Acute dehydrating disease caused by Vibrio cholerae serogroups O1 and O139 induce increases in innate cells and inflammatory mediators at the mucosal surface of the gut.

BACKGROUND AND AIMS: The general concept is that as Vibrio cholerae is not invasive, it mediates a non-inflammatory type of infection. This is being re-evaluated based on available data that natural cholera infection or cholera toxin induces a Th2-type of immune profile and stimulates the humoral immune response, innate cells, and mediators in the host. METHODS: To perform a comprehensive analyses of the inflammatory components, we studied mucosal biopsies from patients, both adults and children with acute watery diarrhoea caused by V cholerae O1 and O139. Patients with cholera, adults (n = 30) and children (n = 18), as well as healthy controls (n = 24) were studied. Histochemical, immunohistochemical, and ultrastructural studies were carried out to elucidate the contribution of the different factors using paraffin and frozen duodenal and/or rectal sections as appropriate. Samples were collected during the acute stage and during early and/or late convalescence. RESULTS: Following natural cholera infection, patients responded with increases in neutrophil polymorphs during the acute stage (p<0.001) compared with healthy controls whereas mucosal mast cells (MMC) (p = 0.008) and eosinophils (p = 0.034) increased in the gut during convalescence. Electron microscopic analyses of duodenal biopsies from adult patients showed increased piecemeal degranulation in both MMC and eosinophils and accumulation of lipid bodies in MMC. Duodenal biopsies from V cholerae O1 infected patients showed upregulation of myeloperoxidase, lactoferrin, PGHS-1, SCF, tryptase, tumour necrosis factor alpha, alpha-defensin, and eotaxin during the acute stage and chymase, interleukin 3 and major basic protein during convalescence. CONCLUSION: We have shown that innate cells and their mediators are upregulated in acute watery diarrhoea. These cells and factors of the innate arm may be important in the host's defence against cholera. Such effects may need to be simulated in a vaccine to achieve long lasting protection from cholera.

Enteroaggregative Escherichia coli infection in a rabbit model.

Type strains of enteroaggregative Escherichia coli EAEC (17-2, serotype O3:H2; JM 221, serotype O92:H33), isolates from an adult and a child with diarrhoea and an asymptomatic colonised child were used to orally infect adult rabbits. The experimental animals were followed up and sacrificed at defined time periods. Colonisation of both small and large intestine was seen with all strains and isolates used. Isolates from an adult patient with diarrhoea (MP 27) and from an asymptomatic colonised child from the community (KM 1337) were recovered from the small intestine during the first week of infection and subsequently from the large intestine. A total of seven rabbits was infected with MP 27; while colonising the gastrointestinal tract of all seven rabbits, this isolate caused diarrhoea in only one. On ultrastructural examination, the rabbits infected with 17-2 showed invasion of lymphoid follicles. Bacteria were seen in intercellular spaces and within M cells, a finding that has not previously been described. It is clearly possible to produce gut colonisation by oral infection with EAEC in adult rabbits with normal flora.

A monkey model for enterohemorrhagic Escherichia coli infection.

Adult Macaca radiata (n=22) were infected intragastrically with 10(12) Escherichia coli O157:H7 strain 84-01, which produces Shiga toxins 1 and 2. Clinical symptoms and bacterial excretion were documented in each monkey for a specified time period before they were killed. At necropsy, samples were obtained for culture and histologic and ultrastructural examination. Seventeen monkeys had diarrhea: E. coli O157 was isolated from postinfection stool samples from all monkeys and from autopsy cultures for 14 of 22 monkeys. Histologic examination showed attaching-effacing lesions, which appeared at 12 h and persisted for 7 days, in 12 monkeys. Widening of the intercellular spaces, degeneration and vacuolization of the epithelial cells, epithelial tufting, extrusion of epithelial cells, and neutrophilic infiltration were characteristic features seen in 20 of the 22 infected monkeys but not in 4 control monkeys. This monkey model closely parallels the early stages of the disease produced by E. coli O157:H7 and would be useful in the further study of pathogenic mechanisms and prevention methods in enterohemorrhagic E. coli infections.

A prospective study of nosocomial enteric pathogen acquisition in hospitalized children in South India.

