Role of probiotic in preventing acute diarrhoea in children: a community-based, randomized, double-blind placebo-controlled field trial in an urban slum.

Acute diarrhoea remains a major public health challenge in developing countries. We examined the role of a probiotic in the prevention of acute diarrhoea to discover if there was an effect directed towards a specific aetiology. A double-blind, randomized, controlled field trial involving 3758 children aged 1-5 years was conducted in an urban slum community in Kolkata, India. Participants were given either a probiotic drink containing Lactobacillus casei strain Shirota or a nutrient drink daily for 12 weeks. They were followed up for another 12 weeks. The primary outcome of this study was the occurrence of first episodes of diarrhoea. We assessed this during 12 weeks of intake of study agent and also for 12 weeks of follow-up. There were 608 subjects with diarrhoea in the probiotic group and 674 subjects in the nutrient group during the study period of 24 weeks. The level of protective efficacy for the probiotic was 14% (95% confidence interval 4-23, P<0·01 in adjusted model). The reduced occurrence of acute diarrhoea in the probiotic group compared to nutrient group was not associated with any specific aetiology. No adverse event was observed in children of either probiotic or nutrient groups. The study suggests that daily intake of a probiotic drink can play a role in prevention of acute diarrhoea in young children in a community setting of a developing country.

Metal mutagens and carcinogens affect RNA synthesis rates in a distinct manner.

Five metal salts (lead, cadmium, cobalt, copper, and manganese),which are mutagenic or carcinogenic, decreasing the fidelity of DNA synthesis in vitro, stimulated chain initiation of RNA synthesis at concentrations that inhibited overall RNA synthesis. In contrast, other metal salts (zinc, magnesium, lithium, sodium,and potassium) not in this category inhibited chain initiation of RNA synthesis at concentrations that inhibited overall RNA synthesis.

Increasing antimicrobial resistance--an emerging problem in the treatment of shigellosis.

Shigellosis is a major cause of diarrhoea-related morbidity and mortality, especially in developing countries. Effective antibiotic treatment reduces the average duration of illness by reducing faecal excretion of the bacterium and preventing further transmission and potentially lethal complications. Treatment of shigellosis is currently limited by the high prevalence of multidrug-resistant strains of Shigella. Although fluoroquinolones are currently effective in treating adults, resistance to fluoroquinolones among Shigella spp. is emerging, and their use in children is subject to limitations. Azithromycin and third-generation cephalosporins are also effective in the treatment of shigellosis, but monitoring of Shigella isolates to detect the emergence of resistance is essential.

Aromaticity at position 37 in human epidermal growth factor is not obligatory for activity.

The third disulfide loop (amino acids 33 to 42) of human epidermal growth factor (hEGF) encompasses the region of highest amino acid conservation among all of the EGF-like family of molecules. The importance of some of these highly conserved residues for the maintenance of biological activity, especially the aromatic amino acid tyrosine at position 37, has until now been considered essential on the basis of previous studies with the EGF-like molecule transforming growth factor alpha. Variants at the Tyr-37 position of hEGF were constructed by site-directed mutagenesis. The substituting amino acids were phenylalanine, histidine, serine, alanine, aspartic acid, arginine, and glycine. The variants were tested for their ability to competitively displace native [125I]hEGF from its receptor and to stimulate the protein-tyrosine kinase activity of the receptor; the order of efficacy of substituting amino acids was Phe greater than His greater than Ser greater than Ala greater than Asp greater than Arg greater than Gly in both assays. All were effective, with no or only moderate reduction in potency, in stimulating the incorporation of [3H]thymidine into acid-insoluble material of quiescent mouse A31 cells. Only Tyr-37----Ala, Tyr-37----Arg and Tyr-37----Gly were slightly less potent in the cell assay. Thus, neither tyrosine nor another aromatic amino acid at position 37 in hEGF is essential for full biological activity.

Engineering epidermal growth factor for enhanced mitogenic potency.

