Hematological investigations in a case of intravascular hemolysis due to Clostridium perfringens infection.

A patient presented with fever, severe pain and edematous tight due to hip trauma and was scheduled for urgent fasciotomy. Following physical examination, laboratory analyses were requested, and results revealed anemia and severe infection. As the patient's condition was serious, a new set of samples was sent to the laboratory four hours later. Following centrifugation, severely hemolyzed dark-colored serum and plasma samples were obtained and in vitro hemolysis was suspected. The collection of samples was repeated, but a new set of samples was also hemolyzed with a significant decrease in the hemoglobin value. At that point, in vivo hemolysis was suspected, and samples were processed according to standard laboratory procedures for hemolytic samples. Following confirmation of the gas gangrene diagnosis by clinicians, the cause of hemolysis was attributed to the cytotoxic activity of α-toxin produced by the anaerobic gram-positive bacterium Clostridium perfringens. An insight into the laboratory procedure that could help to narrow down the causes of hemolysis and single out C. perfringens as a cause of intravascular hemolysis was given.

Systemic neutrophil degranulation and emergency granulopoiesis in patients with Clostridioides difficile infection.

OBJECTIVES: Clostridioides difficile infection (CDI) is characterized by neutrophilia in blood, with a high leukocyte count accompanying severe infection. In this study, we characterized peripheral blood neutrophil activation and maturity in CDI by (i) developing a method to phenotype stored neutrophils for disease-related developmental alterations and (ii) assessing neutrophil-associated biomarkers. METHODS: We stored fixed leukocytes from blood collected within 24 h of diagnosis from a cohort of hospitalized patients with acute CDI. Additional study cohorts included recurrent CDI patients at time of and two months after FMT therapy and a control healthy cohort. We assessed levels of neutrophil surface markers CD66b, CD11b, CD16 and CD10 by flow cytometry. Plasma neutrophil elastase and lipocalin-2 were measured using ELISA, while G-CSF, GM-CSF and cytokines were measured using O-link Proteomic technology. RESULTS: CD66b+ neutrophil abundance assessed by flow cytometry correlated well with complete blood counts, establishing that neutrophils in stored blood are sufficiently well-preserved for phenotyping by flow cytometry. Neutrophil abundance was significantly increased in CDI patients compared to healthy controls. Emergency granulopoiesis in acute CDI patients was evidenced by lower neutrophil surface expression of CD10, CD11b and CD16. CD10+ staining of neutrophils started to recover within 3-7 days of CDI treatment. Neutrophil activation and degranulation were higher in acute CDI as assessed by plasma neutrophil elastase and lipocalin-2. Biomarker levels in immunocompetent subjects were associated with recurrence and fatal outcomes. CONCLUSIONS: Neutrophil activation and emergency granulopoiesis characterize the early immune response in acute CDI, with plasma degranulation biomarkers predictive of disease severity.

Effective Microorganisms (EM) Improve Internal Organ Morphology, Intestinal Morphometry and Serum Biochemical Activity in Japanese Quails under Clostridium perfringens Challenge.

The effect of effective microorganisms (EM) on internal organ morphology, intestinal morphometry, and serum biochemical activity in Japanese quails under Clostridium perfringens challenge was determined. After 30 days of EM addition, one group of quails was orally inoculated with Clostridium perfringens. The second group did not receive EM and was inoculated with C. perfringens. In the gut, EM supplementation reduced the number of lesions, enhanced gut health, and protected the mucosa from pathogenic bacteria. EM showed an anti-inflammatory effect and fewer necrotic lesions in villi. In the internal organs, EM showed a protective effect against a typical lesion of C. perfringens infection. Necrosis and degeneration of the hepatocytes, necrosis of bile ducts, and bile duct proliferation were more severe in the infected group without EM. Morphometric evaluation showed significantly higher villi in the jejunum after EM addition. A greater crypt depth was observed in the C. perfringens group. Biochemical analysis of the blood indicated lower cholesterol on the 12th day of the experiment and between-group differences in total protein, lactate dehydrogenase (LDH), and albumin levels in the EM group. Further studies are needed to improve EM activity against pathologic bacteria as a potential alternative to antibiotics and to develop future natural production systems.

Integrating gut microbiome and host immune markers to understand the pathogenesis of Clostridioides difficile infection.

