Tuberculosis Caused by Mycobacterium orygis in Dairy Cattle and Captured Monkeys in Bangladesh: a New Scenario of Tuberculosis in South Asia.

Mycobacterium orygis, commonly known as the oryx bacillus and a newly proposed Mycobacterium tuberculosis complex subspecies, was isolated from 18 cattle in a dairy farm and two captured rhesus monkeys in a zoo in Bangladesh. All the infected animals had tuberculosis lesions in their lungs, suggesting transmission and infection with M. orygis by an airborne route. The 20 isolates were analysed using a range of conventional and molecular typing methods, and RD-deletion typing and sequencing of selected genes confirmed the isolates as M. orygis. Multiple-locus variable-number tandem repeat analysis (MLVA) allowed the isolates to be divided into three clusters based on the relatedness of their MLVA profiles. The two monkey isolates shared the same MLVA pattern with 15 of the cattle isolates, whereas the remaining three cattle isolates had different patterns, even though the latter animals had been kept in the same dairy farm. The diversity observed among isolates may suggest the bacteria have been established in this area for a long period. This study along with other recent findings that report the detection of M. orygis from animals as well as humans originating from South Asia potentially indicate endemic distribution of M. orygis in South Asia.

Refeeding enteroclysis as an alternative to parenteral nutrition for enteric fistula.

AIM: Refeeding enteroclysis is one method of giving artificial nutritional support to patients with enterocutaneous fistula. This study compares the results of this technique with parenteral nutrition or nutrition given via a proximal stoma. METHOD: All patients admitted to our intestinal failure unit with a proximal enteric fistula and managed with refeeding enteroclysis over a 4-year period were included and compared with a matched group of patients managed without using this technique. RESULTS: Twenty patients (15 men) with a proximal enteric fistula received chyme refeeding down the distal limb of the fistula. This was established at a mean of 14 days after admission to the unit and total parenteral nutrition could be weaned off by 20 days. The mean output from the proximal limb was 1800 ml and the mean volume refed down the distal limb was 1220 ml per day. Additional enteric feed was given to 12 patients. No patient was given pharmacological agents to delay gastrointestinal transit or additional intravenous water and electrolyte for most of the time after refeeding was established. There were no complications or deaths related to chyme refeeding. CONCLUSION: Refeeding enteroclysis is feasible in selected patients with a proximal enteric fistula or stoma. Adequate nutrition, water and electrolyte balance can be achieved without resorting to parenteral infusions.

Molecular epidemiology of tuberculosis in rural Matlab, Bangladesh.

OBJECTIVE: To characterise and classify clinical isolates collected from tuberculosis (TB) patients in rural Bangladesh and to investigate the mode of transmission. DESIGN: An epidemiological study using a combination of conventional and molecular methods was performed in a rural population of Bangladesh. A total of 168 clinical isolates were collected from TB patients. Deletion analysis, used for rapid differentiation of members of the Mycobacterium tuberculosis complex, spoligotyping and variable number tandem repeats of mycobacterial interspersed repetitive units (VNTR-MIRU) typing were used. RESULTS: Deletion analysis identified all isolates as M. tuberculosis and further divided them into 109 strains (65%) carrying the M. tuberculosis deletion region 1 (TbD1-intact or 'ancestral' strains) and 59 strains (35%) lacking this region (TbD1 or 'modern' strains). MIRU analyses showed that 149 strains (89%) had unique patterns, whereas 19 strains (11%) clustered into eight groups. The largest cluster comprised five TbD1 strains of the Beijing type. The rate of recent transmission was estimated to be 6.5%. CONCLUSIONS: Our results suggest that TB in rural Bangladesh is caused primarily by reactivation of latent infections involving TbD1 intact strains, overlaid with the recent emergence of Beijing strain clusters that include multidrug-resistant isolates.

Bacteriostatic effect of Piper betle and Psidium guajava extracts on dental plaque bacteria.

