lactoferrin and Enterocolitis--Pseudomembranous

We investigated patients with Clostridioides difficile-associated diarrhoea to see if clinical resolution correlated with faecal concentrations of metronidazole or markers of inflammation.. Faecal metronidazole, lactoferrin and serum CRP were measured daily. These were then compared with clinical progress.. Metronidazole concentration correlated with lactoferrin (ρ = 0.17, p = 0.015), CRP (ρ = 0.23, p < 0.001) and number of diarrhoeal stools per day (ρ = 0.29, p < 0.001). Lactoferrin correlated with CRP (ρ = 0.57, p < 0.001) and the number of diarrhoeal stools per day (ρ = 0.52, p < 0.001) as did CRP (ρ = 0.52, p < 0.001).. We found no association between cessation of diarrhoea and metronidazole or lactoferrin concentrations. There was a relationship between metronidazole concentrations and markers of inflammation and stool frequency.

Topics: Aged; Aged, 80 and over; Anti-Bacterial Agents; Clostridioides difficile; Enterocolitis, Pseudomembranous; Feces; Female; Humans; Lactoferrin; Male; Metronidazole

Some forms of bovine lactoferrin (bLf) are effective in delaying Clostridioides difficile growth and preventing toxin production. However, therapeutic use of bLf may be limited by protein stability issues. The objective of this study was to prepare and evaluate colon-targeted, pH-triggered alginate microparticles loaded with bioactive bLf and to evaluate their anti-C difficile defense properties in vitro. Different forms of metal-bound bLf were encapsulated in alginate microparticles using an emulsification or internal gelation method. The microparticles were coated with chitosan to control protein release. In vitro drug release studies were conducted in pH-simulated gastrointestinal conditions to investigate the release kinetics of encapsulated protein. No significant release of metal-bound bLf was observed at acidic pH; however, on reaching simulated colonic pH, most of the encapsulated lactoferrin was released. The application of bLf (5 mg/mL) delivered from alginate microparticles to human intestinal epithelial cells significantly reduced the cytotoxic effects of toxins A and B as well as bacterial supernatant on Caco-2 and Vero cells, respectively. These results are the first to suggest that alginate-bLf microparticles show protective effects against C difficile toxin-mediated epithelial damage and impairment of barrier function in human intestinal epithelial cells. The future potential of lactoferrin-loaded alginate microparticles against C difficile deserves further study.

Topics: Alginates; Animals; Caco-2 Cells; Cell Line; Cell Line, Tumor; Chitosan; Chlorocebus aethiops; Clostridioides difficile; Colon; Drug Liberation; Enterocolitis, Pseudomembranous; Epithelial Cells; Humans; Hydrogen-Ion Concentration; Intestinal Mucosa; Lactoferrin; Vero Cells

Clostridium difficile infection (CDI) is a global healthcare problem. Recent evidence suggests that the availability of iron may be important for C. difficile growth. This study evaluated the comparative effects of iron-depleted (1% Fe(3+) saturated) bovine apo-lactoferrin (apo-bLf) and iron-saturated (85% Fe(3+) saturated) bovine holo-lactoferrin (holo-bLf) in a human in vitro gut model that simulates CDI.. Two parallel triple-stage chemostat gut models were inoculated with pooled human faeces and spiked with C. difficile spores (strain 027 210, PCR ribotype 027). Holo- or apo-bLf was instilled (5 mg/mL, once daily) for 35 days. After 7 days, clindamycin was instilled (33.9 mg/L, four times daily) to induce simulated CDI. Indigenous microflora populations, C. difficile total counts and spores, cytotoxin titres, short chain fatty acid concentrations, biometal concentrations, lactoferrin concentration and iron content of lactoferrin were monitored daily.. In the apo-bLf model, germination of C. difficile spores occurred 6 days post instillation of clindamycin, followed by rapid vegetative cell proliferation and detectable toxin production. By contrast, in the holo-bLf model, only a modest vegetative cell population was observed until 16 days post antibiotic administration. Notably, no toxin was detected in this model. In separate batch culture experiments, holo-bLf prevented C. difficile vegetative cell growth and toxin production, whereas apo-bLf and iron alone did not.. Holo-bLf, but not apo-bLf, delayed C. difficile growth and prevented toxin production in a human gut model of CDI. This inhibitory effect may be iron independent. These observations suggest that bLf in its iron-saturated state could be used as a novel preventative or treatment strategy for CDI.

