Oral Nano Vitamin D Supplementation Reduces Disease Activity in Ulcerative Colitis: A Double-Blind Randomized Parallel Group Placebo-controlled Trial.

INTRODUCTION: Vitamin D possesses anti-inflammatory properties and could be beneficial in ulcerative colitis (UC). METHODS: We studied the effect of oral nano vitamin D3 supplementation on disease activity in active UC [ulcerative colitis disease activity index (UCDAI)≥3]. Patients with active UC and vitamin D <40 ng/mL were randomized to receive either oral nano vitamin D (60,000 IU/d×8 d) or placebo. They were evaluated for disease activity (UCDAI scores, C-reactive protein, erythrocyte sedimentation rate, and fecal calprotectin) at baseline and reassessed at 4 weeks. The response was defined as a 3-point reduction in UCDAI score at 4 weeks and reduction in inflammatory markers. RESULTS: The median vitamin D levels increased from 15.4 to 40.83 mg/dL in vitamin D group (P≤0.001) and marginally from 13.45 to 18.85 mg/dL (P=0.027) in controls. The 3-point reduction in UCDAI was seen more often in vitamin D group as compared with the control (53% vs. 13%; P=0.001). Increase in vitamin D levels correlated with reduction in UCDAI score (P≤0.001; ρ=-0.713), C-reactive protein (P≤0.001; ρ=-0.603), and calprotectin (P=0.004; ρ=-0.368). Patients who achieved target vitamin D of >40 ng/mL (n=17) more often had a 3-point reduction in UCDAI (80% vs. 20%; P≤0.001) and reduction in grade of severity from 60% to 35% (P=0.038). Vitamin D administration (odds ratio, 9.17; 95% confidence interval, 2.02-41.67) and baseline histologic activity (odds ratio, 1.92; 95% confidence intervals, 1.2-3.08) independently predicted response. CONCLUSIONS: Oral nano vitamin D supplementation in active UC is associated with a reduction in disease activity and severity grade and is seen more often in those who achieved a target vitamin D level of 40 ng/mL.

Role of Positron Emission Tomography in Assessing Disease Activity in Ulcerative Colitis: Comparison with Biomarkers.

BACKGROUND: Disease activity in ulcerative colitis (UC) is best assessed clinically by Mayo score. 18-Fluorodeoxyglucose positron emission tomography-computerized tomography (FDG PET-CT) is a noninvasive imaging technique to assess extent, disease activity and response to treatment of UC, especially in high risk population or patients unwilling for endoscopy. AIMS: We conducted a prospective observational study with the aim of assessing and correlating UC disease activity by clinical criteria, endoscopy, histology, serum and fecal biomarkers, and FDG PET-CT. METHODS: Sixty eligible patients of UC were enrolled into three groups (26 remission, 24 moderate and 10 severe activity) as per Mayo score and FDG PET-CT was performed within 72 h of colonoscopy. ESR, CRP, and fecal calprotectin (FC) levels were determined for all patients. RESULTS: Of 60 enrolled patients, 10% patients had proctitis, 43.3% left-sided colitis, and 46.7% extensive colitis. ESR, CRP, FC levels, and rectal PET activity were significantly higher in groups with moderate and severe disease activity. Rectal PET activity showed a significant correlation with the Mayo score (k = 0.465, p < 0.001), endoscopic subscore (k = 0.526, p < 0.001), histological score (k = 0.496, p < 0.001), and FC (k = 0.279, p = 0.031). Extent evaluation by FDG PET-CT and colonoscopy showed a significant correlation (k = 0.582, p < 0.001). Besides, FDG PET-CT identified sacroiliitis in one patient and adenocarcinoma in one patient. CONCLUSION: FDG PET-CT is a reliable noninvasive tool for detection of disease activity, extent in UC with good correlation with Mayo score, histology and fecal biomarkers and accurate predictor of disease remission.

Toxigenic Clostridium difficile isolates from clinically significant diarrhoea in patients from a tertiary care centre.