In this study, the bacteriostatic effect of Piper betle and Psidium guajava extracts on selected early dental plaque bacteria was investigated based on changes in the doubling time (g) and specific growth rates (micro). Streptococcus sanguinis, Streptococcus mitis and Actinomyces sp. were cultured in Brain Heart Infusion (BHI) in the presence and absence of the extracts. The growth of bacteria was monitored periodically every 15 min over a period of 9 h to allow for a complete growth cycle. Growth profiles of the bacteria in the presence of the extracts were compared to those in the absence and deviation in the g and micro were determined and analyzed. It was found that the g and mu were affected by both extracts. At 4 mg mL(-1) of P. betle the g-values for S. sanguinis and S. mitis were increased by 12.0- and 10.4-fold, respectively (p < 0.05). At similar concentration P. guajava increased the g-value by 1.8- and 2.6 -fold, respectively (p < 0.05). The effect on Actinomyces sp. was observed at a much lower magnitude. It appears that P. betle and P. guajava extracts have bacteriostatic effect on the plaque bacteria by creating a stressed environment that had suppressed the growth and propagation of the cells. Within the context of the dental plaque, this would ensure the attainment of thin and healthy plaque. Thus, decoctions of these plants would be suitable if used in the control of dental plaque.

Prevalence of sputum smear-positive tuberculosis in a rural area in Bangladesh.

The objective of the study was to determine the prevalence of smear-positive tuberculosis (TB) in a rural area in Bangladesh at Matlab. A TB surveillance system was established among 106,000 people in rural Bangladesh at Matlab. Trained field workers interviewed all persons aged > or = 15 years to detect suspected cases of TB (cough > 21 days) and sputum specimens of suspected cases were examined for acid-fast bacilli (AFB). Of 59,395 persons interviewed, 4235 (7.1%) had a cough for > 21 days. Sputum specimens were examined for AFB from 3834 persons, 52 (1.4%) of them were positive for AFB. The prevalence of chronic cough and sputum positivity were significantly higher among males compared to females (P < 0.001). The population-based prevalence rate of smear-positive TB cases was 95/100,000 among persons aged > or = 15 years. Cases of TB clustered geographically (relative risk 5.53, 95% CI 3.19-9.59). The high burden of TB among rural population warrants appropriate measures to control TB in Bangladesh. The higher prevalence of persistent cough and AFB-positive sputum among males need further exploration. Factors responsible for higher prevalence of TB in clusters should be investigated.

Biological characterization of Aeromonas spp. isolated from the environment.

Cytotoxic enterotoxin (Act) is a key virulence factor in the pathogenesis of infections caused by Aeromonas spp. The cytotoxic enterotoxin gene (act) was detected in 32 out of 69 environmental isolates of Aeromonas spp. by hybridization with the act gene probe. To evaluate the pathogenic potential of the act gene probe-positive isolates, 32 act gene probe-positive and 31 randomly selected act gene probe-negative isolates were tested for enterotoxicity in a suckling mice assay (SMA), for haemolytic activity on sheep blood agar plates, for the presence of CAMP-like factors, and for cytotoxicity in a Vero cell line. The act gene probe-positive isolates significantly differed from the toxin gene probe-negative ones with respect to enterotoxicity in the SMA (P=0.009) and haemolytic activity (P=0.005). The CAMP-haemolysin phenotype was significantly associated with the rabbit ileal loop assay (P= 0.08), Vero cell assay (P= 0.064), and haemolysin production under the microaerophilic conditions (P= 0.056) of the act gene probe-positive isolates of Aeromonas spp. These data indicated the role of Act in the pathogenesis of Aeromonas infections and that the enterotoxic potential of Aeromonas spp. could be assessed by simply performing a CAMP-haemolysin assay.

Association of Vibrio cholerae O1 with the cyanobacterium, Anabaena sp., elucidated by polymerase chain reaction and transmission electron microscopy.

It has been hypothesized that Vibrio cholerae is an autochthonous flora of the estuarine and brackish water environment. Zooplankton and phytoplankton have been considered as possible reservoirs. The present study was carried out in microcosms to confirm the role of a cyanobacterium, Anabaena sp., as a reservoir of V. cholerae O1 using culture, polymerase chain reaction (PCR) and immunoelectron microscopy. Survival of culturable V. cholerae in microcosms was monitored by using tellurite taurocholate gelatin agar. Culturable V. cholerae were detected for up to 1 h in association with Anabaena sp. from a microcosm. However, viable but nonculturable (VBNC) V. cholerae O1 were detected for up to 25 months using PCR and immunoelectron microscopy. Results also showed that VBNC V. cholerae can multiply and maintain their progeny in the mucilaginous sheath of Anabaena sp. This is the first time that PCR and immunoelectron microscopy have been used to detect nonculturable V. cholerae in association with Anabaena sp. This study further clarifies the role of Anabaena sp. as a possible reservoir of cholera.

Factors affecting production of haemolysin by strains of Vibrio fluvialis.