Topics: Anti-Bacterial Agents; Bacterial Load; Clindamycin; Clostridioides difficile; Clostridium Infections; Enterocolitis, Pseudomembranous; Feces; Gastrointestinal Tract; Humans; Intestines; Iron; Lactoferrin; Spores, Bacterial

Number of infection caused by Clostridium difficile in hospitalized children is increasing. Even though children unlike adults seldom develop complications after being ill, cases of persistent diarrhoea triggered by this pathogen and mortality from this origin have been reported. At present the important problem constitute differentiation between the colonization and infection limiting the proper diagnosis of C. difficile infections (CDI) in this age group. The aim of this study was to evaluate the presence of lactoferrin in faeces as the inflammatory marker confirming C. difficile infections in children.. Seventy seven samples of faeces where examined. Among them in 55 toxin A/B or C. difficile toxinogenic strain and in 15 nontoxinogenic C. difficile had been detected, 7 were collected from healthy children. Stool samples were tested with the use of method routinely applied in laboratory: automatic VIDAS C. difficile Toxin A&B test (bioMerieux, France), culture and GDH test. Lactoferrin in stool has been identified with ELISA IBD-SCAN test (Techlab, Blacksburg, VA). The CRP protein was detected with VITROS 5600.. Among 55 children with CDI lactoferrin was detected in 45,5% (25/55) of them. In 30 (54,5%) CDI patients the inflammatory biomarker was not identified. In 15 persons with nontoxinogenic strain cultured, one child had lactoferrin present. CDI was detected most frequently (51%) in 6-11 years old children. The increase of CDI cases was observed in period 2013-2014. Neither differences in frequency of raised CRP level in examined groups of children nor correlation between presence of lactoferrin and CRP was observed.. Lactoferrin an intestinal inflammatory biomarker may be a useful tool in distinguishing between C. difficile infection and colonization. More studies including clinical observations are needed.

Topics: Adolescent; Biomarkers; Child; Child, Preschool; Clostridioides difficile; Enterocolitis, Pseudomembranous; Feces; Humans; Lactoferrin

Measurement of both calprotectin and lactoferrin in faeces has successfully been used to discriminate between functional and inflammatory bowel conditions, but evidence is limited for Clostridium difficile infection (CDI). We prospectively recruited a cohort of 164 CDI cases and 52 controls with antibiotic-associated diarrhoea (AAD). Information on disease severity, duration of symptoms, 30-day mortality and 90-day recurrence as markers of complicated CDI were recorded. Specimens were subject to microbiological culture and PCR-ribotyping. Levels of faecal calprotectin (FC) and lactoferrin (FL) were measured by ELISA. Statistical analysis was conducted using percentile categorisation. ROC curve analysis was employed to determine optimal cut-off values. Both markers were highly correlated with each other (r2 = 0.74) and elevated in cases compared to controls (p<0.0001; ROC>0.85), although we observed a large amount of variability across both groups. The optimal case-control cut-off point was 148 mg/kg for FC and 8.1 ng/µl for FL. Median values for FL in CDI cases were significantly greater in patients suffering from severe disease compared to non-severe disease (104.6 vs. 40.1 ng/µl, p = 0.02), but were not significant for FC (969.3 vs. 512.7 mg/kg, p = 0.09). Neither marker was associated with 90-day recurrence, prolonged CDI symptoms, positive culture results and colonisation by ribotype 027. Both FC and FL distinguished between CDI cases and AAD controls. Although FL was associated with disease severity in CDI patients, this showed high inter-individual variability and was an isolated finding. Thus, FC and FL are unlikely to be useful as biomarkers of complicated CDI disease.