Screening for enteric pathogens in stool samples from 249 children under the age of 36 months, admitted to hospital for non-gastrointestinal disorders, was positive at admission in 41 (16.4 per cent) in a prospective study of enteric pathogen acquisition and diarrhoea in hospitalized children. Infection with multiple organisms was found in 31/41 (75.6 per cent) children who were positive when screened at admission. Of 194 children who had no enteric pathogens on admission and could be followed up for 3 days after discharge, clinical or laboratory data showed nosocomial enteric infections in 39 (20.1 per cent). Presumed nosocomial infection with more than one organism was seen in only two patients and no pathogens were isolated in 14 (35.8 per cent). Children presenting to hospital may asymptomatically carry enteric pathogens and potentially act as a source of nosocomial infections. One in five children admitted into hospital without an enteric infection is at risk of developing a nosocomial gastrointestinal infection, with rotavirus being the most common aetiological agent.

Glomerular injury induced in mice by intraperitoneal injection of Shiga-like toxins.

BACKGROUND AND OBJECTIVES: Shiga-like toxins I and II (Stx1 and Stx2) play an important role in the pathogenesis of renal disease by causing renal microvascular injury. A murine model was used to study glomerular lesions produced by Stx1 and Stx2. METHODS: Swiss albino mice of the Rockefeller strain were inoculated intraperitoneally with LD(50) doses of endotoxin-free Stx1 of Stx2 and observed for signs of disease. Samples of renal cortical tissue from mice were examined with the electron microscope. RESULTS: the mice developed systemic and neurological symptoms including hind limb paralysis and generalised convulsions. Renal arteriolar damage and glomerular endothelial cytoplasmic swelling, vacuolation, lysis and intravascular coagulation were present and resembled the microangiopathy seen in renal biopsies from patients. INTERPRETATION AND CONCLUSIONS: these experiments establish the role of Stx1 and Stx2 in glomerular vascular injury and provide a model for studying the pathogenesis of Shiga-like toxin related microangiopathy.

Innate immune responses in children and adults with Shigellosis.

An array of pro- and anti-inflammatory mediators of the innate immune system was analyzed in stool, urine, and rectal mucosa samples from adults and children with shigellosis to better understand their role in recovery from and in the immunopathogenesis of the disease. Increased concentrations of lactoferrin (Lf), myeloperoxidase (MPO), prostaglandin E(2), and leukotriene B(4) (LTB(4)) in stool during acute shigellosis in both children and adults indicated that activated cells of the innate defense system at the mucosal site were secreting the mediators. Increased concentration of MPO and 8-iso-prostaglandin F(2alpha) and lower levels of superoxide dismutase (SOD) activity in stool during acute Shigella infection suggested increased formation of reactive oxygen species, free radical-catalyzed peroxidation of membrane lipids, and decreased scavenging of the reactive oxygen radicals. In children, lower expression of SOD in tissue with severe inflammation and lower levels of SOD activity in stool for longer periods compared to adults may further worsen the tissue damage and predispose the children to a lowered defense. Both adult and pediatric patients had significantly higher expression of inducible nitric oxide synthase (iNOS) in the rectum with severe inflammation, compared to that seen with mild inflammation, accompanied by persistently up-regulated iNOS mRNA, reflecting increased production of nitric oxide at the local site. However, in contrast to adults, reduced urinary nitrate levels in pediatric patients during acute shigellosis suggested lower production of nitric oxide in the renal compartment. Persistent production of Lf in pediatric patients may contribute to chronic inflammation in the rectum. In addition, increased production of proinflammatory mediators in the rectum of patients with severe histology suggested contribution of these molecules to the immunopathogenesis of severe colitis caused by shigellae.

Prevalence of enteroaggregative and other HEp-2 cell adherent Escherichia coli in asymptomatic rural south Indians by longitudinal sampling.

Enteroaggregative and other HEp-2 cell adherent Escherichia coli can produce acute and persistent diarrhoea in children and adults, but their prevalence in asymptomatic individuals in the community is not known. In this study, faecal specimens were obtained at 3-4 monthly intervals from 349 subjects constituting a 20% age-stratified sample of a rural community for a period of two years. HEp-2 cell adherent E. coli were found in 210 subjects, and repeat isolations of enteroaggregative E. coli belonging to the same serogroup were found in 12.6% of children less than 12 years of age, indicating that this organism can asymptomatically colonise the intestinal tract. These children may act as a reservoir of infection for the community.