The in vitro production of haemolysin by Vibrio fluvialis was studied using sheep erythrocyte. The effect of the composition of various media and different concentrations of sodium chloride on the production of haemolysin and heat-stability was investigated. Comparatively higher titre of haemolysin production was noted in brain heart infusion (BHI) broth. Adding 0.5% NaCl to BHI broth reduced the production of haemolysin; adding 5.0% NaCl to the medium totally inhibited it. The highest titre of haemolysin was produced at 30 degrees C and 37 degrees C, whereas no haemolysin was produced at 50 degrees C. Haemolytic activity was totally destroyed when heated at 56 degrees C for 30 minutes. Haemolysin could be assayed easily following this method.

Enterotoxigenicity, hemolytic activity and antibiotic resistance of Aeromonas spp. isolated from freshwater prawn marketed in Dhaka, Bangladesh.

Aeromonas spp. were isolated from gills, swimmerets, eggs, stomachs and ventral muscles of freshwater prawns (Macrobrachium malcolmsonii) available in the local fish market of Dhaka, Bangladesh. The density of Aeromonas spp. on these different body parts of the prawn samples ranged from 1.1 +/- 0.2 x 10(4) to 1.5 +/- 0.16 x 10(7) cfu per gram. The viable counts of aeromonads, fecal coliforms (FC) and Escherichia coli gradually increased in prawn samples when stored at 4 C. At -20 C, the viable counts gradually decreased and became zero on the 12th day of storage. The isolation of A. sobria (56%) was more frequent than that of A. hydrophila (31%) and A. caviae (13%). In the rabbit ileal loop (RIL) model, fluid accumulation induced by live cultures and cell-free culture filtrates of 11 strains ranged from 0.5 to 1.5 and 0.5 to 1.7 ml/cm of gut, respectively. Of 11 enterotoxigenic strains, 7 were A. sobria and 4 were A. hydrophila. Enterotoxigenicity correlated with hemolytic activity on blood agar. All enterotoxigenic strains were uniformly sensitive to chloramphenicol and gentamicin and resistant to novobiocin and vancomycin. Isolation of enterotoxigenic and antibiotic-resistant Aeromonas from these prawn samples indicates possible public health problems for their handlers as well for raw prawn consumers.

Evaluation of a probe hybridisation serotyping method for group A rotavirus.

Serotype-specificity and sensitivity of oligonucleotide probes to serotype human rotaviruses was assessed. Probes could detect as little as 6.3 ng of homologous RNA and none reacted with as much as 100 ng of heterologous RNA. Northern-blot analysis revealed that probes reacted with one of genomic segments 7, 8 or 9 of corresponding serotypes.

Localization of myosin heavy chain A in the human pathogen Entamoeba histolytica.

To recognize myosin II in trophozoites of the human pathogen Entamoeba histolytica, a specific antimyosin polyclonal serum was raised against a fusion protein consisting of a 146-amino-acid fragment of the myosin II heavy chain A of E. histolytica (MhcA) fused with beta-galactosidase. The hybrid protein was encoded by a chimera gene formed by a DNA fragment, from the mhcA gene, amplified by polymerase chain reaction and fused with the lacZ gene of Escherichia coli. Polymerase chain reaction-amplified DNA is located within the region encoding the tail domain of myosin. This antibody recognized a 250-kDa protein in extracts of E. histolytica trophozoites. Confocal microscope analysis of antibody-labelled trophozoites indicated that MhcA localizes at the posterior pole of locomoting cells and concentrates within the uroid. These results might indicate that MhcA is involved in movement and in the uroid formation which help amoebas to escape the host immune response. These data are the first evidence indicating that myosin exists in E. histolytica. In addition, two other peptides were found in myosin-enriched extracts of amoebas, indicating that other myosins may be present in this parasite.

A one-year survey of rotavirus strains from three locations in Bangladesh.

Group A rotavirus strains from 3 locations in Bangladesh collected over one year were examined. Serotypes 1-4 were found throughout in 2 locations, whereas an epidemic of RV diarrhoea due to a single rotavirus strain occurred in one location.

Isolation of enterotoxigenic Vibrio cholerae non-01 from the Buriganga river and two ponds of Dhaka, Bangladesh.

