Intradermal injection of methylene blue for the treatment of refractory pruritus ani.

OBJECTIVE: Idiopathic pruritus ani is a common perianal condition that can be refractory to diligent perineal care. We wished to evaluate the efficacy and side effects of intradermal methylene blue for the treatment of refractory pruritus ani. METHOD: A prospective series of 49 patients with idiopathic pruritus ani, who had failed to improve with perineal care, were treated by a single surgeon. All patients received intradermal injections of methylene blue. Endpoints were patient symptom score, and complications (pain, dysaesthesia, skin necrosis, incontinence and anaphylaxis). RESULTS: Symptoms improved in 96% and resolved in 57% of patients after one treatment. All four patients who had a second treatment became symptom-free. Seven patients noticed changes in continence, all resolved between 10 days and 6 weeks. Two patients were distressed by their decrease in their perianal sensation. There was no skin necrosis or anaphylaxis. CONCLUSION: Treatment of refractory pruritus ani by intradermal injection of methylene blue is effective and generally well tolerated.

Bacillus cereus is common in the environment but emetic toxin producing isolates are rare.

AIMS: To determine the incidence of emetic toxin producing Bacillus cereus in soil, animal faeces and selected vegetable produce to compare the results with the previously reported high incidence in rice paddy fields. To examine whether the emetic toxin has antibiotic activity. METHODS AND RESULTS: The incidence of emetic toxin producing B. cereus was evaluated by plating on selective agar 271 samples of soils, animal faeces, raw and processed vegetables. Overall, 45.8% of samples were positive for B. cereus. One hundred and seventy-seven B. cereus isolates were recovered at 30 degrees C with the grand mean spore count being 2.6 +/- 1.7 log(10) CFU g(-1) and 148 B. cereus isolates were recovered at 7 degrees C with the grand mean spore count being 2.2 +/- 1.2 log(10) CFU g(-1) of the 177 B. cereus isolated at 30 degrees C, only 3 were positive for emetic toxin production at a titre of 1/64, 1/32, 1/16, respectively. Also, 1 of 148 B. cereus isolated at 7 degrees C was positive for emetic toxin production to a titre of 1/128. All positive isolates came from washed or unwashed potato skins, one was psychrotrophic as determined by PCR and growth at 7 degrees C on subculture. The emetic toxin was not shown to have any antibiotic effects in growth inhibition studies. CONCLUSIONS: While B. cereus was a common isolate, the incidence of the emetic strain was rare. This is in contrast to previous findings of the high incidence in rice paddy fields and the processing environment, which may suggest rice is a selective area for growth of the emetic strain of B. cereus. SIGNIFICANCE AND IMPACT OF STUDY: The finding that a psychrotrophic isolate of B. cereus can produce emetic toxin is the first ever such observation and suggests the possibility that psychrotrophic isolates could grow in refrigerated fresh foods and cause emesis. The incidence of emetic B. cereus strains in rice paddy fields now requires further study for comparison with the low incidence found in other soils. The emetic toxin failed to inhibit the growth of other bacterial, fungal and yeast species. Whether the toxin (which is similar in structure to the antibiotic valinomycin) plays a competitive role in the environment therefore remains unclear.

Bacillus cereus produces most emetic toxin at lower temperatures.

Seven emetic toxin-producing strains of Bacillus cereus were examined for toxin production in Skim Milk Medium at incubation temperatures ranging from 10 to 50 degrees C. Minimum and maximum growth temperatures were found to be 12 and 46 degrees C, respectively. At 12 and 15 degrees C, levels of toxin production were significantly higher (P < 0.01) than that observed at 30 degrees C, while no toxin was produced above 37 degrees C. Increased levels of sporulation were observed at increased temperatures, and no correlation was found between levels of sporulation and toxin production (R(2) = 0.086).

Semiautomated metabolic staining assay for Bacillus cereus emetic toxin.