Clostridioides difficile (C.difficile) infection is the most common cause of healthcare-associated infection and an important cause of morbidity and mortality among hospitalized patients. A comprehensive understanding of C.difficile infection (CDI) pathogenesis is crucial for disease diagnosis, treatment, and prevention. Here, we characterized gut microbial compositions and a broad panel of innate and adaptive immunological markers in 243 well-characterized human subjects (including 187 subjects with both microbiota and immune marker data), who were divided into four phenotype groups: CDI, Asymptomatic Carriage, Non-CDI Diarrhea, and Control. We found that the interactions between gut microbiota and host immune markers are very sensitive to the status of C.difficile colonization and infection. We demonstrated that incorporating both gut microbiome and host immune marker data into classification models can better distinguish CDI from other groups than can either type of data alone. Our classification models display robust diagnostic performance to differentiate CDI from Asymptomatic carriage (AUC~0.916), Non-CDI Diarrhea (AUC~0.917), or Non-CDI that combines all other three groups (AUC~0.929). Finally, we performed symbolic classification using selected features to derive simple mathematic formulas that explicitly quantify the interactions between the gut microbiome and host immune markers. These findings support the potential roles of gut microbiota and host immune markers in the pathogenesis of CDI. Our study provides new insights for a microbiome-immune marker-derived signature to diagnose CDI and design therapeutic strategies for CDI.

Fecal Microbiota Transplantation for Recurrent Clostridioides difficile Infection Associates With Functional Alterations in Circulating microRNAs.

BACKGROUND AND AIMS: The molecular mechanisms underlying successful fecal microbiota transplantation (FMT) for recurrent Clostridioides difficile infection (rCDI) remain poorly understood. The primary objective of this study was to characterize alterations in microRNAs (miRs) following FMT for rCDI. METHODS: Sera from 2 prospective multicenter randomized controlled trials were analyzed for miRNA levels with the use of the Nanostring nCounter platform and quantitative reverse-transcription (RT) polymerase chain reaction (PCR). In addition, rCDI-FMT and toxin-treated animals and ex vivo human colonoids were used to compare intestinal tissue and circulating miRs. miR inflammatory gene targets in colonic epithelial and peripheral blood mononuclear cells were evaluated by quantitative PCR (qPCR) and 3'UTR reporter assays. Colonic epithelial cells were used for mechanistic, cytoskeleton, cell growth, and apoptosis studies. RESULTS: miRNA profiling revealed up-regulation of 64 circulating miRs 4 and 12 weeks after FMT compared with screening, of which the top 6 were validated in the discovery cohort by means of RT-qPCR. In a murine model of relapsing-CDI, RT-qPCR analyses of sera and cecal RNA extracts demonstrated suppression of these miRs, an effect reversed by FMT. In mouse colon and human colonoids, C difficile toxin B (TcdB) mediated the suppressive effects of CDI on miRs. CDI dysregulated DROSHA, an effect reversed by FMT. Correlation analyses, qPCR ,and 3'UTR reporter assays revealed that miR-23a, miR-150, miR-26b, and miR-28 target directly the 3'UTRs of IL12B, IL18, FGF21, and TNFRSF9, respectively. miR-23a and miR-150 demonstrated cytoprotective effects against TcdB. CONCLUSIONS: These results provide novel and provocative evidence that modulation of the gut microbiome via FMT induces alterations in circulating and intestinal tissue miRs. These findings contribute to a greater understanding of the molecular mechanisms underlying FMT and identify new potential targets for therapeutic intervention in rCDI.

Novel immunoassay for diagnosis of ongoing Clostridioides difficile infections using serum and medium enriched for newly synthesized antibodies (MENSA).