Use of serology, the urease test & histology in diagnosis of Helicobacter pylori infection in symptomatic & asymptomatic Indians.

Age-specific prevalence of IgA and IgG antibodies in 714 subjects without gastrointestinal complaints aged 6 months to 90 yr was measured by an enzyme linked immunoassay using an acid-glycine extract of H. pylori as the antigen. The urease test and histology were used for the diagnosis of H. pylori infection in 83 subjects with a clinical diagnosis of dyspepsia, and these results were compared with measurement of IgG, IgA and IgM antibodies. The age specific prevalence of IgG and IgA antibodies respectively was 57 and 43 per cent for subjects aged 6 months to 4 yr and showed an increase with age to a maximum of 90 per cent for IgG in subjects > 60 yr of age and to 87 per cent for IgA in subjects between 51 and 60 yr. In symptomatic patients, there was a high degree of correlation between severity of H. pylori infection on histopathological examination and IgG (P < 0.02) levels. The use of IgG and IgA estimation could have identified H. pylori infection without endoscopy in 50 of the 83 patients. Serology for IgG and IgA antibodies against H. pylori may play a major role in decreasing the need for endoscopy, but cut-off values must be determined for each assay based on the prevalence of antibodies in the population.

Cytomegalovirus infection of the human gastrointestinal tract.

BACKGROUND: Current interest in cytomegalovirus (CMV) is largely due to an increase in the number of cases of acquired immunodeficiency syndrome and organ transplantation in recent years. The proper recognition of CMV-infected cells in gastrointestinal mucosal biopsies is critical for effective treatment of this condition. METHODS: A total of 6580 endoscopic mucosal biopsies from 6323 patients in the 8-year period (1989-1996) were examined for CMV inclusion bodies. The endoscopic appearance and particularly the presence of ulcers were also analysed. RESULTS AND CONCLUSIONS: The prevalence of cytomegalovirus (CMV) inclusions was 9 per thousand in the gastrointestinal mucosal biopsies from an unselected group of patients. Of the 54 patients with CMV infection, 37 were immunocompromised and 17 apparently immunocompetent. Typical Cowdry inclusions and atypical inclusions were present, the latter more frequently in immunocompromised patients. The maximum prevalence of inclusions was in the oesophageal mucosa in immunocompromised individuals.

Endoscopic mucosal biopsies are useful in distinguishing granulomatous colitis due to Crohn's disease from tuberculosis.

BACKGROUND: Intestinal tuberculosis and Crohn's disease are chronic granulomatous disorders that are difficult to differentiate histologically. AIMS: To characterise distinctive diagnostic features of tuberculosis and Crohn's disease in mucosal biopsy specimens obtained at colonoscopy. METHODS: Selected histological parameters were evaluated retrospectively in a total of 61 biopsy sites from 20 patients with tuberculosis and 112 biopsy sites from 20 patients with Crohn's disease. The patients were chosen on the basis of clinical history, colonoscopic findings, diagnostic histology, and response to treatment. RESULTS: The histological parameters characteristic of tuberculosis were multiple (mean number of granulomas per section: 5.35), large (mean widest diameter: 193 microm), confluent granulomas often with caseating necrosis. Other features were ulcers lined by conglomerate epithelioid histiocytes and disproportionate submucosal inflammation. The features characteristic of Crohn's disease were infrequent (mean number of granulomas per section: 0.75), small (mean widest diameter: 95 microm) granulomas, microgranulomas (defined as poorly organised collections of epithelioid histiocytes), focally enhanced colitis, and a high prevalence of chronic inflammation, even in endoscopically normal appearing areas. CONCLUSIONS: The type and frequency of granulomas, presence or absence of ulcers lined by epithelioid histiocytes and microgranulomas, and the distribution of chronic inflammation have been identified as histological parameters that can be used to differentiate tuberculosis and Crohn's disease in mucosal biopsy specimens obtained at colonoscopy.

Enteric pathogens in southern Indian HIV-infected patients with & without diarrhoea.