Successful use of growth factors in therapeutic and bioprocessing applications requires overcoming two attenuation mechanisms: growth factor depletion and receptor down-regulation. Current ameliorative strategies use physiologically inappropriate high growth-factor concentrations, along with periodic media refeeding in vitro and reinjection or controlled-release devices in vivo. We demonstrate a new approach derived from understanding how these attenuation mechanisms arise from ligand/receptor trafficking processes. Specifically, a recombinant epidermal growth factor (EGF) mutant with reduced receptor binding affinity is a more potent mitogenic stimulus for fibroblasts than natural EGF or transforming growth factor alpha because of its altered trafficking properties.

Phage typing, biotyping & antimicrobial resistance profile of Salmonella enterica serotype Typhi from Kolkata.

BACKGROUND & OBJECTIVE: Kolkata and its suburbs in eastern India are known to be endemic for typhoid fever. The objective of this study was to determine phage types, biotypes and antimicrobial resistance patterns of Salmonella enterica serotype Typhi isolated during the period 2003-2005 from a prospective surveillance for typhoid fever in two urban slums in Kolkata. METHODS: A total of 195 Salmonella enterica serotype Typhi isolated from blood cultures were phage typed, biotyped and tested for their antimicrobial susceptibility profile. RESULTS: Phage type E1 was the most common (60.3%) followed by phage type A among five phage types identified. Biotype I (95%) was predominant, 28 isolates were multidrug resistant (MDR) and most of the MDR strains belonged to phage type E1 and biotype I. INTERPRETATION & CONCLUSION: A single phage type and biotype were prevalent among the Salmonella enterica serotype Typhi isolates studied from Kolkata, India.

Molecular epidemiology of human astrovirus infections in Kolkata, India.

UNLABELLED: The study is aimed to determine the seasonal distribution and clinical characteristics of astroviruses associated with acute watery diarrhoea among children in Kolkata and characterize them at the molecular level. METHOD OF STUDY: Faecal specimens of acute watery diarrhoea cases (n=857) and non-diarrhoeic samples (n=211) from the hospitals and a nearby field community were screened with IDEIA Astrovirus detection kit; astrovirus co-infections with rotavirus and/or picobirnavirus were detected by RNA-PAGE and silver staining. Further RT-PCR was carried out using specific primers, viz. Mon340 (+) and Mon348 (-) targeting a highly conserved domain of ORF1a (289 bp) of human astroviruses. RESULTS: Astrovirus infection was detected in 50 cases (50/857); astroviruses were detected mostly in children aged 6-12 months (50%); all non-diarrhoeic samples (n=211) were negative for astrovirus. In 52% of astrovirus positive cases, the virus was detected as the sole agent; mixed infections were also detected with other diarrhoeic pathogens such as rotavirus (32%), picobirnavirus (2%), rotavirus and picobirnavirus (2%), picobirnavirus and Enterotoxigenic E. coli (ETEC) (2%), rotavirus and ETEC (2%), rotavirus and Enteroaggregative E. coli (EAEC) (2%), Enteropathogenic E. coli (EPEC) (2%), Shigella flexneri type 3a (2%) and Ascaris (2%). RT-PCR and sequencing of amplicons of astroviruses from Kolkata, with specific primers targeted to the conserved domain of ORF1a (289 bp) of the astrovirus genome, showed maximum homology to the astrovirus strain ("5-158") from Seoul (98%). RESULTS AND CONCLUSIONS: Clinical characteristics of the diarrhoeic children in Kolkata indicated that astrovirus infections were detected throughout the year and were associated with varying degree of dehydration and acute watery diarrhoea. In-depth molecular epidemiological surveillance of astroviruses in Kolkata is essential for better understanding of their overall genetic nature.

Epidemiological, microbiological & electron microscopic study of a cholera outbreak in a Kolkata slum community.