Topics: Aged; Biomarkers; Case-Control Studies; Clostridioides difficile; Clostridium Infections; Enterocolitis, Pseudomembranous; Feces; Female; Humans; Lactoferrin; Leukocyte L1 Antigen Complex; Male; Prospective Studies; Ribotyping

To identify specific fecal biomarkers for symptomatic Clostridium difficile infection and predictors of poor outcomes.. We enrolled 65 children with positive C difficile testing (cases) and 37 symptomatic controls. We also analyzed stool samples from colonized and non-colonized asymptomatic children. We performed enzyme immunoassays to determine fecal interleukin (IL)-8, lactoferrin, and phosphorylated-p38 protein concentrations, and quantitative polymerase chain reaction to determine IL-8 and chemokine ligand (CXCL)-5 RNA relative transcript abundances, and C difficile bacterial burden.. Of 68 asymptomatic controls, 16 were colonized with C difficile. Phosphorylated-p38 was specific for C difficile infection but lacked sensitivity. Fecal cytokines were elevated in samples from symptomatic children, whether cases or controls. In children with C difficile infection, fecal CXCL-5 and IL-8 messenger RNA abundances at diagnosis correlated with persistent diarrhea after 5 days of C difficile infection therapy and with treatment with vancomycin. When children with concomitant viral gastroenteritis were excluded, these correlations persisted. Time-to-diarrhea resolution was significantly longer in patients with elevated fecal cytokines at diagnosis. A logistic regression model identified high CXCL-5 messenger RNA abundance as the only predictor of persistent diarrhea. Conversely, fecal C difficile bacterial burden was not different in symptomatic and asymptomatic children and did not correlate with any clinical outcome measure.. Fecal inflammatory cytokines may be useful in distinguishing C difficile colonization from disease and identifying children with C difficile infection likely to have prolonged diarrhea.

Topics: Biomarkers; Case-Control Studies; Child; Enterocolitis, Pseudomembranous; Feces; Female; Humans; Interleukin-8; Lactoferrin; Male; p38 Mitogen-Activated Protein Kinases; Prospective Studies

Long-term care facilities (LTCF) residents have been estimated to have the highest incidence of diarrheal illness among adults living in the developed world. This study describes undiagnosed diarrhea, intestinal inflammation, and Clostridium difficile colonization in a LTC population and explores whether these are associated with functional decline, as defined by weight loss or a change in cognitive or ADL status.. An observational study of a convenience sampling of residents in a 180-bed LTCF was obtained; evaluation of stool and medical records was done. Stool specimens were evaluated for consistency, gross blood, inflammation (via quantitative fecal lactoferrin, IBD-SCAN), and C difficile (via PCR for gdh). SPSS and STATA were used and significance was set at P < .05.. There were 46 stools collected; 13 of the subjects were male, 28 were older than 65 years, and 35 were prescribed 5 to 15 medications. Twenty-six of the 46 stools collected had elevated quantitative fecal lactoferrin levels. Although only 5 subjects were reported to have diarrhea (4 with elevated lactoferrin), 28 stool specimens were observed to be liquid or semi-solid (19 with elevated lactoferrin), and these liquid/ semisolid stools were significantly correlated with lactoferrin positivity (P = .017). In analysis of functional status, there was no statistically significant association between change in ADL (n = 17) or cognitive status (n = 5) and elevated lactoferrin. However, all 3 subjects who had significant weight loss had elevated lactoferrin, although the mean fecal lactoferrin was not statistically different from those without weight loss. Of the 2 samples with C difficile, both were liquid and, when compared with all other liquid stools (n = 22), the mean lactoferrin was statistically higher (134.1 versus 28.8 microg/mL, P = .008). These 2 subjects had neither weight loss nor change in cognitive status, but 1 had a change in ADL status.. Diarrhea in LTCF residents is underdiagnosed. Diarrhea and the presence of C difficile in the stool are associated with intestinal inflammation, as detected by fecal lactoferrin. With our small numbers, we were not able to identify a specific link; however, we were able to identify a correlation between weight loss and intestinal inflammation, but, with just 2 samples, not C difficile colonization. This relationship highlights the importance of larger studies to further examine the rate of diarrhea in LTCF; the effect of diarrhea and intestinal inflammation on weight loss; and the interaction of C difficile colonization with weight loss, malnutrition, and functional decline.

