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.

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.

Cytokines Are Markers of the Clostridium difficile-Induced Inflammatory Response and Predict Disease Severity.

The host immune response affects pathogen virulence in Clostridium difficile infection (CDI). Thus, cytokine responses to CDI likely are associated with disease initiation and progression. Understanding the molecular drivers of inflammation and biochemical markers of disease severity is important for developing novel therapies and predicting disease prognosis. In this study, we investigated cytokine production in patients with CDI and evaluated the potential of cytokines to serve as biomarkers for CDI and predictors of disease severity. The systemic cytokine profiles of 36 CDI patients (20 with severe disease) and 8 healthy donors and the toxin-induced cytokine profiles of peripheral blood mononuclear cells (PBMC) were determined. Further, we evaluated glucosyltransferase (GT) activity in regulation of toxin-induced cytokine expression. We found upregulation of the majority of measured cytokines (11/20, 55%) in CDI patients. Interleukin-1ß (IL-1ß), IL-6, IL-8, IL-17A, and IL-16 were the most upregulated. High serum levels of IL-2 and IL-15 were associated with a poor prognosis in CDI patients, whereas high levels of IL-5 and gamma interferon (IFN-γ) were associated with less severe disease. Both TcdA and TcdB were potent inducers of cytokine responses, as demonstrated by stimulation of a greater number and amount of cytokines. In addition to confirming prior reports on the role of IL-8, IL-1ß, and IL-6 in CDI, our data suggest that IL-16 and IL-17A, as well as the IL-1ß/Th17 axis, play a key role in driving inflammatory responses in CDI. A functional GT domain of C. difficile toxins was required for the induction of a majority of cytokines investigated.

Laboratory and Clinical features of EIA Toxin-positive and EIA Toxin-negative Community-acquired Clostridium difficile Infection.

Studies have described the clinical course of patients with Clostridium difficile infection (CDI) with positive enzyme immunoassay (EIA) for toxins A and B. Limited information is available for the patients with negative EIA but positive for the toxin B gene (TcdB) by the PCR. The aim of our study is to determine if there are any differences that exist among the clinical and laboratory parameters in the patients tested to be positive by EIA for toxin and those who were negative. This is a retrospective cohort study conducted in a 700-bed teaching hospital. We reviewed charts of the patients with presumptive CDI between January 2006 and July 2013. We divided these patients into two groups, EIA-positive and EIA-negative, based on result of EIA for toxins A and B and the requirement for a positive PCR analysis of the TcdB gene. The EIA-positive group had significantly higher white blood cell counts (p<0.001), with a significantly greater percentage of bands (p<0.0001). Albumin and total protein both exhibit significantly (p<0.0001, both comparisons) lower values in the EIA-positive group. Among clinical findings, the EIA-positive group had significantly longer length of hospital stay (p=0.010). These data suggest that an infection with an EIA-negative strain of C. difficile presents laboratory markers closer to those of healthy subjects and clinical features suggesting considerably less severe than infection with EIA-positive C. difficile.

Risk factors, outcomes and epidemiology associated with Clostridium difficile infection in patients with haematological malignancies in a tertiary care hospital in China.

The purpose of this study was to evaluate the risk factors, outcomes and epidemiology associated with Clostridium difficile infection (CDI) in patients with haematological malignancies in a tertiary care hospital in China. C. difficile screening was performed on patients admitted for chemotherapy or haematopoietic stem cell transplantation between 2009 and 2013. C. difficile isolates were analysed by multilocus sequence typing, and a retrospective chart review was performed on all patients with a positive toxin assay. CDI was diagnosed in 21 haematology-oncology ward patients and 14 marrow transplantation service patients for a cumulative incidence of 1.89/1000 and 3.69/1000 patient-days, respectively. Univariate analyses showed that patients who received etoposide had an increased risk of CDI (odds ratio 4.25, 95 % confidence interval 1.32-13.64). There was only one patient death, for which CDI was not the primary cause. Ten sequence types (STs) were identified, of which ST-3 and ST-54 were the most common; the hypervirulent ST-1 (ribotype 027) and ST-11 (ribotype 078) C. difficile strains were not detected in the patients in this study. The incidence of CDI did not differ between patients receiving chemotherapy and those receiving haematopoietic stem cell transplantation. The only risk factor for chemotherapy patients was treatment with etoposide.