This paper describes a specific, sensitive, semiautomated, and quantitative Hep-2 cell culture-based 3-(4,5-dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide assay for Bacillus cereus emetic toxin. Of nine Bacillus, Brevibacillus, and Paenibacillus species assessed for emetic toxin production, only B. cereus was cytotoxic.

Total 24-hour melatonin secretion in adolescent idiopathic scoliosis. A case-control study.

STUDY DESIGN: A case-control study of 24-hour urinary melatonin production in patients with adolescent idiopathic scoliosis. OBJECTIVES: To address the controversy over the role of melatonin deficiency in adolescent idiopathic scoliosis by measuring total melatonin production over a 24-hour period. SUMMARY OF BACKGROUND DATA: An association between melatonin deficiency and experimental scoliosis has been suggested in several animal species. Recent work has failed to show a deficiency in humans with scoliosis. However, this conclusion was based on single urinary estimations. In this study the period assayed was standardized to 24-hours for all patients to include the full diurnal cycle of melatonin excretion. METHODS: Consecutive patients at an outpatient clinic for adolescent idiopathic scoliosis were recruited as subjects for this study, and patients from a fracture clinic who were of similar age and gender were recruited as controls at their final follow-up examination after the healing of their fracture. Patients and control individuals collected urine over a 24-hour period that was divided into consecutive day and night collections of 12 hours each. Total urinary excretion of 6-sulphatoxy melatonin was determined by radioimmunoassay for each 12-hour period in patients and control individuals. RESULTS: No significant difference in diurnal, nocturnal, or total urine 6-sulphatoxy melatonin excretion was found between adolescent patients with idiopathic scoliosis and controls of similar age and gender. There was also no difference between the two groups when 6-sulphatoxy melatonin excretion was corrected for body weight, body surface area, and body mass index. Nor was there a significant difference between 6-sulphatoxy melatonin excretion of patients with scoliosis whose curves failed to progress over the course of a year and the excretion of those who underwent surgery. CONCLUSIONS: In adolescent idiopathic scoliosis, neither the presentation with a stable spinal deformity, nor presentation with a severe deformity requiring surgery is associated with melatonin deficiency.

Temperature characterization of psychrotrophic and mesophilic Bacillus species from milk.

A total of 50 isolates of Bacillus spp. and one reference strain were investigated for their growth at 6.5 degrees C for 10 d, 30 degrees C for 3 d and 40 degrees C for 2 d. The results obtained differentiated three physiological groups: one clearly psychrotrophic (able to grow at 6.5 degrees C in 10 d, but not at 40 degrees C in 2 d), one intermediate in psychrotrophy (it grew at both 40 and 6.5 degrees C) and one mesophilic (capable of growth at 30 and 40 degrees C, but not at 6.5 degrees C). The proportion of strains in the second group was higher among isolates of B. cereus than for other Bacillus spp. However, the proportion of real mesophilic strains was lower for B. cereus isolates. Psychrotrophic B. cereus grew better at both 6.5 and 30 degrees C than other psychrotrophic Bacillus spp. Using eight strains, a correlation between differential growth at mesophilic temperatures (count at 30 degrees C minus count at 40 degrees C) and a standard psychrotrophic count at 6.5 degrees C for 10 d (r = 0.95) was obtained in mixed cultures when the psychrotrophic flora count was < or = 1 log (cfu/ml) lower than the mesophilic count.

Spinal fusion in Duchenne's muscular dystrophy.