Topics: Activities of Daily Living; Aged; Aged, 80 and over; Clostridioides difficile; Diarrhea; Enterocolitis, Pseudomembranous; Female; Humans; Lactoferrin; Male; Medical Audit; Middle Aged; Observation; Residential Facilities; Virginia; Weight Loss

Faecal samples from 1007 patients suspected of having diarrhoea caused by Clostridium difficile infection are investigated for the presence of toxins A and B and for the presence of C. difficile-specific glutamate dehydrogenase (GDH). Toxigenic culture is performed on all samples and is used as the 'gold standard' for the purpose of the study. A marker for intestinal inflammation, faecal lactoferrin, is used on any samples that give a positive result in any of the above tests. Part of the study also involves an assessment of six commercial toxin kits to detect the presence of C. difficile toxins in faecal samples. This study revealed that the commercial toxin detection kits used can give rise to false-positive and false-negative results and that all demonstrated poor sensitivity when compared to the gold standard of toxigenic culture. Testing of faecal samples for GDH can be useful as a negative screening method as the results of this test show high correlation with culture. Faecal toxin testing can then be performed on all GDH-positive samples (GDH positivity is independent of toxigenicity in strains of C. difficile). The combined use of GDH and toxin testing, coupled with toxigenic culture, revealed that some patients with diarrhoea who harboured toxigenic strains of C. difficile were faecal toxin-negative. Lactoferrin appears to be a useful marker for the presence of inflammatory diarrhoea.

Topics: Aged; Anti-Bacterial Agents; Bacterial Proteins; Bacterial Toxins; Bacteriological Techniques; Carrier State; Clostridioides difficile; Enterocolitis, Pseudomembranous; Enterotoxins; Enzyme-Linked Immunosorbent Assay; Feces; Glutamate Dehydrogenase; Humans; Infant, Newborn; Lactoferrin; Reagent Kits, Diagnostic; Recurrence; Sensitivity and Specificity

Inflammation is the hallmark of Clostridium difficile associated diarrhoea and lactoferrin is produced by inflammatory cells. The aim of this study was to find out whether faecal lactoferrin latex agglutination (FLLA) assay done simultaneously with Clostridium difficile toxin (CDT) assay would help in the diagnosis of C. difficile infection in paediatric patients. One hundred and fifty faecal samples were obtained from paediatric group of patients. Both FLLA and CDT assays were done in conjunction on these samples. The data were expressed by descriptive statistics. One hundred and nineteen patients received antibiotics while 31 did not receive it. Of the former group 89 (74.8%) had diarrhoea while 30 (25.2%) did not have it. No significant relationship (p=0.287) was seen between antibiotic usage and occurrence of diarrhoea. However, CDT positivity was seen to be influenced by prior antibiotic usage as 51 (42.9%) patients receiving antibiotics were CDT positive when compared to 4 (7.3%) of those who did not receive antibiotics (p=0.002). A highly statistically significant (p<0.001) relationship was seen between CDT and FLLA positivity. FLLA appears to be an useful adjunct for C. difficile associated intestinal diseases in children when both the tests are done simultaneously and when other enteropathogens causing inflammatory diarrhoeas are ruled out.

Topics: Bacterial Toxins; Child; Child, Preschool; Clostridioides difficile; Diarrhea; Enterocolitis, Pseudomembranous; Enterotoxins; Feces; Female; Humans; Infant; Lactoferrin; Latex Fixation Tests; Male

Clostridium difficile is the primary aetiological agent of antibiotic-associated diarrhoea. The faecal lactoferrin (FL) assay is a simple in vitro test which is highly sensitive to the presence of a marker of polymorphonuclear cells. We evaluated the use of the FL assay in conjunction with the C. difficile toxin assay in faecal samples obtained from 231 adult patients. The relationship between C. difficile toxin and FL in both negative and positive status was highly significant statistically (P < 0.001). Therefore, the FL assay performed simultaneously with the C. difficile toxin assay can help rule out asymptomatic carriage of C. difficile.

