Translocation of gut flora and its role in sepsis.

Bacterial translocation is the invasion of indigenous intestinal bacteria through the gut mucosa to normally sterile tissues and the internal organs. Sometimes instead of bacteria, infl ammatory compounds are responsible for clinical symptoms as in systemic infl ammatory response syndrome (SIRS). The difference between sepsis and SIRS is that pathogenic bacteria are isolated from patients with sepsis but not with those of SIRS. Bacterial translocation occurs more frequently in patients with intestinal obstruction and in immunocompromised patients and is the cause of subsequent sepsis. Factors that can trigger bacterial translocation from the gut are host immune defi ciencies and immunosuppression, disturbances in normal ecological balance of gut, mucosal barrier permeability, obstructive jaundice, stress, etc. Bacterial translocation occurs through the transcellular and the paracellular pathways and can be measured both directly by culture of mesenteric lymph nodes and indirectly by using labeled bacteria, peripheral blood culture, detection of microbial DNA or endotoxin and urinary excretion of non-metabolisable sugars. Bacterial translocation may be a normal phenomenon occurring on frequent basis in healthy individuals without any deleterious consequences. But when the immune system is challenged extensively, it breaks down and results in septic complications at different sites away from the main focus. The factors released from the gut and carried in the mesenteric lymphatics but not in the portal blood are enough to cause multi-organ failure. Thus, bacterial translocation may be a promoter of sepsis but not the initiator. This paper reviews literature on the translocation of gut fl ora and its role in causing sepsis.

Anti-ganglioside antibodies in patients with Guillain Barré syndrome and other neurological disorders.

A study was performed on 59 Guillain-Barré syndrome (GBS) cases, 58 neurological controls (NC) and 60 non-neurological controls (NNC) to investigate the association of anti-ganglioside antibodies in GBS and other neurological disorders. Campylobacter jejuni was isolated from 5.7% of GBS patients. Anti-ganglioside immunoglobulin G was present in 82% and immunoglobulin M in 46% in acute inflammatory demyelinating polyneuropathy patients, 70% and 44% respectively in acute motor axonal neuropathy subgroup and 38% each in acute motor sensory axonal neuropathy subgroup. Though high intensity of anti-gangliosides was present in the GBS patients, the NC patients also had adequate anti-gangliosides compared with the NNC group.

Effect of biotherapeutics on antitoxin IgG in experimentally induced Clostridium difficile infection.

Purpose: Recurrent diarrhoea after successful treatment of primary Clostridium difficile associated disease (CDAD) occurs due to bowel flora alterations and failure to mount an effective antibody response. Apart from antibiotics, risk factors include immunosuppressive and acid-suppressive drug administration. Biotherapeutics such as probiotic and epidermal growth factor (EGF) may offer potential effective therapy for CDAD. Materials and Methods: The effect of biotherapeutics in mounting an antibody response against C. difficile toxins was studied in BALB/c mice challenged with C. difficile after pre-treatment with ampicillin, lansoprazole or cyclosporin. Sera from sacrificed animals were estimated for antitoxin IgG by enzyme linked immunosorbent assay. Results: Antitoxin IgG was significantly higher (P<0.05) in C. difficile challenged groups compared to unchallenged controls, but insignificant (P>0.05) in animals in which C. difficile was given after pre-treatment with cyclosporin compared to those without any pre-treatment, or pre-treatment with antibiotic or lansoprazole. In inter-subgroup comparisons also significant anomaly in production of antitoxin IgG was found. The antitoxin IgG levels were raised in animals administered C. difficile after pre-treatment with ampicillin, but lower in animals administered cyclosporin. High levels of antitoxin IgG were also found in the serum samples of animals receiving lansoprazole and C. difficile. Conclusions: Probiotics showed their beneficial effect by boosting the immune response as seen by production of antitoxin IgG. Oral administration of EGF did not affect the immune response to C. difficile toxins as significant increase was not observed in the serum antitoxin IgG levels in any of the groups investigated.