BACKGROUND: Clostridioides difficile infections (CDI) have been a challenging and increasingly serious concern in recent years. While early and accurate diagnosis is crucial, available assays have frustrating limitations. OBJECTIVE: Develop a simple, blood-based immunoassay to accurately diagnose patients suffering from active CDI. MATERIALS AND METHODS: Uninfected controls (N = 95) and CDI patients (N = 167) were recruited from Atlanta area hospitals. Blood samples were collected from patients within twelve days of a positive CDI test and processed to yield serum and PBMCs cultured to yield medium enriched for newly synthesized antibodies (MENSA). Multiplex immunoassays measured Ig responses to ten recombinant C. difficile antigens. RESULTS: Sixty-six percent of CDI patients produced measurable responses to C. difficile antigens in their serum or MENSA within twelve days of a positive CDI test. Fifty-two of the 167 CDI patients (31%) were detectable in both serum and MENSA, but 32/167 (19%) were detectable only in MENSA, and 27/167 (16%) were detectable only in serum. DISCUSSION: We describe the results of a multiplex immunoassay for the diagnosis of ongoing CDI in hospitalized patients. Our assay resolved patients into four categories: MENSA-positive only, serum-positive only, MENSA- and serum-positive, and MENSA- and serum-negative. The 30% of patients who were MENSA-positive only may be accounted for by nascent antibody secretion prior to seroconversion. Conversely, the serum-positive only subset may have been more advanced in their disease course. Immunocompromise and misdiagnosis may have contributed to the 34% of CDI patients who were not identified using MENSA or serum immunoassays. IMPORTANCE: While there was considerable overlap between patients identified through MENSA and serum, each method detected a distinctive patient group. The combined use of both MENSA and serum to detect CDI patients resulted in the greatest identification of CDI patients. Together, longitudinal analysis of MENSA and serum will provide a more accurate evaluation of successful host humoral immune responses in CDI patients.

Advanced age and increased CRP concentration are independent risk factors associated with Clostridioides difficile infection mortality.

Clostridioides difficile (C.difficile) is a Gram-positive, spore-forming, toxin-producing anaerobic bacillus, which is one of the most common causes of health-care-associated infection developed mainly by elderly patients. The objective of this study was to assess mortality among the patients of the Hospital for Infectious Diseases in Warsaw related to C.difficile infection. Analysis was conducted of 1638 records reporting the medical histories of patients hospitalized for the first time due to Clostridioides difficile infection (CDI) in the Hospital for Infectious Diseases in Warsaw from 2010 to 2017. The inclusion criteria were any (principal or secondary) discharge diagnosis code for CDI according to ICD-10 and being an adult (≥ 18 years). 108 out of 1638 (7%) of the patients died. The median age in this group was 83 years. The largest number of deaths (90%) occurred in the group of patients aged 65 years or older and 81-90 years old (53% of all the deaths). In the multivariate logistic regression model relevant only to the age groups, not to sepsis-age over 80 and over 90 were independent predictors of death, increasing the risk of death by 3.4 and 1.8 times, respectively. The result of the receiver operating curve (ROC) analysis determined the age of 77 years as the threshold value, indicating the increased risk of death (AUC 0.727, standard error 0.025, 95% CI 0.678-0.776, p < 0.0001). In addition, other quantitative variables, namely CRP, creatinine and leucocytes were studied and turned out to be independent death predictors as well. The diagnosis of sepsis increased the risk of death fourfold (OR = 4.042; 95% Cl 2.4-6.7; p < 0.001). Increased inflammatory parameters, namely CRP and white blood cell count, advanced age, particularly over the age of 80, as well as a diagnosis of sepsis are independent risk factors for death and could be used as predictive markers of poor outcome in CDI.

Systemic Inflammatory Mediators Are Effective Biomarkers for Predicting Adverse Outcomes in Clostridioides difficile Infection.