Topics: Adolescent; Adult; Aged; Aged, 80 and over; Anti-Bacterial Agents; Bacterial Toxins; Carrier State; Clostridioides difficile; Diarrhea; Enterocolitis, Pseudomembranous; Enterotoxins; Feces; Humans; Lactoferrin; Latex Fixation Tests; Middle Aged

Twenty-two patients with Clostridium difficile colitis as determined by positive enzyme immunoassay for toxin A were evaluated for fecal inflammatory markers and their relationship to the severity of illness. Fourteen of 22 specimens were positive for fecal lactoferrin (FLF), with titers from 1:50 to 1:800. Nine of 10 stools tested had ratios of interleukin-1beta (IL-1beta) to IL-1 receptor antagonist (IL-1ra) of >0.01. Seventeen of 22 specimens also had elevated IL-8 concentrations, and 12 of 14 had elevated IL-1beta concentrations. A review of the 18 available patient records revealed that fecal IL-8 concentrations, IL-1beta/IL-1ra ratios, and FLF titers were significantly higher in patients with moderate to severe disease than in patients with mild disease. These findings suggest that the proinflammatory effects of C. difficile may directly influence clinical characteristics of human disease.

Topics: Adult; Aged; Aged, 80 and over; Child; Clostridioides difficile; Enterocolitis, Pseudomembranous; Feces; Female; Humans; Interleukin-1; Interleukin-8; Lactoferrin; Male; Middle Aged; Receptors, Interleukin-1

The accurate and sensitive diagnosis of Clostridium difficile-related diarrhea, normally treated with vancomycin, has become increasingly important in light of the emergence of dangerous new strains of vancomycin-resistant enterococci. In order to improve the threshold for C. difficile diagnosis and treatment, a number of commonly used assays for the diagnosis of C. difficile diarrhea were examined. These included an enzyme-linked immunosorbent assay for C. difficile toxin A (ToxA), a CHO cell culture assay for fecal C. difficile (cyto)toxin B, and a lactoferrin latex agglutination assay for fecal lactoferrin (LFLA). We studied 722 fecal specimens submitted by physicians for C. difficile toxin testing at the Salem, Va., Veterans' Affairs Hospital and at the University of Virginia Medical Center in Charlottesville. Charts were reviewed from 123 Veterans' Hospital patients and 114 University of Virginia patients for clinical criteria indicative of C. difficile diarrhea. An increasing titer of CHO cell cytotoxicity was correlated with an increasing likelihood of ToxA positivity (5 to 90%), LFLA positivity (39 to 77%), and clinical agreement (28 to 85%). However, some data indicate that the CHO cell cytotoxicity assay may be nonspecific when positive only at low titers. When the CHO assay result is positive at high titers, it remains the best diagnostic tool. Yet, when it is positive at a low titer, careful interpretation of the results in conjunction with other assays and the clinical setting is warranted, especially in light of new drug-resistant strains of microorganisms.

Topics: Animals; Bacterial Proteins; Bacterial Toxins; Biological Assay; CHO Cells; Clostridioides difficile; Cohort Studies; Cricetinae; Enterocolitis, Pseudomembranous; Enterotoxins; Enzyme-Linked Immunosorbent Assay; Feces; Humans; Lactoferrin; Latex Fixation Tests; Sensitivity and Specificity

The fecal lactoferrin assay was more sensitive (75%) than methylene blue microscopy (40%) for the detection of leukocytes in Clostridium difficile toxin-positive fecal samples. Although limited sensitivity and specificity precludes its use as a laboratory screening test, it may be a more useful initial test in an algorithm for clinically suspected C. difficile-associated disease.

Topics: Bacterial Proteins; Bacterial Toxins; Bacteriological Techniques; Enterocolitis, Pseudomembranous; Feces; Humans; Lactoferrin; Latex Fixation Tests; Leukocytes; Methylene Blue; Sensitivity and Specificity