This study was undertaken to determine the carriage rate of various enteric pathogens in southern Indian patients with HIV infection, both with and without diarrhoea. Stool from 111 consecutive HIV-positive patients (50 without and 61 with diarrhoea) was examined by microscopy and culture. Jejunal biopsy and fluid examination were carried out if diarrhoea persisted, with negative stool examination. Enteric pathogens were detected from stool in 57.4 per cent of diarrhoeal patients compared to 40 per cent of those without diarrhoea (P > 0.05). Jejunal biopsy and fluid examination provided 11 additional diagnoses. Protozoa accounted for 71.8 per cent of all pathogens isolated. Isospora was significantly more common in patients with (11/61) than in those without (2/50) diarrhoea (P < 0.05). Bacterial pathogens were isolated more commonly from patients with diarrhoea (12/61 compared to 2/50, P < 0.05). Isolation rate of pathogens was higher from patients with diarrhoea for more than 2 wk, compared to those with less than 2 wk duration. Remission of diarrhoea either spontaneously or with symptomatic therapy was observed in 22 patients with acute diarrhoea. A high enteric carriage of a number of pathogens was noted in HIV patients without diarrhoea, but I. belli and bacterial enteropathogens were more likely to be associated with diarrhoea.

Collagenous gastritis and collagenous colitis: a report with sequential histological and ultrastructural findings.

The case is reported of a young adult man with collagenous gastritis, an extremely rare disorder with only three case reports in the English literature, who subsequently presented with collagenous colitis. Sequential gastric biopsies showed a notable increase in thickness of the subepithelial collagen band. Ultrastructural study of gastric and rectal mucosa showed the characteristic subepithelial band composed of haphazardly arranged collagen fibres, prominent degranulating eosinophils, and activated pericryptal fibroblasts.

Salicylate inhibits fimbriae mediated HEp-2 cell adherence of and haemagglutination by enteroaggregative Escherichia coli.

Enteroaggregative Escherichia coli (EAggEC) are associated with both acute and persistent diarrhoea in children. Bowel colonisation due to fimbrial adherence factors appears to play a major role in the disease process. In this study, we investigated the effect of sodium salicylate and 5-aminosalicylic acid on adherence of a type strain and 40 clinical isolates of EAggEC to HEp-2 cells and erythrocytes from different species. Growth in the presence of 10 mM salicylate resulted in markedly decreased adherence to tissue culture cells with 33/40 (82.5%) isolates, and was also associated with inhibition of haemagglutination in 20/33 (60.6%) isolates. Complete or partial inhibition of adherence was also seen in two of five isolates showing localised adherence and three of five isolates with diffuse adherence. Decrease in adherence was associated with decreased or absent expression of fimbriae in 28/40 (70%) of the EAggEC isolates, although production of outer membrane proteins was not affected. Salicylates appear to inhibit adherence mediated by fimbrial adhesins.

Quantitative and ultrastructural analysis of rectal mucosal mast cells in acute infectious diarrhea.

The role of mast cells, potential mediators of mucosal immunity and inflammation, was studied morphologically in the rectal mucosa in two acute diarrheal diseases, cholera and shigellosis. Quantitation of mucosal mast cells showed that they were significantly higher in the deeper lamina propria where blood vessels and nerves were more abundant. There was no difference in mast cell counts or degranulation in the mucosa in both groups of patients and controls. Intraepithelial mast cells were decreased in the patients. The prevalence of lipid bodies was significantly higher in mast cells from patients with cholera and shigellosis (P < 0.01). These findings suggest that mast cell populations are more dense around blood vessels and nerves and that inflammatory mediators derived from arachidonic acid metabolites, as indicated by the lipid bodies, are the response of mast cells to the alterations in diarrhea, despite differences in the etiology of diarrhea.

Prevalence of intestinal parasites in rural Southern Indians.

OBJECTIVE: To determine the prevalence of intestinal protozoal and helminthic infection in a rural population. METHOD: Seventy-eight members of 15 families from a village were studied. Stool samples from all subjects were examined on alternate days for one month. RESULTS: The overall prevalence rate of various parasitic infections was 97.4%, with only 2 of 78 subjects not excreting parasites in any of their 15 samples. Eighteen (23.1%) persons had only one type of parasite, while 58 (74.3%) excreted multiple parasites. Giardia and Cryptosporidium were the commonest protozoan infections, affecting 42/78 (53.8%) and 31/78 (39.7%), respectively. Hookworm infestations were the commonest helminthic infections, seen in 48/78 (61.5%). Based on excretion patterns, the asymptomatic individuals could be divided into 2 groups of infrequent and frequent excretors, indicating that the host response may determine the level of parasite replication in the gut.