BACKGROUND & OBJECTIVES: Clostridium difficile is the primary cause of hospital-acquired colitis in patients receiving antibiotics. The pathogenicity of the organism is mainly due to the production of toxins. This study was conducted to investigate the presence of toxigenic C. difficile in the faecal samples of hospitalized patients suspected to have C. difficile infection (CDI) and corroborating the findings with their clinical and demographic data. METHODS: Diarrhoeic samples obtained from 1110 hospitalized patients were cultured for C. difficile and the isolates confirmed by phenotypic and molecular methods. Toxigenicity of the isolates was determined using enzyme-linked immunosorbent assay for toxins A and B. Details of patients included in the study were noted and analyzed. RESULTS: Of the 1110 patients (mean age 39±19.6 yr), 63.9 per cent were males and 36.1 per cent were females. The major antibiotics received by the patients were nitazoxanide (23.9%), penicillins/penicillin combinations (19.0%), quinolones including fluoroquinolones (13.1%), carbapenems (11.5%), glycopeptides (11.0%) and cephalosporins (8.4%). The clinical symptoms predominantly present were watery diarrhoea (56.4%), fever (40.0%) and abdominal pain (35.3%). The underlying diseases were gastrointestinal disorders (52.6%), followed by cancers (13.2%), surgical conditions (8.3%), and hepatic disorders (8.0%). Of the 174 C. difficile isolates, 54.6 per cent were toxigenic. Toxigenic C. difficile was present in all patients with surgical conditions, 65.2 per cent with cancers and 57.1 per cent with gastrointestinal disorders. INTERPRETATION & CONCLUSIONS: C. difficile was found to be an important cause of gastrointestinal infections in hospitalized patients with underlying diseases and on antibiotics. Clinical conditions of the patients correlating with toxigenic culture can be an important tool for establishing CDI diagnosis.

Fidaxomicin--the new drug for Clostridium difficile infection.

Clostridium difficile is one of the many aetiological agents of antibiotic associated diarrhoea and is implicated in 15-25 per cent of the cases. The organism is also involved in the exacearbation of inflammatory bowel disease and extracolonic manifestations. Due to increase in the incidence of C. difficile infection (CDI), emergence of hypervirulent strains, and increased frequency of recurrence, the clinical management of the disease has become important. The management of CDI is based on disease severity, and current antibiotic treatment options are limited to vancomycin or metronidazole in the developing countries. this review article briefly describes important aspects of CDI, and the new drug, fidaxomicin, for its treatment. Fidaxomicin is particularly active against C.difficile and acts by inhibition of RNA synthesis. Clinical trials done to compare the efficacy and safety of fidaxomicin with that of vancomycin in treating CDI concluded that fidaxomicin was non-inferior to vancomycin for treatment of CDI and that there was a significant reduction in recurrences. The bactericidal properties of fidaxomicin make it an ideal alternative for CDI treatment. However, fidaxomicin use should be considered taking into account the potential benefits of the drug, along with the medical requirements of the patient, the risks of treatment and the high cost of fidaxomicin compared to other treatment regimens.

In vitro and in vivo activities of the novel Ketolide RBx 14255 against Clostridium difficile.

The MIC(90) of RBx 14255, a novel ketolide, against Clostridium difficile was 4 µg/ml (MIC range, 0.125 to 8 µg/ml), and this drug was found to be more potent than comparator drugs. An in vitro time-kill kinetics study of RBx 14255 showed time-dependent bacterial killing for C. difficile. Furthermore, in the hamster model of C. difficile infection, RBx 14255 demonstrated greater efficacy than metronidazole and vancomycin, making it a promising candidate for C. difficile treatment.

Evaluation of fecal myeloperoxidase as a biomarker of disease activity and severity in ulcerative colitis.