BACKGROUND & OBJECTIVE: Epidemics of cholera caused by Vibrio cholerae O1 or O139 have been reported from different parts of India. Factors such as unsafe water supply, poor environmental sanitation, indiscriminate defaecation and lack of personal hygiene are mainly responsible for continued transmission of this disease. We report here epidemiological and microbiological findings of a localized outbreak of cholera, which occurred during March and April 2004 in the eastern part of Kolkata city. METHODS: The affected slum area has a population of 4409, predominantly muslims. Patients suffering from acute watery diarrhoea attended the health outposts organized by National Institute of Cholera and Enteric Diseases, Kolkata and International Vaccine Institute, South Korea as part of a routine surveillance programme at the locality as well as the emergency medical camp organized by the Kolkata Municipal Corporation. Stool and water samples were collected and tested for diarrhoeagenic pathogens in the laboratory. Bacteriophages specific for V. cholerae were isolates and studied electron microscopically for morphology. RESULTS: A total of 89 diarrhoea cases were reported giving an attack rate of 2 per cent. V. cholerae O1 biotype ElTor, serotype Ogawa was isolated as a sole pathogen from 15 (15.8%) of 89 stool samples screened. Water samples (2 from tube wells, 3 from municipal taps and 1 from well) showed presence of coliform bacilli with high MPN (Most Probable Number) count. Bacteriophages specific to V. cholerae were isolated from 2 of 6 water samples examined. A leakage was detected in the main pipeline supplying drinking water to that area. INTERPRETATION & CONCLUSION: The outbreak was caused by V. cholerae O1 (Ogawa) biotype ElTor. The presence of phages in the water samples was an additional indicator for V. cholerae contamination in this community. Occurrences of such outbreaks support vaccination against cholera as an alternative strategy.

Epidemiology of HIV infection among brothel-based sex workers in Kolkata, India.

A cross-sectional study was carried out during January-June 2004 to assess the status of HIV infection among brothel-based sex workers in Kolkata city, India. Six hundred and twenty-two sex workers, selected from six brothels, were included in the study to assess their HIV status, and 362 sex workers, a subset of the above population, were interviewed to study their risk behaviour and practices. Blood samples were collected from each sex worker for testing HIV by an unlinked anonymous method. The prevalence of HIV infection was 9.6%, but was much higher among younger sex workers aged 20 years or less (27.7%) compared to the older age group (8.4%). This difference was statistically significant, indicating an association of younger ages with HIV infection [p = 0.006 and odds ratio (OR) = 4.18; (95% confidence interval (CI) 1.2-13.8)]. HIV was not associated with duration of sex work, average number of clients entertained per day, condom use, practice of sex during menstruation, pre-coital examination of penis for visible ulcer/discharge, suffering from sexually transmitted infections, and entertaining clients outside the brothel. There was a big gap between the reported and the evaluated condom use by sex workers. The results suggest that there is a need to develop suitable HIV intervention strategies, considering the socioeconomic and cultural aspects of city sex workers, with a provision for continuous monitoring and evaluation.

Analogs of human epidermal growth factor which partially inhibit the growth factor-dependent protein-tyrosine kinase activity of the epidermal growth factor receptor.

Three site-directed mutants of human epidermal growth factor, Leu-26----Gly, Leu-47----Ala, and Ile-23----Thr, were examined for their ability to stimulate the protein-tyrosine kinase activity of the epidermal growth factor receptor. The receptor binding affinities of the mutant growth factors were 20- to 50-fold lower, as compared to wild-type growth factor. At saturating concentrations of growth factor, the velocities of the phosphorylation of exogenously added substrate and receptor autophosphorylation were significantly lower with the mutant analogs, suggesting a partial 'uncoupling' of signal transduction. The mutant analogs were shown to compete directly with the binding of wild-type, resulting in a decrease in growth factor-stimulated kinase activity.

Human epidermal growth factor. Distinct roles of tyrosine 37 and arginine 41 in receptor binding as determined by site-directed mutagenesis and nuclear magnetic resonance spectroscopy.