Clostridioides difficile infection (CDI) can result in severe disease and death, with no accurate models that allow for early prediction of adverse outcomes. To address this need, we sought to develop serum-based biomarker models to predict CDI outcomes. We prospectively collected sera ≤48 h after diagnosis of CDI in two cohorts. Biomarkers were measured with a custom multiplex bead array assay. Patients were classified using IDSA severity criteria and the development of disease-related complications (DRCs), which were defined as ICU admission, colectomy, and/or death attributed to CDI. Unadjusted and adjusted models were built using logistic and elastic net modeling. The best model for severity included procalcitonin (PCT) and hepatocyte growth factor (HGF) with an area (AUC) under the receiver operating characteristic (ROC) curve of 0.74 (95% confidence interval, 0.67 to 0.81). The best model for 30-day mortality included interleukin-8 (IL-8), PCT, CXCL-5, IP-10, and IL-2Rα with an AUC of 0.89 (0.84 to 0.95). The best model for DRCs included IL-8, procalcitonin, HGF, and IL-2Rα with an AUC of 0.84 (0.73 to 0.94). To validate our models, we employed experimental infection of mice with C. difficile Antibiotic-treated mice were challenged with C. difficile and a similar panel of serum biomarkers was measured. Applying each model to the mouse cohort of severe and nonsevere CDI revealed AUCs of 0.59 (0.44 to 0.74), 0.96 (0.90 to 1.0), and 0.89 (0.81 to 0.97). In both human and murine CDI, models based on serum biomarkers predicted adverse CDI outcomes. Our results support the use of serum-based biomarker panels to inform Clostridioides difficile infection treatment.IMPORTANCE Each year in the United States, Clostridioides difficile causes nearly 500,000 gastrointestinal infections that range from mild diarrhea to severe colitis and death. The ability to identify patients at increased risk for severe disease or mortality at the time of diagnosis of C. difficile infection (CDI) would allow clinicians to effectively allocate disease modifying therapies. In this study, we developed models consisting of only a small number of serum biomarkers that are capable of predicting both 30-day all-cause mortality and adverse outcomes of patients at time of CDI diagnosis. We were able to validate these models through experimental mouse infection. This provides evidence that the biomarkers reflect the underlying pathophysiology and that our mouse model of CDI reflects the pathogenesis of human infection. Predictive models can not only assist clinicians in identifying patients at risk for severe CDI but also be utilized for targeted enrollment in clinical trials aimed at reduction of adverse outcomes from severe CDI.

Validation of the SHEA/IDSA severity criteria to predict poor outcomes among inpatients and outpatients with Clostridioides difficile infection.

OBJECTIVE: To determine whether the Society for Healthcare Epidemiology of America (SHEA) and the Infectious Diseases Society of America (IDSA) Clostridioides difficile infection (CDI) severity criteria adequately predicts poor outcomes. DESIGN: Retrospective validation study. SETTING AND PARTICIPANTS: Patients with CDI in the Veterans' Affairs Health System from January 1, 2006, to December 31, 2016. METHODS: For the 2010 criteria, patients with leukocytosis or a serum creatinine (SCr) value ≥1.5 times the baseline were classified as severe. For the 2018 criteria, patients with leukocytosis or a SCr value ≥1.5 mg/dL were classified as severe. Poor outcomes were defined as hospital or intensive care admission within 7 days of diagnosis, colectomy within 14 days, or 30-day all-cause mortality; they were modeled as a function of the 2010 and 2018 criteria separately using logistic regression. RESULTS: We analyzed data from 86,112 episodes of CDI. Severity was unclassifiable in a large proportion of episodes diagnosed in subacute care (2010, 58.8%; 2018, 49.2%). Sensitivity ranged from 0.48 for subacute care using 2010 criteria to 0.73 for acute care using 2018 criteria. Areas under the curve were poor and similar (0.60 for subacute care and 0.57 for acute care) for both versions, but negative predictive values were >0.80. CONCLUSIONS: Model performances across care settings and criteria versions were generally poor but had reasonably high negative predictive value. Many patients in the subacute-care setting, an increasing fraction of CDI cases, could not be classified. More work is needed to develop criteria to identify patients at risk of poor outcomes.

Clostridium difficile-associated disease and Helicobacter pylori seroprevalence: A case-control study.

BACKGROUND: Helicobacter pylori inhabits the stomach and causes persistent inflammation, with changes in gastric acidity. However, it is unclear whether the presence of H pylori plays a role in Clostridium difficile-associated disease (CDAD). The study's aim was to examine relationships of H pylori seroprevalence and serum pepsinogens (PGs), as markers of gastric inflammation, with CDAD. MATERIALS AND METHODS: A case-control study was conducted among 49 CDAD cases and 54 controls (median age 82 years). Using enzyme-linked immunosorbent assays, sera were tested for H pylori IgG antibody, and PGI and PGII levels. Helicobacter pylori-positive samples were tested for IgG antibody to recombinant cytotoxin-associated gene A (CagA) virulent protein. Logistic regression models were fitted. RESULTS: Cases and controls were comparable in age (P = .5) and sex distribution (females 62% vs 57%, P = .6). Helicobacter pylori IgG seroprevalence was 47%, of whom 23% were CagA seropositives. Among cases compared to controls, 43% vs 28% were H pylori seropositive but lacking CagA IgG antibody: adjusted odd ratio (OR) 3.43 (95% confidence intervals [CI] 1.29-9.10); 18% vs 4% were positive for CagA phenotype: adjusted OR 9.32 (95% CI 1.61-53.76). This association was not affected by PG levels. CONCLUSIONS: Helicobacter pylori infection, especially with CagA virulent phenotype, might predispose to C difficile infection in elderly patients.