BACKGROUND: Subclinical inflammation in ulcerative colitis (UC) can predispose to relapses and biomarkers can detect mucosal inflammation. AIMS: To study the role of fecal myeloperoxidase (FMPO) in assessing disease activity and response to therapy in UC. METHODS: Patients with UC attending our hospital from July 2005 to September 2006 were studied. All patients underwent clinical, endoscopic, and histological assessment for disease extent and severity. Estimation of FMPO levels at baseline and on follow-up was carried out. Age-matched healthy controls were studied for FMPO levels. RESULTS: A total of 55 patients of UC (30 males, 25 females, mean age 38.6 ± 12 years) and 54 age-matched controls (mean age 37.6 ± 13.6 years) were studied. Cases had higher median MPO levels than controls (0.42 [IQR 0.84] vs. 0.06 [IQR 0.12]); (p < 0.001). Cases with endoscopically more severe disease (Gr III & IV; n = 18) had higher median FMPO levels compared to those with milder disease (Gr II, n = 37), [0.075 (IQR 1.315) vs. 0.315 (IQR 0.813); p = 0.02]. The median MPO level in 27 patients was 0.58 [IQR 0.89] units/ml at presentation which on follow-up decreased significantly to 0.18 [IQR 0.42] units/ml (p value 0.002). However, there was no significant association between FMPO and endoscopic extent and histological scores of activity and chronicity. CONCLUSIONS: Fecal MPO is an effective biomarker for assessing disease activity and response to therapy in patients with ulcerative colitis.

Effect of Lactobacillus acidophilus & epidermal growth factor on experimentally induced Clostridium difficile infection.

BACKGROUND & OBJECTIVES: Clostridium difficile-associated disease (CDAD) remains an important nosocomial ailment. Antimicrobial therapy used for CDAD gives inconsistent results. This experimental study was planned to investigate the beneficial effects of Lactobacillus acidophilus and epidermal growth factor (EGF) for CDAD management. METHODS: Among 10 groups of BALB/c mice (6 in each), group 1 served as controls receiving no inoculum. Animals in groups 2-10 received C. difficile, those in groups 3, 6 and 9 received L. acidophilus and those in groups 4, 7 and 10 received EGF after C. difficile inoculation. Animals in groups 5-7 were pre-treated with ampicillin and those in groups 8-10 with lansoprazole prior to C. difficile. The animals were killed and investigated for colonisation by C. difficile and toxin production, myeloperoxidase (MPO) activity and histopathology. RESULTS: Colonisation by C. difficile was found to be significantly different (P<0.001) in the various groups. C. difficile toxin titres and MPO activity were significantly lower in animals given L. acidophilus and EGF after ampicillin (groups 6 and 7) and lansoprazole (groups 9 and 10). The severity of acute inflammation was also significantly less (P<0.05) in caecal and colonic segments of animals in groups 6 and 7 compared to those in group 5. Although the severity of acute inflammation was less in the caecal and colonic segment of animals in groups 9 and 10, the reduction was not significant compared to group 8. INTERPRETATION & CONCLUSIONS: Our findings showed that the administration of L. acidophilus and EGF reduced the severity of C. difficile infection in the experimental animals.

Can High-Sensitivity C-Reactive Protein Levels Predict Functional Outcome Following Epidural Steroid Injection in Patients with Lumbar Disc Disease?

STUDY DESIGN: Prospective cohort study. PURPOSE: Inflammatory cytokines produced at the site of disc herniation are considered as pain generators in patients with lumbar disc disease. Whether a high-sensitivity C-reactive protein (hs-CRP) assay can be used in order to predict the quantum of inflammation surrounding nerve roots is a matter of investigation. This study aimed to evaluate the association of hs-CRP level and functional outcomes measured by the Modified Oswestry Low Back Pain Disability Questionnaire (MODY) before and after epidural steroid injection (ESI) in patients with lumbar disc disease. OVERVIEW OF LITERATURE: Although many studies examining the role of hs-CRP levels and lumbar pain have been published previously, the results are equivocal, and there is no clear consensus regarding which patients will benefit from an ESI. METHODS: This was a prospective study, with 77 patients in the study group and 23 participants in the control group. Baseline hs-CRP levels were obtained for both groups. Study group patients received a single ESI and were subjected to detailed pre- and postprocedure evaluation using MODY scores. For this group, hs-CRP levels were measured at 1 and 2 months after injection. RESULTS: Out of 77 patients, 52 had acute and 25 had chronic low back pain. Thirty-six patients with acute pain obtained significant improvement, while 16 had an insignificant response to the ESI. None of the chronic cases had a significant response. The mean baseline hs-CRP (mg/L) among the study group (29.83±10.43) was significantly higher than for the controls (10.26±2.783). The baseline hs-CRP among acute cases, where post ESI MODY score at 2 months had significant reduction, was 32.19±5.126, and those with insignificant reduction was 18.13±7.949 (p<0.001). CONCLUSIONS: Baseline hs-CRP levels can be used to prognosticate the outcome following ESI in patients with acute lumbar disc disease, with radicular pain refractory to physiotherapy and analgesics.