The Women's and Children's Hospital experience with Luque spinal fusion in Duchenne's muscular dystrophy was reviewed from its commencement in 1983 to the present with a view to assessing the clinical and radiologic outcome and safety of the procedure. Seventeen boys have undergone spinal fusion. L-rod instrumentation was used in 10, six of whom had significant problems with sitting imbalance or progression of the scoliosis or both. In seven cases, distal instrumentation was taken to the pelvis with a Galveston construct and rigid crosslinking. Apart from some progression and sitting imbalance in the L-rod group, there were few complications. In the Galveston group, pelvic obliquity was corrected by a mean of 63%, and there was better maintenance of correction. There were no pseudoarthroses or instrument failures in the Galveston group. Of the total group, four patients had forced vital capacity (FVC) values < 25% predicted, and two required ventilation postoperative (< 48 h). There were no other respiratory complications. The effect of surgery on respiratory function remains uncertain. Spinal fusion with the Luque rod construct and pelvic fixation is a safe procedure. It provided a mean correction of 60% and control of pelvic obliquity without significant postoperative deterioration. In our experience, surgery can be safely performed with FVC value down to 20% predicted. On the basis of these data, one current practice is to instrument to the pelvis with a Galveston construct and Texas Scottish Rite Hospital cross-linking.

Effect of spinal surgery on lung function in Duchenne muscular dystrophy.

BACKGROUND: The effect on subsequent respiratory function of spinal stabilisation for scoliosis in Duchenne muscular dystrophy is unclear. In order to clarify this clinical problem, changes in the forced vital capacity of a group of children with Duchenne muscular dystrophy who had undergone spinal surgery were measured and compared with a group of children with Duchenne muscular dystrophy who had not had surgery. METHODS: In this retrospective study 17 boys with Duchenne muscular dystrophy who underwent spinal stabilisation at a mean age of 14.9 years (surgical group) were compared with 21 boys with Duchenne muscular dystrophy who had not had surgery (non-surgical group). The mean (SD) Cobb angle of the surgical group at 14.9 years was 57 (16.4) degrees, and of the non-surgical group at 15 years was 45 (29.9) degrees. Forced vital capacity expressed as percentage predicted (% FVC) was measured in total over a seven year period in the surgical group and over 6.5 years in the non-surgical group, and regression equations were calculated. Survival curves for both groups were also constructed. RESULTS: No difference was found between spinal stabilisation (surgical group) and the non-surgical group in the rate of deterioration of % FVC which was 3-5% per year. There was no difference in survival in either group. CONCLUSIONS: Spinal stabilisation in Duchenne muscular dystrophy does not alter the decline in pulmonary function, nor does it improve survival.

Seasonal occurrence of psychrotrophic Bacillus species in raw milk, and studies on the interactions with mesophilic Bacillus sp.

Mesophilic and psychrotrophic isolates of Bacillus species displayed seasonal incidences in raw and pasteurised milk. The incidence of mesophilic isolates was highest in the winter and lowest in the summer/autumn while pschrotroph incidence was conversely lowest in the winter and highest in the late summer/autumn. Spores of Bacillus sp. were isolated from raw milk taken from farm milk machines and bulk tanks, milk tankers, diary silos and pasteurised milk. A consistent seasonal fluctuation in incidence throughout these samples suggested that spores of Bacillus sp. derived from the farm environment survived as important contaminants right through the milk chain to the pasteurised product. Up to seven mesophilic Bacillus sp. were isolated from a single sample with three species commonly occurring in most samples. The predominant mesophilic species isolated were B. pumilus, B. licheniformis and B. subtilis. The dominant psychrotrophic isolate was B. cereus. Selected mesophilic isolates were examined for possible antagonistic effects on the growth of psychrotropic B. cereus and B. pumilus isolates. Bacillus subtilis and B. licheniformis were found to produce antagonistic factors. It was considered that these factors may influence the incidence and growth of psychrotrophic isolates in the farm environment or in milk but the factors are not yet fully characterised or identified.

Phenotypic analysis of lymphocytes obtained by bronchoalveolar lavage of normal sheep.

Lungs were excised from apparently healthy sheep at slaughter and lymphocytes obtained by bronchoalveolar lavage were separated by buoyant density and characterised phenotypically. In most animals T-lymphocytes (CD5+ cells) accounted for almost the entire population, with B-lymphocytes (sIg+ cells) generally accounting for less than 10%. In the main, CD4+ T cells exceeded CD8+ T cells by at least a factor of three and CD4-/CD8- (gamma delta) T cells represented around 6% of the population. Proportions of subtypes of lymphocytes obtained by bronchoalveolar lavage differed from those in peripheral blood. The normal range of lymphocyte phenotypes in the ovine lung established in this study will be of value in subsequent work on animals with defined respiratory disease.