Spontaneous Non-Traumatic Clostridium perfringens Sepsis.

Clostridium species are gram-positive, spore-forming, anaerobic rods normally found in the soil and gastrointestinal tract of humans and animals. Spontaneous sepsis due to C. perfringens is not caused by injury, which sets it apart from the classical gas gangrene that typically follows trauma. Spontaneous C. perfringens sepsis often develops as a rapidly progressive intravascular hemolysis and metabolic acidosis, with high mortality rates of over 70% with standard intensive care. In such cases, alpha toxin secreted by C. perfringens is considered the main toxin responsible for intravascular hemolysis, disseminated intravascular coagulopathy, and multiple organ failure. Theta-toxin causes a cytokine cascade, which results in peripheral vasodilation similar to that seen in septic shock. For C. perfringens infections, antibiotics, such as high-dose penicillin, and surgical drainage as early as possible are the principal treatments of choice. However, considering the current mortality rate of sepsis, outcomes have not improved with the current standard treatment for C. perfringens infections. Monoclonal antibody against theta toxin in combination with gas gangrene antitoxin presents a promising therapeutic option.

The Prognostic Value of Inflammatory Markers in Clostridium difficile-associated Diarrhea.

BACKGROUND: The incidence of Clostridium difficile-associated diarrhea (CDAD) is increasing and is associated with significant morbidity and mortality. Therefore, there is a need to find new tools to determine the severity of the disease. OBJECTIVES: To investigate the prognostic values of inflammatory markers such as mean platelet volume (MPV), neutrophil-lymphocyte ratio (NLR), and C-reactive protein (CRP) in patients with CDAD. METHODS: The study comprised of 100 patients diagnosed with CDAD. The study included an additional control group of 69 patients with diarrhea who were negative for C. difficile toxin. The control group was age- and sex-matched and hospitalized at the same time period. NLR and MPV were obtained from complete blood count results. Serum CRP levels were measured by the latex particle enhanced immunoturbidimetric assay. Blood samples for all inflammatory markers were collected at time of diagnosis and prior to initiating the antibiotic therapy. Demographic, clinical, laboratory, and prognostic data were collected from medical records for a period of 90 days from the initial diagnosis of CDAD. RESULTS: The mean age of the CDAD group was 68.6 ± 21.5 years compared to 65.6 ± 24.5 in the control group (P = 0.29). Our findings show that patients with CDAD had significantly higher NLR, MPV and serum CRP levels compared to the control group (P < 0.001)). Moreover, significantly higher levels were observed when CDAD was fatal (P < 0.001). CONCLUSIONS: Elevated NLR, MPV, and serum CRP levels may serve as biomarkers for prediction of recurrence and mortality in patients with CDAD.

Elevated serum CXCL10 in patients with Clostridium difficile infection are associated with disease severity.

Clostridium difficile infection (CDI) is the primary cause of community- and health care-associated diarrhea. CXCL10, also known as IFN-γ-inducible protein of 10 (IP-10), is involved in various inflammatory diseases, but its role in CDI remains unknown. In this study, We determined the serum concentration of CXCL10 in 80 CDI patients and 76 sex & age-matched diarrhea patients by enzyme-linked immunosorbent assay (ELISA) and analyzed the correlation between CXCL10 levels and CDI disease severity parameters. Besides, we also measured the level of other cytokines and/or chemokines in CDI patients, such as IL-1ß, IL-6, TNF-α and CXCL9. We found that serum CXCL10 in CDI patients was significantly higher compared with those in non-C. difficile diarrhea patients, especially in the moderate disease. Elevated serum CXCL10 correlated positively and significantly with severity score index (SSI) score in all CDI patients. CXCL10 levels were also positively correlated with WBC count, creatinine and inflammatory cytokines including, IL-1ß, IL-6 and CXCL9, but negatively correlated with albumin. Furthermore, serum CXCL10 concentration could be significantly decreased after effective treatment of CDI. Therefore, the above results suggest that the up-regulated release of CXCL10 is important in the immunopathogenesis of CDI, and may be served as a potential alternative biomarker for the monitoring of CDI disease severity and therapeutic efficacy.