Vibrio cholerae 01 is usually considered the most toxigenic member of the Vibrionaceae and V. cholerae non-01 isolated from the environment is non-toxigenic. In our survey of the pollution of some aquatic environments in and around Dhaka, Bangladesh, we wanted to investigate the toxigenicity of V. cholerae non-01 isolated from water and sediment samples of the Buriganga river and two ponds in Dhaka, in the rabbit ileal loop (RIL) model. Fluid accumulation was induced by 18 of 28 live cultures and five of 18 cell-free culture filtrates in RIL. Seven of ten V. cholerae non-01 which failed to induce fluid in RIL were subjected to repeated passage in rabbit gut. Within two consecutive passages, all the strains could induce fluid in rabbit gut. Both toxigenic and non-toxigenic strains were uniformly sensitive to chloramphenicol and gentamicin but resistant to neomycin, novobiocin, polymyxin-B, streptomycin and vancomycin. Tetracycline sensitivity was found among eight of 17 toxigenic and six of 12 non-toxigenic strains. Sensitivity to trimethoprime-sulfa-methoxazole was noted among seven of 17 toxigenic and six of 12 non-toxigenic strains. Occurrence of enterotoxigenic and drug-resistant V. cholerae non-01 in the surface water is a public health hazard.

Incidence of Aeromonas isolated from diarrhoeal children and study of some virulence factors in the isolates.

Stool samples from 305 children with diarrhoea and equal number of age and sex matched non-diarrhoeal control children, less than 5 years of age, were examined during the period from Sept 1988 to April 1989. Aeromonas spp. were isolated from 37 (12.1%) diarrhoeal and 05 (1.6%) control cases. Out of 37 diarrhoeal isolates 13 (35.1%) were A. hydrophila, 19 (51.1%) A. sobria and 05 (13.5%) A. caviae. All the isolated strains were tested for haem agglutination property and haemolysin production. Seventeen diarrhoeal and 05 control isolates were tested for cytotoxin production in He La cell line and enterotoxin production in rat ileal loop model and suckling mouse model. Chinese hamster ovary cell (CHO) assay and Gm-1 ELISA methods were also employed. Cytotoxin production was found in 82.5% of diarrhoeal and 40% of control isolates. Haemagglutination was found in 62.1% of Aeromonas isolated from diarrhoeal children and 20% from control children. Enterotoxin production was detected in 58.8% diarrhoeal and none of the control isolates by either of the methods. Of the virulence factors enterotoxin production was found to correlate well with enteropathogenicity but haemolysin, cytotoxin and haemagglutinin did not.

Electrophoretic detection of salivary alpha-amylase activity.

Fresh samples of human whole saliva containing approximately 20-40 micrograms protein were analyzed using SDS-polyacrylamide slab gel electrophoresis systems. More than 20 protein bands were revealed by Coomassie Brilliant Blue R 250 staining. Some of the protein bands were shown to be glycoprotein-positive with PAS (periodic acid-Schiff) reagent. The protein bands with alpha-Amylase activity appeared within a molecular weight range of 120,000-180,000, which is 2 to 2.8 times higher than the normal molecular weight reported for alpha-Amylase from parotid saliva, and showed positive staining with PAS reagent. These results show that the alpha-Amylase in whole saliva appears to exist in a macromolecular form which is not dissociated in the presence of sodium dodecyl sulfate (SDS).

Prevalence of Plesiomonas shigelloides among diarrhoeal patients in Bangladesh.

The incidence of Plesiomonas shigelloides among diarrhoeal patients attending the Dhaka Treatment Centre of the International Centre for Diarrhoeal Disease Research, Bangladesh (ICDDR, B) from January through December 1987, has been reported. Using bile peptone broth (pH 8.8) as an enrichment medium and Salmonella-Shigella agar to isolate the organism, P. shigelloides was isolated from 838 (6.4%) of 13,142 patients, 523 (4.0%) of whom had no other pathogen isolated. The percentage of isolation was higher from stool (9.2%) than from rectal swab (5.9%) specimens (P < 0.005). The incidence of P. shigelloides was higher among male (64.0%) than female (36.0%) patients (P < 0.005). Isolation was highest among children less than five years. P. shigelloides was isolated most often in March (11.0%) and September (7.7%), indicating two seasonal peaks of incidence before and after the monsoons. All the strains were uniformly sensitive to chloramphenicol, furazolidon, gentamicin and trimethoprim-sulfamethoxazole. Sensitivity of the organism to ampicillin and tetracycline was 27.0% and 89.0%, respectively. This indicates that P. shigelloides may be an important agent of diarrhoea in our patient population.

Diffusion of buffered glutaraldehyde and formocresol from pulpotomized primary teeth.

The medicaments used in this study were buffered glutaraldehyde and formocresol. Schiff's reagent was used in the quantification of aldehyde released into the collecting medium. The results of this study clearly show that formocresol diffused throughout the dentine and cementum within fifteen minutes following a pulpotomy procedure, whereas no diffusion of buffered glutaraldehyde was observed.