Effect of biotherapeutics on cyclosporin-induced Clostridium difficile infection in mice.

BACKGROUND AND AIM: Immunosuppressive therapy may precipitate Clostridium difficile associated disease (CDAD). We evaluated the role of cyclosporin in the development of CDAD in the experimental mouse model and studied the effect of probiotic and epidermal growth factor (EGF) as biotherapeutics measures. METHODS: BALB/c mice (n = 24) were divided into four groups. Group I animals not given any inoculum served as controls. Animals in the remaining three groups (Group II, III and IV) were given cyclosporin daily from days 1-7 followed by C. difficile inoculum on day 8. Additionally, the animals received Lactobacillus acidophilus (Group III) and EGF (Group IV) for one-week post C. difficile challenge. The animals were evaluated for colonization and toxin production by C. difficile, myeloperoxidase (MPO) activity and histopathological changes. RESULTS: Clostridium difficile was colonized and elaborated its toxins in animals receiving cyclosporin and C. difficile. MPO activity was significantly higher (P < 0.05) and histopathological epithelial damage, cryptitis and acute inflammatory changes were seen in the cecum and colon. C. difficile count, toxins A and B titers and MPO activity were significantly lowered (P < 0.05) in animals receiving probiotic and EGF. Histopathologically, mucodepletion and inflammatory infiltrate were decreased in the biotherapeutic receiving animals. CONCLUSIONS: Cyclosporin led to the development of mild to moderate CDAD in animals. Administration of biotherapeutics reduced the severity of CDAD. Future clinical trials are needed for further investigation of these potential biotherapeutic measures.

Clinical spectrum & pathogenesis of Clostridium difficile associated diseases.

Clostridium difficile is the major aetiological agent of antibiotic associated diarrhoea and colitis. The majority of hospitalized patients infected by C. difficile are asymptomatic carriers who serve as silent reservoirs for continued C. difficile contamination of the hospital environment. C. difficile associated disease (CDAD) is a serious condition with mortality up to 25 per cent in frail elderly people. C. difficile infection may present itself in several forms with both colonic and extracolonic manifestations. Several factors are involved in determining whether or not a patient develops C. difficile infection. These include factors related to the pathogen as well as the host. Transmission of C. difficile can be endogenous or exogenous. Colonization of the pathogen occurs when the gut flora gets disrupted due to various factors. The main virulence factors for CDAD are the two potent toxins: toxin A and toxin B which share 63 per cent of amino acid sequence homology and act on small guanosine triphosphate binding proteins. The emergence of the global hypervirulent C. difficile strain has been a cause of concern. Diagnosis of CDAD infection can be done by detection of C. difficile toxin in the stool specimen. Vancomycin is the drug of choice for severely ill patient, whereas metronidazole can be used for mild to moderately ill patients. Clinical spectrum, the factors precipitating CDAD, pathogenesis, diagnostic assay and treatment of the disease are reviewed.

Fecal lactoferrin, myeloperoxidase and serum C-reactive are effective biomarkers in the assessment of disease activity and severity in patients with idiopathic ulcerative colitis.