Preliminary investigation of environmental prevalence of Clostridium difficile affecting inpatients in a north Indian hospital.

A preliminary study was conducted to see the prevalence of Clostridium difficile in patients and their environment in a tertiary care hospital. Seventy-nine fecal specimens from hospitalized patients, 176 swab samples from beds and 48 from hands of hospital personnel were investigated. Sixty-three patients received antibiotics and 14 proton pump inhibitors. Abdominal pain was observed in 16 patients with fever in 15 of them. C. difficile culture was positive in 12.6% patients at initial sampling but none were toxin-positive. Eight patients developed diarrhea and five were both culture and toxin-positive. Fifty-one percent of bed swab samples and 62.5% of hand swab samples were culture positive. Similarly 8.5% of bed swab samples and 4.2% of hand swab samples were positive for toxins A and B. The environmental cross-infection between patients and carriage by hospital personnel are plausible sources of C. difficile infection and spread in our hospital.

Cost-effective screening of pooled faecal specimens from patients with nosocomial diarrhoea for Clostridium perfringens enterotoxin.

Purpose: Clostridium perfringens is a significant cause of nosocomial AAD. The prevalence of C. perfringens enterotoxin (CPE)-positive stool specimens in hospitalised patients is very low in the Indian setting making the diagnostics very expensive. Therefore, a cost-effective diagnostic approach to screen faecal specimens for CPE was devised. Materials and Methods: Faecal specimens from 540 hospitalised patients with various ailments and from 340 healthy subjects were investigated for CPE. An aliquot of pooled faecal supernatants was made by mixing 100 μl each of 10 specimens to be tested. Each aliquot was investigated for the presence of CPE by an enzyme immunoassay. A repetition of the assay was done with individual specimens of the pooled aliquots from each positive well as seen visually by colour development. Results: Of the 540 patient specimens tested, 405 (75%) patients were on antibiotics, the predominant ones being cephalosporins, penicillin, quinolones, aminoglycosides, etc. During the time of sampling, diarrhoea was present in 481 (89%), abdomen pain in 203 (37.6%) and fever in 242 (44.8%) patients. C. perfringens enterotoxin was positive in nine wells of the 540 pooled test specimens whereas all of the pooled 340 control samples were negative. Repeat of individual specimens comprising the nine wells with positive samples helped to identify the individual patients positive for CPE. Conclusion: Only two CPE kits were needed for a total of 880 faecal specimens tested. The cost-effective diagnostic approach to screen faecal specimens for CPE, as described herein will help to save institutional resources.

Established and potential risk factors for clostridum difficile infection.

Clostridium difficile is the aetiological agent for almost all cases of pseudo membranous colitis and 15-25% of antibiotic associated diarrhoea. In recent years, C. difficile associated disease (CDAD) has been increasing in frequency and severity due to the emergence of virulent strains. Severe cases of toxic mega colon may be associated with mortality rates of 24-38%. The prevalence of CDAD is global and the incidence varies considerably from place to place. In the initial stages of its discovery, C. difficile infection was regarded mainly as an outcome of antibiotic intake and not as a life threatening disease. Intervention by man has produced conditions making C. difficile a significant cause of morbidity and mortality. The recent outbreak of CDAD in Quebec has sent the alarm bells ringing. Apart from a threefold increase in the incidence of CDAD, clinicians have also reported a higher number of cases involving toxic mega colon, colectomy or death. Among all the risk factors, inclusive of the host and the environmental factors, antibiotics are the most important ones. Surgical patients comprise 55-75% of all patients with CDAD due to the fact that perioperative prophylaxis requires the use of antibiotics. However, other drugs such as immunosuppressants and proton pump inhibitors are also important risk factors. Thus CDAD is a growing nosocomial and public health challenge. Additionally, the recognition of community acquired CDAD signals the presence of several risk factors. In this review, the established and potential risk factors of CDAD, along with the epidemiology, diagnostic modalities, management and preventive measures of the disease have been elaborated.