Site-directed mutagenesis was employed to examine the function of two highly conserved residues, Tyr-37 and Arg-41, of human EGF (hEGF) in receptor binding. Both a conservative change to phenylalanine and a semi-conservative change to histidine at position 37 yield proteins with receptor affinity similar to wild-type hEGF. A non-conservative change to alanine results in a molecule with about 40% of the receptor affinity, indicating that an aromatic residue is not essential at this position. Both conservative (to lysine) and non-conservative (to alanine) substitutions at position 41 drastically reduced receptor binding to less than 0.5% of the wild-type activity. 1D-NMR data indicate that the replacement of Arg-41 by lysine does not significantly alter the native protein conformation. Thus, Arg-41 may be directly involved in ligand receptor interaction, whereas the side chain of Tyr-37, although possibly important structurally, is not essential for receptor binding.

Prevalence of the sat, set and sen genes among diverse serotypes of Shigella flexneri strains isolated from patients with acute diarrhoea.

New virulence factors associated with the genus Shigella have been described recently. These comprise enterotoxins ShET-1 and ShET-2, encoded by the set and sen genes, respectively, and the secreted autotransporter toxin Sat, encoded by the sat gene. The present study aimed to determine the prevalence of genes encoding these toxins among a collection of Shigella flexneri clinical isolates belonging to different serotypes. While the set gene was detected only in S. flexneri serotypes 2a and 2b, the sen and sat genes were present in diverse serotypes of S. flexneri.

Aetiological studies on hospital in-patients with acute diarrhoea in Calcutta.

Bacterial enteropathogens and rotavirus were sought in 356 cases with acute diarrhoea admitted to the Infectious Diseases Hospital, Calcutta. One or more pathogens were isolated from 74.7% of the cases. Single enteropathogens could be detected from 66% and multiple enteropathogens from 8.7% of the patients. Vibrio cholerae biotype El Tor, rotavirus, V. parahaemolyticus, and enteropathogenic and enterotoxigenic Escherichia coli were the major pathogens detected. Rotavirus was detected from 7.6% of the cases. A higher rate of detection of rotavirus was seen in children younger than two years. Campylobacter jejuni could be isolated from the faeces of six (15%) of 40 cases either as a single pathogen or in association with V. cholerae biotype El Tor.

Fluorescence studies with human epidermal growth factor.

Steady-state and time-resolved fluorescence studies have been performed with human epidermal growth factor, a small globular protein having two adjacent tryptophan residues near its C-terminus. Based on the relatively red fluorescence and accessibility to solute quenchers, the two tryptophan residues are found to be exposed to solvent. Anisotropy decay measurements show the dominant depolarizing process to have a sub-nanosecond rotational correlation time indicating the existence of rapid segmental motion of the fluorescing tryptophan residues. From an analysis of the low-temperature excitation anisotropy spectrum of the protein (and in comparison with that of tryptophan, the peptide melittin, and the dipeptide trp-trp), it is concluded that homo-energy transfer and/or exciton interaction occurs between the adjacent tryptophan residues. A thermal transition in the structure of the protein, which is observed by circular dichroism measurements, is not sensed by the steady-state fluorescence of the protein. This result, in conjunction with the anisotropy decay results, indicates that the two tryptophan residues are in a highly flexible C-terminus segment, which is not an integral part of the three-dimensional structure of the protein. Fluorescence measurements with three site-directed mutants also show very little variation.

Selective effects of metal ions on RNA synthesis rates.