BACKGROUND AND AIM: Disease activity and severity of ulcerative colitis (UC) is assessed using colonoscopy, which is invasive, costly and has poor patient acceptability. The role of non-invasive biomarkers of intestinal inflammation in the evaluation of patients with UC is not known. The aim of the study was to examine the role of serum C-reactive protein (SCRP), fecal myeloperoxidase (FMPO) and fecal lactoferrin (FLF) in assessing disease severity, activity and response to therapy. METHODS: Consecutive patients with idiopathic UC (IUC) attending our hospital from July 2005 to September 2006 were studied. All underwent clinical, endoscopic and histological assessment for disease activity, extent, severity and estimation of SCRP, FMPO and FLF levels at baseline and follow up (FU). An equal number of healthy age-matched controls were studied for biomarker levels. RESULTS: A total of 37 patients (mean age 37 +/- 12 years) were studied. All three biomarkers were elevated more often in the cases than in the controls (all P = 0.000). Cases with severe IUC had higher CRP, MPO and FLF titers than those without severe IUC. At FU, a significant fall in biomarker levels paralleled the reduction in Mayo's scores. All three biomarkers showed a high degree of correlation with each other. The areas under the curve for FLF, MPO and CRP were 1.00, 0.867 and 0.622, respectively. The sensitivity and specificity of markers were: FLF (94%, 100%), FMPO (89%, 51%) and SCRP (24%, 100%). CONCLUSION: Biomarkers are useful in assessing disease activity, severity and response to therapy in patients with UC. They showed a high degree of correlation with each other.

Pancreatic disease patients are at higher risk for Clostridium difficile infection compared to those with other co-morbidities.

BACKGROUND: Surveillance of Clostridium difficile infection (CDI) in patients with underlying diseases is important because use of prophylactic antibiotics makes them prone to CDI. Epidemiology of CDI in this high-risk population is poorly understood. A study was conducted to evaluate the impact of CDI in patients with specific underlying co-morbidities. METHOD: A total of 2036 patients, whose fecal samples were processed for C. difficile toxin A and B assay by ELISA formed the basis of study. Patients with underlying diseases were classified based on the organ/kind of disease as pancreatic (n = 340), renal (n = 408), hepatic (n = 245), malignant (n = 517) and miscellaneous disease (n = 526). Laboratory records of clinical and demographic details were reviewed. The association of CDI with age, gender, antibiotic receipt, clinical symptoms and underlying co-morbidities was analyzed. Variation in CDI cases based on age groups was also investigated. RESULT: Clostridium difficile toxin positivity was 21.6% in general, whereas it was 30.6% in the pancreatic, 17.9% in the renal, 19.6%, in the hepatic, 21.3% in the malignancy and 20.0% in the miscellaneous disease groups. Toxin positivity was the lowest (14.8%) for female gender under renal disease and the highest (31.8%) for patients aged 40 to < 60 years, under pancreatic disease. Bloody diarrhea was a significant predictor for C. difficile toxin positivity. C. difficile toxin status irrespective to the underlying diseases was neither dependent on gender, age-groups or the number of antibiotics used. Association between patients' gender, age and antibiotics receipt with underlying disease conditions, respective to C. difficile toxin status showed significance in relation to male gender (p < 0.05), age 40 to < 60 years (p = 0.03) and those receiving single (p = 0.09) or multiple antibiotics (p = 0.07). CONCLUSION: Pancreatic disease patients are at a higher risk for developing CDI, and particularly male gender, age 40 to < 60 years and those receiving antibiotics are at significant risk.

Vitamin D deficiency in adult patients with ulcerative colitis: Prevalence and relationship with disease severity, extent, and duration.

BACKGROUND: Vitamin D plays a key role in gut immunity and maintenance of the mucosal barrier. Vitamin D deficiency (VDD) worsens ulcerative colitis (UC) and its supplementation ameliorates the disease in mouse models. The prevalence and predictors of VDD in UC are not known. METHODS: Consecutive patients with UC (n = 80) underwent clinical, endoscopic, and histological evaluation to assess the extent, severity using UC disease activity index (UCDAI) score, and duration of illness. An equal number of age and gender-matched healthy adults without any features of inflammatory bowel disease (IBD) living in the same latitude were identified as controls. The serum 25-hydroxy vitamin D3 level was estimated. The subjects were classified as deficient (< 20 ng/mL), insufficient (20-32 ng/mL), sufficient (32-80 ng/mL), and optimal (> 80 ng/mL) based on vitamin D levels. Chi-square test and Mann-Whitney U test were done to identify factors associated with vitamin D deficiency. RESULTS: The patients and controls were similar in age and gender (40 ± 11.4 years, 51% male vs. 40 ± 12 years, 51% male; p = 1.000). Median vitamin D levels among patients were lower than the controls (18.1 ng/mL [IQR 14] vs. 32.5 ng/mL [IQR 36]; p < 0.001). Patients were more often VDD (56% vs. 40%) or insufficient (34% vs. 9%) and less often sufficient (9% vs. 40%) or optimal (1% vs. 11%), in contrast to controls (p < 0.001). Median vitamin D levels were lower in those with UCDAI > 6 (15 vs. 21 ng/mL; p = 0.01), having pancolitis (13 vs. 21 ng/mL, p = 0.01), and longer duration of illness > 2 years (13.8 vs. 20.8; p = 0.025). Vitamin D levels showed a negative correlation with frequency of stools (rho = - 0.244, p = 0.05), disease duration (rho = - 0.244, p = 0.007) and UCDAI score (r = - 0.348, p = 0.002). CONCLUSION: VDD is highly prevalent among patients with UC. Patients with longer disease duration, more severe symptoms, and pancolitis are likely to have lower vitamin D levels.