Faecal excretion of brush border membrane enzymes in patients with clostridium difficile diarrhoea.

PURPOSE: To look for the presence of intestinal brush border membrane (BBM) enzymes in the faecal samples of patients with Clostridium difficile association. METHODS: One hundred faecal samples were investigated for C.difficile toxin (CDT). Simultaneous assays for faecal excretion of intestinal BBM enzymes viz., disaccharidases, alkaline phosphatase (AP) and leucine aminopeptidase (LAP) were also done. RESULTS: C.difficile toxin was detected in 25 (25%) of the samples with a titre ranging from 10 to 160. No significant difference (p>0.05) was seen between the CDT positive and negative groups with any of the disaccharidases studied. However, significant increase (p< 0.05) in the AP excretion was observed in the CDT positive patients compared to the CDT negative group. In contrast to this, a significant decrease (p< 0.001) in the LAP enzyme excretion was observed in the latter group. CONCLUSIONS: The results of this study suggest that there is a significant disturbance in the intestinal BBM enzymes in patients with C.difficile diarrhoea.

Bacterial profile of dairy products sold in Chandigarh.

Gastrointestinal infections are known to occur due to bacterial contamination of dairy products. A total of 141 dairy products viz. kulfi, ice cream and softy samples were investigated bacteriologically. Staphylococcus was the predominant organism isolated followed by Klebsiella pneumoniae, Escherichia coli and Campylobacter. Two of the samples also yielded Yersinia. The total bacterial counts obtained ranged from 2 x 10(1) to 5.2 x 10(11) colony forming units per ml (CFU/ml) for kulfi, 4 x 10(1) to 9 x 10(9) CFU/ml for ice cream and 2 x 10(1) to 2 x 10(10) CFU/ml for softy samples. The high degree of bacterial contamination seen indicates poor hygienic conditions and faults in manufacturing/handling of dairy products during and after processing and production.

Bacteriological study of Indian cheese (paneer) sold in Chandigarh.

A study was conducted to isolate and identify bacterial pathogens/contaminants in paneer samples sold in Chandigarh. Fifty eight samples of paneer bought at random were cultured on several media. Bacterial colony counts were also done. The predominant organisms isolated were Staphylococcus species, aerobic spore bearers, Klebsiella pneumoniae, Campylobacter jejuni, Acinetobacter species and Streptococcus species. The viable bacterial counts obtained ranged from 3 x 102 to 9.7 x 1010 CFU/mL. Contamination of paneer by pathogenic bacteria could be an important factor of gastrointestinal illnesses in the consumers.

Indigenous rapid diagnostic method for Escherichia coli O157:H7.

Rapid methods for detection of Escherichia coli O157: H7 diarrhoea are preferred to limit differential diagnostic evaluation including invasive procedures. Fifty stool samples from children and adult patients having diarrhoea and ten control samples from volunteers without diarrhoea were collected. Each sample was inoculated into two tubes containing peptone water and one containing MacConkey broth. They were incubated for 5 hours or longer till growth occurred. The first tube of peptone water with growth was tested for production of indole. All samples were tested for enzyme beta-glucuronidase in the pellet of the second peptone water growth. The MacConkey broth growth was tested with latex beads sensitized separately with antisera to E. coli O157 and E. coli H7. All indole positive and beta glucuronidase negative samples with positive agglutination with E. coli O157 and H7 coated latex beads were taken as E. coli O157: H7 positive samples. Three out of 50 diarrhoeic samples were found to be positive for E. coli O157:H7. Confirmation of the results of our rapid assay was done by parallel conventional culture of faecal specimens on sorbitol MacConkey agar. Our rapid assay required only 7-10 hours compared to the conventional technique where the report is available only on the third day. It can therefore be used routinely for initial screening of faecal specimens for E. coli O157: H7.