Effects of salinomycin and Bacillus subtilis on growth performance and immune responses in broiler chickens.

The present study was undertaken to compare the effect of salinomycin and Bacillus subtilis on growth performance, serum antibody levels against Clostridium spp. and Eimeria spp., and cytokine mRNA expression levels in broiler chickens raised in the used litter. Broiler chickens fed a diet containing salinomycin showed lower (P < 0.05) body weights compared with the control diet-fed counterparts. Serum nitric oxide levels were significantly (P < 0.05) elevated in chickens fed the B. subtilis-enriched diet compared with those on either the salinomycin-fed or control diet-fed chickens. None of the dietary treatments affected (P > 0.05) serum antibody levels against Clostridium perfringens toxins. Both salinomycin and B.subtilis significantly lowered (P < 0.05) the serum levels of Eimeria-specific antibodies compared with the control group. Salinomycin, but not B. subtilis, significantly modulated (P < 0.05) the expression of cytokines encoding interferon-γ (IFN-γ), interleukin10 (IL-10) and tumor necrosis factor superfamily 15 (TNFSF15) compared with the control group. In conclusion, dietary salinomycin and B. subtilis affected serum anticoccidial antibody and intestinal cytokine expression, but failed to improve growth performance in broiler chickens. Further study is warranted to investigate the mode of action of salinomycin on host immune response and growth performance in broiler chickens.

Clostridium hathewayi bacteraemia and surgical site infection after uterine myomectomy.

A 42-year-old woman with uterine fibroids underwent myomectomy. She developed postoperative sepsis and bloodstream infection with Clostridium hathewayi secondary to an infected haematoma. The patient was readmitted after failure of oral antibiotic therapy and underwent intrauterine drainage followed by prolonged parenteral antibiotic therapy. The patient was followed for 1 year and did not have any relapse of infection.

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.

Clostridium difficile infection and limitations of markers for severity in patients with hematologic malignancy.

OBJECTIVE: To describe characteristics of Clostridium difficile infection (CDI) and markers of severe CDI among patients with hematologic malignancies. DESIGN: Case-control study. SETTING: Tertiary care teaching hospital. PATIENTS AND METHODS: Inpatients with hematologic malignancies and CDI were age and time matched with 2 control inpatients without hematologic malignancies. Chart reviews were performed, and C. difficile isolates were strain typed. RESULTS: Case patients (n = 41) and control patients (n = 82) patients were different in respect to receipt of immunosuppressive agents within 2 months (92.7% vs 25.6%; P < .0001); neutropenia within 2 months (75.6% vs 3.7%; P < .0001) and mean (± standard deviation) white blood cell (WBC) count at diagnosis (vs 4.9 ± 14.1 vs 11.8 ± 6.8 x 10(3) cells/mL; P <.0001); baseline mean creatinine level (0.89 ± 0.1 vs 1.6 ± 2.4 mg/dL; P = .003), mean creatinine level at diagnosis (0.83 ± 0.4 vs 1.85 ± 1.9 mg/dL; P = .004), and creatinine increases of 1.5 times over baseline (2.4% vs 15.1%; P = .02). Immunosuppressive agents and creatinine level remained significant in multivariable analysis (P = .03 for both variables). Severity correlated with mortality when measured by alternate severity criteria but not when measured by the Society for Healthcare Epidemiology of America/Infectious Diseases Society of America criteria, which are based solely on WBC count and creatinine elevation. The prevalence of the epidemic BI/NAP1/027 strain was similar in both groups. CONCLUSIONS: Patients with hematologic malignancies had lower creatinine levels at the time of CDI diagnosis compared with control patients. WBC counts also tended to be lower in case patients. CDI severity criteria based on WBC count and creatinine level may not be applicable to patients with hematologic malignancies.

PPI therapy and albumin are better predictors of recurrent Clostridium difficile colitis than choice of antibiotics.