Speciation of fecal Candida isolates in antibiotic-associated diarrhea in non-HIV patients.

Candida is the most frequently encountered fungal infection of the gastrointestinal tract after antibiotic exposure. The pathogenesis of Candida probably varies with each species. The speciation of fecal Candida after antibiotic use is not well investigated. One hundred and eleven fecal samples negative for Clostridium difficile toxin and for other enteric pathogens formed the basis of our investigation. The diarrheic samples came from patients receiving antibiotics in a hospital setting. In addition, samples from 30 age-matched healthy participants who did not receive antibiotics and did not have diarrhea were also studied. Initially, a Gram stain identification for yeasts was performed for each fecal sample, then each sample was cultured on Sabouraud's dextrose agar. Candida was isolated as pure growth (>10(5) cfu/ml) from the stools of 32 (28.8%) patients. The identification of the yeast was done based upon a combination of morphological, physiological and biochemical criteria. The predominant isolates were C. tropicalis (n=16), C. albicans (n=14) and C. krusei (n=2). Candida isolated from healthy participants (n=4) was sparse and therefore not speciated. Different Candida spp. may play an important role in precipitating antibiotic-associated diarrhea.

Clostridium perfringens enterotoxin in antibiotic-associated diarrhea.

Clostridium perfringens type A is associated with 5-20% cases of antibiotic-associated diarrhea (AAD) even though Clostridium difficile is implicated in the most severe cases. Fecal specimens from one hundred hospitalized patients, who developed diarrhea regardless of antibiotic intake and who were negative for C. difficile toxin assay, were investigated for C. perfringens enterotoxin (CPE). Simultaneously, cultures were set up for other possible aetiological factors. Ten healthy controls were also similarly investigated. CPE was positive in 2/100 (2%) of the patients and the samples were also positive for the organism in culture. Other organisms isolated were non-toxigenic C. difficile (4%), staphylococci (6%), Candida (18%) and Klebsiella pneumoniae (1%). Stool samples from healthy controls grew mixed growth of no significance and CPE was negative in all of them. Detection of CPE is not part of routine laboratory investigation due to resource implication. Criteria for initiating investigations have to be therefore established by understanding the true burden of C. perfringens-associated AAD by further research.

Characterization of biofilms in biliary stents and potential factors involved in occlusion.