Enteropathogenic Escherichia coli isolates in paediatric diarrhoea.

Enteropathogenic Escherichia coli (EPEC) are known to cause infantile enteritis. We studied the prevalence of EPEC in paediatric patients with acute and persistent diarrhoea. A total of 56 stool samples from paediatric patients were studied. There were 28 significant bacterial isolates. Of these 21 were untypable E. coli, 5 were typable E. coli, four of which belonged to members considered to be enteropathogenic. Non E. coli isolates grown in pure culture were one each of Pseudomonas aeruginosa and Citrobacter freundi. The study reveals the definitive role of EPEC in childhood diarrhoea at all age groups and emphasizes the need for characterisation of all significant E. coli isolates in this age group.

Detection of Clostridium difficile toxin by an indigenously developed latex agglutination assay.

An indigenously developed latex agglutination assay using C. sordelli antitoxin was used to screen 211 stool samples received from hospitalized patients. Of 126 samples from patients receiving single to multiple antibiotics for various ailments, 38 (30%) were positive by the toxin assay, whereas only 6/85 (7%) of samples of patients not receiving antibiotics were also positive. Thus, of 211 samples a total of 44 (20.8%) were positive by our toxin assay, giving titers ranging from 1 in 5 to 1 in 320. The test developed by us is simple, rapid, easy and reliable and can be easily adapted to all microbiology laboratories.

Survival of enteropathogenic Escherichia coli exposed to adverse conditions.

Enteropathogenic Escherichia coli (EPEC) was exposed to different adverse conditions for varying period of time to assess its survival under such circumstances. From the results it is extrapolated that EPEC survive for a very long time when shielded from sunlight and after several hours of exposure to UV irradiation.

Antibiotic associated diarrhoea and enterocolitis.

C. difficile is the major aetiological agent of AAD and PMC and results from overgrowth of C. difficile already present endogenously or of newly acquired exogenous organisms after suppression of competing gut flora. C. difficile produces two kinds of toxins A and B. These toxins attack the colonic mucosa which becomes necrotic with the formation in fulminating cases of an exudative pseudomembrane. Toxigenic and non-toxigenic strains of C. difficile may be present together in an individual suffering from AAD. There is substantial variation among strains with respect to the quantity of lethal toxin produced. There are several strategies available for the investigation of C. difficile associated disease. Detection of toxins by neutralization with C. sordelli antitoxin is an easy, simple and sensitive method. Methods to deal effectively with silent carriers are not known because the routine administration of antibiotic treatment in an attempt to eradicate the carrier state would in fact boomerang by promoting C. difficile associated enteric disease rather than eliminating C. difficile.

Bacillaemia and Mycobacterium leprae cell wall antigen in paucibacillary leprosy.

A study was undertaken to estimate bacillaemia and M. leprae antigen detection in 54 paucibacillary leprosy patients (TT, BT). Acid-fast bacilli were detected in the blood of 14.8% patients of borderline tuberculoid (BT) leprosy. M. leprae antigen was demonstrated in 48.2% patients of BT leprosy. Slit-skin smears were negative in all these patients. At the end of treatment (6 months of WHO-MDT) all the follow-up blood samples were negative for both bacillaemia and M. leprae antigen in the serum.

Adenosine triphosphate content of Mycobacterium leprae by percoll buoyant density centrifugation.

Thirty nine untreated patients of bacilliferous leprosy with a mean bacteriological index of 4.8 and morphological index of 1.3% formed the study group. Adenosine triphosphate assay was carried out by (i) enzyme treatment method in 18 patients and (ii) percoll buoyant density gradient method in 21 patients. ATP content obtained by percoll buoyant density gradient method was significantly higher than that obtained by enzyme treatment method. Percoll buoyant density centrifugation for purification and isolation of bacilli from human leproma is simplier, quicker and can serve as an alternate method of enzyme treatment.