Hypoalbuminemia as predictor of recurrence of Clostridium difficile infection.

BACKGROUND: Novel drugs for Clostridium difficile (C. difficile) infections have been proven to reduce recurrent infections. Because of their high financial costs, identification of patients at high risk for recurrence is essential to provide optimal treatment. The ATLAS score's ability to predict 90-day recurrence, disease complications and 1­year all-cause mortality was evaluated. METHODS: 144 consecutive symptomatic patients with positive stool test for C. difficile were enrolled. The ATLAS score (consisting of the variables age, temperature, leukocyte count, albumin, systemic antibiotics, serum creatinine) was calculated and patients were stratified into 4 subgroups according to their scores. A Cox regression model was used to estimate the extent to which ATLAS was associated with 90-day recurrence. Furthermore, the score was correlated with disease complications and one-year all-cause mortality. RESULTS: ATLAS was unable to predict 90-day recurrence (p = 0.064, HR 1.134 [0.993;1.295]), but performed well for disease complications (D = 0.382, p < 0.001, HR 1.547 [1.266;1.889]) and mortality (p < 0.001, HR 1.374 [1.194;1.583]). Serum albumin was the only parameter able to predict 90-day recurrence (p = 0.016, HR 0.958 [0.926;0.992]) and was also a predictor of disease complications (p < 0.001, HR 0.865[0.809;0.924]) and one-year all-cause mortality (p < 0.001, HR 0.923 [0.896;0.950]). A threshold of 33.1g/L (sensitivity = 56%, specificity = 80%, AUC 0.683) and 29.2g/L (sensitivity = 75%, specificity = 70%, AUC 0.763) of serum albumin could be identified to be predictive for 90-day recurrence and one-year all-cause mortality, respectively. CONCLUSIONS: Serum albumin and ATLAS are predictors of disease complications and mortality, while only serum albumin is significantly associated with 90-day disease recurrence.

Correlation between Serological Biomarkers and Disease Activity in Patients with Inflammatory Bowel Disease.

BACKGROUND: Current biomarkers have been routinely used noninvasive methods for assessing disease activity of inflammatory bowel disease (IBD), but none of them are specific. This study was aimed to determine the performance of the serological biomarkers for detecting disease activity in patients with IBD. METHODS: A prospective study that included 73 ulcerative disease (UC) subjects, 141 Crohn's disease (CD) subjects, and 30 of them complicated with C. difficile infection (CDI) were diagnosed at a single-institution IBD center. Disease activity was assessed using by Truelove and Witts criteria for UC and Harvey Bradshaw Simple Index for CD. Serological inflammatory biomarkers were compared in different severity groups. Receiver operator curve analyses assessed the performance of each biomarker in discriminating disease states. RESULTS: For UC patients, elevated monocyte counts, C-reactive protein (CRP), and decreased lymphocyte counts and lymphocyte/monocyte ratio (LMR) significantly differed between subjects with active and inactive UC. LMR of 3.1 was 76% sensitive and had a specificity of 67% for active UC. For CD patients, higher values of neutrophils, monocytes, neutrophil/lymphocyte ratio, CRP, fibrinogen, and lower values of LMR and hemoglobin were significantly different between subjects with active and inactive CD. None of the biomarkers included had a good correlation with disease activity (area under the ROC Curve < 0.70). CONCLUSIONS: A low LMR represents an inexpensive, readily available test with a promising value to identify disease activity in UC patients, whereas none of the inflammatory biomarkers showed a discriminative value in disease activity of CD.

Prospective evaluation of the adaptive immune response to SlpA in Clostridium difficile infection.

Clostridium difficile is a major cause of healthcare-associated diarrhea. SlpA is the precursor of the S-layer of C. difficile. The aim of this work was to evaluate the humoral immune response of hospitalized patients to SlpA and its potential role on CDI outcome. Sera of 87 included patients were collected the day of CDI diagnosis as well as at early and late periods after infection. SlpA appeared to be immunogenic with a heterogeneous response between patients after a CDI. Patients with a single episode had a significantly higher anti-SlpA IgG antibody level than patients with recurrent CDI (p = 0.0197). These preliminary results will be useful to understand better the inter-individual variability of immune responses to C. difficile as well as for the development of new therapeutics.