Case report: gastric carcinoma as a complication of dyskeratosis congenita in an adolescent boy.

Dyskeratosis congenita (DC), or the Zinsser-Engman-Cole syndrome, is a rare X-linked heritable disorder, affecting primarily the ectodermal tissues, with hyperpigmentation of the skin, leukoplakia of the buccal and anal mucosa, and nail dystrophy (1, 2). Aplastic anemia (3) and a variety of neoplasms (4, 5) are some of the extraectodermal manifestation of this disorder, which although X-linked recessive, has also been described in a few females (6, 7). Mental retardation, diarrhea, and gastrointestinal bleeding have been considered to be less frequent features (8). We report an adolescent Indian male who presented with all the ectodermal manifestations, as well as mental retardation, bone marrow aplasia, and gastrointestinal hemorrhage secondary to adenocarcinoma of the stomach.

Role of viruses in acute gastroenteritis in infants and young children at Vellore, south India.

The aetiological contribution of viral agents in the causation of acute diarrhoea was studied prospectively in 915 infants and young children aged 1-35 months. This was a hospital-based study, selection being carried out by proportionate random sampling; 587 matched controls were also investigated simultaneously. Methods of detection included direct electron microscopy, ELISA for rotaviruses and immune electron microscopy. Rotaviruses emerged as the single most important cause with highest prevalence in the 6-11-month age group. The contribution of other agents was negligible. Electron microscopy, though a "catch-all' technique, is not a cost-effective diagnostic method for developing countries.

A comparison of five staining methods for detection of Cryptosporidium oocysts in faecal specimens from the field.

Five staining methods for identification of Cryptosporidium oocysts in faecal smears were evaluated using samples obtained from asymptomatic south Indian villagers of all ages. The safranine-methylene blue technique was used as the gold standard and compared with 2 methods each using auramine and mepacrine, with potassium permanganate and carbol fuchsin as counterstains. All five methods identified all samples containing cryptosporidium oocysts, however false positive identifications were obtained by the standard auramine-phenol-potassium permanganate method. The auramine-carbol fuchsin and the mepacrine-carbol fuchsin techniques are the simplest and most rapid of the five methods compared. The mepacrine-carbol fuchsin technique is recommended for screening large numbers of stools, since it does not stain yeasts, is cheaper and less toxic than auramine.

Viability of Mycobacterium leprae in skin and peripheral nerves and persistence of nerve destruction in multibacillary patients after 2 years of multidrug therapy.

The pathological changes, bacterial load, and viability of Mycobacterium leprae in the skin and nerves of nine lepromatous leprosy patients who had undergone 2 years of multidrug therapy (MDT) were studied. M. leprae and varying amounts of their remnants were present in the nerves and skin of all but one patient. M. leprae isolated from skin biopsies of six patients and nerve biopsies of nine patients were inoculated into mouse foot pads. No growth was obtained from any one of them. During the electron-microscopic examination of three nerve biopsies, only one specimen showed a small number of solid-staining M. leprae. These findings would explain the low relapse rate in patients treated with 2 years of fix-duration MDT. Results of a long-term follow up of patients is awaited with interest. The possibility of nerve paralysis due to intraneural microreaction and fibrosis consequent to the continued presence of dead bacterial remnants should be seriously considered.

Molecular epidemiology of nosocomial rotavirus infection.

The molecular epidemiology of nosocomial rotavirus infection in children admitted to the paediatric ward of a large hospital in southern India was studied by transmission electron microscopy and RNA electrophoresis on stool samples. Six hundred and twenty-six samples were collected and rotaviruses were detected in 35 of them. A predominant electropherotype was found in all the samples, and there was no seasonal variation in the incidence of infection. The predominant electropherotype pattern in this study was different from the electropherotype patterns of strains isolated elsewhere in the country and also from the pattern of the strains isolated earlier from children with acute diarrhoea at the same hospital.