPs from RTs 027 and 078 induced a more potent response in macrophages, with increased levels of IL-6, IL-12p40, IL-10, MIP-1α, MIP-2 production relative to RT 001 and 014. Furthermore, RTs 027 and 078 induced higher expression of CD40, CD80 and MHC II on macrophages with decreased ability to phagocytose relative to LPS.ConclusionsThese results tightly link sequence differences in C. difficile SLPs to disease susceptibility and severity, and suggest that positively selected sites in the SLPs may play a role in driving the emergence of hyper-virulent strains.Electronic supplementary materialThe online version of this article (doi:10.1186/s12862-017-0937-8) contains supplementary material, which is available to authorized users.

g 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), ribotypespecific 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. C lostridium difficile is the leading worldwide infective cause of hospital-acquired and antibiotic-associated diarrhea, imposing a considerable financial burden on health service providers in both Europe and the United States (1-3). Infection causes a spectrum of clinical presentations ranging from an asymptomatic carrier state to severe fulminant colitis and death (4). Following successful treatment, an estimated 20% to 30% of patients with primary C. difficile infection (CDI) develop recurrence of symptoms, caused by either relapse of the original infection or reinfection with a new strain (5).This anaerobic and spore-forming bacterium exerts its major pathological effects through two proinflammatory and cytotoxic protein exotoxins, TcdA (toxin A) and TcdB (toxin B) (6). Nontoxin virulence factors, such as surface layer proteins (SLPs) and cell wall proteins, have also been described and may play a role in disease expression (7-9).The majority of healthy adults have detectable antibodies to C. difficile TcdA and TcdB in their sera that are thought to arise from colonization in infancy or from repeated exposure to C. difficile in adulthood from the environment (10, 11). Several clinical studies suggest that adaptive humoral immune responses, in particular to TcdA and TcdB, may influence clinical outcomes of CDI (12). Most notably, a landmark study in 2000 reported that a low IgG titer to TcdA, but not TcdB, at the time of infection is associated with development of symptomatic disease (13). More recently, the same group demonstrated an association between median IgG titers to TcdA and 30-day all-cause mortality (14). Several r...

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