The effects of 14 metal ions (chlorides) on the transcription of calf thymus DNA and phage T4 DNA with Escherichia coli RNA polymerase were tested. These assays were conducted under improved conditions of lower pH and in the absence of 2-mercaptoethanol to permit greater stability of the metal ions in solution. Among the divalent metal ions tested, the concentration-dependent order of inhibition of overall transcription is Pb2+ greater than Zn2+ greater than Cu2+ greater than Be2+ greater than Cd2+ greater than Ni2+ greater than Ca2+ greater than Co2+ greater than Mn2+ greater than Mg2+ greater than Sr2+ and is the same with either template. At pH 7.4 and in the absence of 2-mercaptoethanol, considerably lower concentrations of several of the divalent metal ions are needed for inhibition of overall transcription than at pH 8.1 and in the presence of 2-mercaptoethanol. Ca2+, Mg2+, Sr2+, Zn2+, Li+, Na+, and K+--considered to be non-mutagenic and non-carcinogenic--decrease chain initiation (measured with T4 DNA) at concentrations that inhibit overall transcription. Pb2+, Cd2+, Co2+, Be2+, and Mn2+--all mutagenic or carcinogenic--stimulate chain initiation (although at widely different rates) at concentrations that inhibit overall transcription. Cu2+ and Ni2+--both carcinogenic--stimulate initiation only at very low concentrations, followed by a progressive decrease in initiation at concentrations that inhibit overall transcription.

Comparison of selective media for optimal recovery of Clostridium difficile from diarrhoeal stools.

Five selective media were compared for their efficacy in the recovery of C. difficile from stool specimens. Of 341 diarrhoeic stool samples, 38 (11%) yielded C. difficile. Eighty per cent of the isolates were detected on modified taurocholate cycloserine cefoxitin fructose agar (MTCCFA) and 73 per cent were detected on taurocholate cycloserine cefoxitin fructose agar (TCCFA). MTCCFA was also found superior to the other four media as it supported better growth of C. difficile colonies, by effectively suppressing the competing microflora. These results suggest that the recovery rate of C. difficile could be enhanced when routine media, incorporated with taurocholate and lower concentration of cycloserine and cefoxitin, is used for the isolation of C. difficile from diarrhoeic stool.

Enteroaggregative Klebsiella pneumoniae in association with childhood diarrhoea.

A total of 19 strains of Klebsiella pneumoniae isolated as sole pathogen from children with diarrhoea were used to study their virulence mechanism using different assays. Eith strains of K. pneumoniae exhibited aggregative adherence that was distinct from the stacked brick enteroaggregative pattern shown by Escherichia coli. The study suggests the presence of a new virulence mechanism in the pathogenesis of Klebsiella-associated diarrhoea.

Multi-drug resistant non-typhoidal Salmonella spp. associated with acute diarrhoeal disease.

The prevalence of different serotypes of non-typhoidal Salmonella spp. among patients suffering from acute diarrhoea admitted to the Infectious Diseases Hospital, Calcutta was investigated. The predominant serogroup was C and Salmonella infantis was the major serotype isolated followed by S. worthington, S. enteritidis, S. typhimurium, S. weltevereden and S. newport. All the Salmonella strains were isolated from adults. Multidrug resistance to various antimicrobial agents was observed in 37.5 per cent of the strains. All the strains were sensitive to ciprofloxacin, norfloxacin and gentamycin.

Association of enterotoxigenic Bacteroides fragilis with childhood diarrhoea.

This study was conducted in a hospital setting to determine whether enterotoxigenic strains of Bacteroides fragilis (ETBF) were associated with childhood diarrhoea. ETBF was isolated from 6 (2.6%) of 226 patients and 3 (1.7%) of 172 controls and was found mostly in children between 1-5 yr of age. The syndrome associated with ETBF was secretory in nature with watery diarrhoea and of mild severity. ETBF may be associated with diarrhoeal illness in children but is not a major problem in this part of the country.

Frequency of isolation of toxigenic Clostridium difficile from healthy adults.

An attempt was made to isolate Clostridium difficile from 129 faecal specimens from 87 young and 42 elderly healthy adults. C.difficile was isolated using bacterial culture from 7 (5.4%) specimens and the frequency of isolation of toxigenic C. difficile as detected by cytotoxin (tissue culture) assay was 1.1 per cent and 2.4 per cent in healthy young and elderly adults respectively. The carrier rate of toxigenic C. difficile amongst healthy adults is relatively low in this part of our country.