BACKGROUND: Recurrent Clostridium difficile colitis (RCDC) is common, but data regarding recurrence rates and predisposing factors are sparse. METHODS: A retrospective case-control study was performed, identifying all inpatients and outpatients ≥18 years of age with C. difficile colitis (CDC) confirmed by a positive stool sample collected at our institution from January 2008 to August 2011. Factors associated with RCDC, the number of RCDC episodes, and the need for admission for RCDC were sought. RESULTS: A total of 739 patients (male, 47 %) were studied, of whom 527 (71 %) received inpatient treatment for their index episode of CDC. There was no difference (p = 0.53) between RCDC rates for inpatients (17.6 %) and outpatients (19.8 %). While severity score and albumin were associated with RCDC in our population, use of proton pump inhibitors (PPIs) correlated with decreased RCDC (p = 0.006) and decreased need for admission (p = 0.005). The addition of vancomycin to metronidazole therapy did not lower RCDC rates (p = 0.52) or decrease the need for admission (p = 0.78). CONCLUSIONS: Hypoalbuminemia strongly correlated with higher recurrence rates, while PPI therapy actually reduced RCDC, representing previously underappreciated potential therapeutic targets for lowering CDC recurrence. The addition of vancomycin to metronidazole did not improve RCDC rates.

Epidemiology and outcomes of clostridial bacteremia at a tertiary-care institution.

A review was performed to determine the epidemiology and outcomes of clostridial bacteremia (CB) at our institution. Ninety-two percent of patients had monomicrobial CB. Patients with Clostridium innocuum bacteremia had a significantly higher 2-day mortality rate compared to patients with C. septicum and C. perfringens. CB is associated with high and rapid mortality, especially in patients with malignancy. Early mortality was significantly lower in patients receiving antibiotics with adequate coverage for Clostridium species.

Pneumorachis caused by metastatic gas gangrene.

Pneumorachis has previously been described only after spread from a contiguous site or after a traumatic event. Our patient experienced sepsis due to multiple enteric organisms, and gas was identified within the spinal canal on computed tomographic imaging. We present the 1st case of pneumorachis caused by disseminated infection.

Features of hemolysis due to Clostridium perfringens infection.

Infection by Clostridium perfringens can be an unsuspected cause of hemolysis in emergency room patients. Historically, this condition has been associated with wound contamination and other tissue infections. We report the case of an autistic patient who presented to our emergency department with a distended abdomen and hemolysis of unknown etiology. The patient had no history of recent surgery. Exploration of the abdomen revealed a hepatic abscess. Blood cultures tested culture positive for C. perfringens. We present images demonstrating the salient features of the peripheral blood smear in cases of this uncommon but deadly cause of hemolysis.

Predictors of mortality and limb loss in necrotizing soft tissue infections.

HYPOTHESIS: Necrotizing soft tissue infections are associated with a high mortality rate. We hypothesize that specific predictors of limb loss and mortality in patients with necrotizing soft tissue infection can be identified on hospital admission. DESIGN: A retrospective cohort study. SETTING: A tertiary care center. PATIENTS: Patients with a diagnosis of necrotizing soft tissue infection during a 5-year period (1996-2001) were included. Patients were identified with International Classification of Diseases, Ninth Revision hospital discharge diagnosis codes, and diagnosis was confirmed by medical record review. INTERVENTIONS: Standard current treatment including early and scheduled repeated debridement, broad-spectrum antibiotics, and physiologic and nutritional support was given to all patients. MAIN OUTCOME MEASURES: Limb loss and mortality. RESULTS: One hundred sixty-six patients were identified and included in the study. The overall mortality rate was 16.9%, and limb loss occurred in 26% of patients with extremity involvement. Independent predictors of mortality included white blood cell count greater than 30 000 x 10(3)/microL, creatinine level greater than 2 mg/dL (176.8 micromol/L), and heart disease at hospital admission. Independent predictors of limb loss included heart disease and shock (systolic blood pressure <90 mm Hg) at hospital admission. Clostridial infection was an independent predictor for both limb loss (odds ratio, 3.9 [95% confidence interval, 1.1-12.8]) and mortality (odds ratio, 4.1 [95% confidence interval, 1.3-12.3]) and was highly associated with intravenous drug use and a high rate of leukocytosis on hospital admission. The latter was found to be a good variable in estimating the probability of death. CONCLUSIONS: Clostridial infection is consistently associated with poor outcome. This together with the independent predictors mentioned earlier should aid in identifying patients on hospital admission who may benefit from more aggressive and novel therapeutic approaches.

Massive intravascular hemolysis: a fatal complication of clostridium perfringens septicemia in a patient with acute myeloid leukemia.

We describe a fatal case of a 74-year old man with acute myeloid leukemia complicated by massive intravascular hemolysis due to Clostridium perfringens sepsis.