AIM: To quantify the components in biofilms and analyze the predisposing factors involved in occlusion of biliary stents. METHODS: In a prospective study conducted from April 2011 to March 2014 at a tertiary care hospital, all consecutive patients who required endoscopic biliary stent exchange/removal were included. Etiology of the biliary disease was diagnosed by imaging, cytology and on follow-up. Clinical details of patients with biliary stent retrieval were noted. All extracted stents were collected in sterile containers and immediately processed for quantification of biofilm proteins and polysaccharides. Molecular identification of commonly known and unknown bacteria was performed by polymerase chain reaction and density gradient gel electrophoresis methods. RESULTS: Eighty one patients (41 males) with age range of 20-86 years were studied. The underlying causes for stent insertion were bile duct stones (n = 46; 56.8%) benign stricture (n = 29; 35.8%) and malignancy (n = 6; 7.4%) with cholangitis in 50 (61.7%) patients. The retrieved stent sizes were 7 Fr (n = 62; 76.5%) and 10 Fr (n = 19; 23.5%) with 65 days median insertion duration. Polybacterial consortia were detected in 90.1% of the stents. The most common bacteria identified by polymerase chain reaction alone and/or sequencing were Pseudomonas (n = 38), Citrobacter (n = 23), Klebsiella (n = 22), Staphylococcus (n = 20), Serratia (n = 16), Escherichia coli (n = 14), Streptococcus (n = 13), Enterococcus (n = 13), Aeromonas (n = 12), Proteus (n = 10) and Enterobacter (n = 9). Protein concentration according to gender (0.547 ± 0.242 mg/mL vs 0.458 ± 0.259 mg/mL; P = 0.115) as well as age > 60 years and < 60 years (0.468 ± 0.295 mg/mL vs 0.386 ± 0.238 mg/mL; P = 0.205) was non-significant. However, polysaccharide concentration was significant both according to gender (0.052 ± 0.021 mg/mL vs 0.049 ± 0.016 mg/mL; P < 0.0001) and age (0.051 ± 0.026 mg/mL vs 0.038 ± 0.016 mg/mL; P < 0.011). Protein concentration in the biofilm was significantly higher (0.555 ± 0.225 mg/mL vs 0.419 ± 0.276 mg/mL; P = 0.018) in patients with cholangitis, lower (0.356 ± 0.252 mg/mL vs 0.541 ± 0.238 mg/mL; P = 0.005) in the 10 Fr group than the 7 Fr group, and significantly higher (0.609 ± 0.240 mg/mL vs 0.476 ± 0.251 mg/mL; P = 0.060) in stents of ≥ 6 mo of indwelling time. However presence/absence of cholangitis, size of stent, indication of stent insertion and indwelling time did not affect the quantity of polysaccharide concentration. CONCLUSION: Plastic stents retrieved from patients with biliary tract disease showed polymicrobial organisms with higher protein content among patients with cholangitis and those with smaller diameter stents. Longer indwelling duration had more biofilm formation.

Quantification of major constituents of biofilms in occluded pancreatic stents.

PURPOSE: Biofilms comprise bacterial populations enclosed in a matrix that attaches to surfaces such as medical stents. We characterized the biofilm components in occluding pancreatic stents and investigated potential factors for the formation of the biofilms. METHODOLOGY: The clinical details of 24 patients (M : F, 15 : 9) undergoing pancreatic stent retrieval were noted and the retrieved stents were processed for the quantification of biofilm proteins and polysaccharides and the molecular identification of bacteria. RESULTS: The patients' ages ranged from 16 to 62 years. The underlying indications for stent insertion were bile duct stone prophylaxis against post-endoscopic retrograde cholangiopancreatography (ERCP) pancreatitis (n=7; 29.1 %) and pancreatic ductal leaks (n=17; 70.9 %). The retrieved stent sizes were 5 Fr (n=5; 20.8 %) and 7 Fr (n=19; 79.2 %), with a mean insertion duration of 103 days. The polybacteria detected by PCR in 95.8 % of the stents were Pseudomonas (n=8), Staphylococcus (n=8), Serratia (n=5), Aeromonas (n=4), Proteus (n=4), Klebsiella (n=4), Escherichia coli (n=4), Enterococcus (n=4), Streptococcus (n=4), Citrobacter (n=3), Bacillus (n=2), Enterobacter (n=1), Vibrio (n=1) and Clostridium (n=1). Several other organisms were identified by sequencing. The mean protein concentration was 0.585±0.29 mg ml-1 and the mean polysaccharide concentration was 0.054±0.03 mg ml-1. No significant differences were observed in the quantity of proteins and polysaccharides (P=0.933) for various factors, namely gender, presence of cholangitis, indications for stenting, stent sizes and duration of indwelling stents. Age was found to be a significant (P=0.013) factor for protein deposition for those aged >50 years. CONCLUSION: The majority of the pancreatic stents grew polymicro-organisms, and those from patients aged >50 years showed significant deposition of protein, which is a key element in biofilm formation. Understanding the constituents of the biofilms in pancreatic stents could be very useful in developing future strategies for the prevention of biofilm formation.

Toxinotyping and Sequencing of Clostridium difficile Isolates from Patients in a Tertiary Care Hospital of Northern India.

BACKGROUND: Clostridium difficile is an important cause of infectious colitis among hospitalized patients across the globe. The pathogenic potential of C. difficile in producing significant morbidity and mortality is mainly due to production of toxins A and B. The outbreaks of C. difficile infection (CDI) are due to changes in the genetic sequences of the organism. There is hardly any molecular study reported on the prevalent types of C. difficile strains in India. Toxinotyping and sequencing of locally circulating C. difficile isolates from patients presenting to our tertiary care center of North India were done. MATERIALS AND METHODS: C. difficile strains (n = 174) isolated from 1,110 fecal samples from patients with suspected CDI were subjected to toxinotyping and partial sequencing of tcdA and tcdB genes. Comparison of nucleotide sequences with reference C. difficile 630 strain using BLAST was made and translated into corresponding amino acid sequences by ExPASy. RESULTS AND DISCUSSION: Of 174 C. difficile isolates, 121 were toxigenic, belonging to toxinotype 0 (n = 76) and VIII (n = 45). Partial sequencing of toxin genes using bioinformatics approaches revealed changes in toxin A sequences of five (50%) C. difficile isolates, but the translated nucleotide sequences showed substitution in only three of them. No variation was seen in the toxin B nucleotide sequences. Interstrain variations were found in the clinical C. difficile isolates in our region. CONCLUSION: PCR amplified toxigenic genes followed by sequencing can help to identify genetic changes and pathogenicity of varied collection of C. difficile isolates.

Evaluation of an in-house rapid diagnostic method for detection of Salmonella enterica serovar Typhi in fecal specimens.

Salmonella enterica serovar Typhi is the etiological agent of typhoid fever. Laboratory diagnosis requires isolation and identification of the organism from the patient's blood or feces. Feces is the specimen most commonly submitted to laboratories. Detection of bacterial antigens is an important adjunct to laboratory diagnosis. We carried out an in-house diagnostic method by preparing test reagents comprising of latex beads coated with specific antisera to detect Vi, O9 and H-d antigens of S. typhi. Fecal specimens from one hundred patients with diarrhea and fever as well as from twenty healthy controls were incubated for enrichment in Selenite F broth for 6 hours or overnight. Latex agglutination tests to detect antigens of S. typhi were carried out on centrifuged broth supernatants. Parallel cultures on media selective for S. typhi were also set up. Nine of the supernatants were positive for two or more specific antigens and S. typhi grew in three of the corresponding cultures. None of the samples from 20 healthy controls were positive by either the diagnostic method or by culture. The result of the in-house diagnostic assay can be obtained overnight and may help in directing immediate antimicrobial therapy.

Estimation of endotoxin in infected bile from patients with biliary diseases.

Diseases of the biliary tract can get complicated by infection. Endotoxin may theoretically be responsible for damage to the gall bladder due to its numerous pathophysiological effects. The aim of the present study was to detect and semi-quantitate the amount of endotoxin present in the bacteriologically positive bile samples and to correlate the endotoxin levels with the clinical profile of the patients. One hundred patients with gall bladder diseases and with infected bile constituted the population for investigation. The clinical profile included presence of fever, jaundice, abdominal pain and gall bladder stones. Endotoxin detection and semi-quantitation in the bile samples were carried out using the Limulus amoebocyte assay: Of 100 infected bile samples investigated, 9 samples (9%) were positive for endotoxin ranging from 1.9 EU/ml to 15 EU/ml. Four of them had Klebsiella pneumoniae, 2 had Acinetobacter anitratus and one each of the remaining 3 samples was positive for (i) Escherichia coli and Serratia marcescens (ii) Pseudomonas aeruginosa and (iii) Salmonella enteritidis. The stool sample of the patient with S. enteritidis in the bile also grew the same microorganism. Statistical analysis showed a significant increase in the presence ofjaundice (p<0.05) and abdominal pain (p<0.01) in the endotoxin positive patients compared to the endotoxin negative ones. Hitherto this is the first report that investigated the endotoxin levels in the bile of patients with gall bladder and biliary tract diseases, along with their biliary bacterial profile. Further research is warranted on the effects of endotoxin on gall stone formation.