Toxin production by Bacillus cereus in dairy products.

Spores of a known toxigenic and psychrotrophic dairy isolate of Bacillus cereus (HRM 44) were unable to grow and produce diarrhoeagenic toxin at 6 degrees C in creams and dairy-based products. These findings suggest that the production of B. cereus diarrhoeagenic toxin is unlikely to occur in creams and dairy-based products maintained within the cold chain. Growth and toxin production were readily demonstrated in creams and some desserts stored at 21 degrees C. Growth in creams was associated with obvious spoilage. However, in the flavoured desserts, spoilage was not always obvious before significant growth of B. cereus and toxin production had occurred. Dairy desserts with high sugar content and/or low pH did not support toxin production and these findings are discussed.

Influence of pH and sugars on the growth and production of diarrhoeagenic toxin by Bacillus cereus.

Broth cultures supplemented with high levels of sugars, particularly glucose at > 50 milligrams, did not support diarrhoeagenic toxin production by psychrotrophic Bacillus cereus despite growth to high counts (approximately 10(7)/ml) over a 4 d period of incubation at 21 degrees C. In contrast, starch levels of 10 and 50 milligrams actually enhanced toxin production. Toxin production was also affected by pH levels of broth cultures, and was concomitant with alterations in bacterial growth. These findings help to explain variations in toxin levels previously found in some dairy desserts, which were thought to be associated with pH and sugar content (Sutherland, 1993).

Effect of heat treatment on Listeria monocytogenes and gram-negative bacteria in sheep, cow and goat milks.

Sheep milk, compared with cow and goat milk, had a protective effect on Gram-negative bacteria and Listeria spp. heated at 65 degrees C in a test-tube method. This effect was not solely due to fat content as cow milk artificially reconstituted to 10% homologous fat was not as protective. Listeria monocytogenes in whole sheep, cow and goat milks at an inoculum level of 1 x 10(6) cfu ml-1 was heated at 68 degrees C for 15 s in the plate pasteurizer and survival was only detected in whole sheep milk after heating. Whole sheep, cow and goat milks containing high levels of L. monocytogenes (1 x 10(6) cfu ml-1) could not survive the current HTST plate pasteurization protocol.

An experimental anti-idiotype vaccine mimicking lipopolysaccharide gives protection against Pasteurella multocida type A infection in mice.

An anti-idiotype strategy was employed which showed that polyclonal anti-idiotype antibodies could be produced which could mimic a linear Pasteurella multocida lipopolysaccharide (LPS) molecule. These antibodies when used as vaccine antigens, induced antibodies which recognised LPS and imparted acquired protection upon syngeneic vaccinates challenged with homologous organisms.

Characterisation and biological activity of monoclonal antibodies specific for Pasteurella haemolytica A1 capsule and lipopolysaccharide.

Monoclonal antibodies (mAb) against both Pasteurella haemolytica A1 capsule and lipopolysaccharide (LPS) were produced. Anti-capsule mAb reacted with the homologous A1 serotype only, whereas mAb against LPS reacted with P. haemolytica serotypes A2, A5, A8, A12, A14 and A16 but not with 33 bacterial species or rough LPS mutant strains tested. Both capsule and LPS antigens were visualised on the surface of bacteria by immunogold electron microscopy. Neither of the mAbs demonstrated antibody-dependent complement-mediated killing in vitro but both facilitated phagocytosis in vitro.

Occurrence and diversity of plasmids in ovine isolates of Pasteurella haemolytica.

160 ovine isolates of Pasteurella haemolytica, representing each of the 16 serotypes and also untypable strains, were examined for plasmid content. Plasmid DNA was identified in, and prepared from, strains of serotypes A2, T3, A14 and A16 and also from an untypable strain. The relationship between the plasmids present in the different strains was examined both by restriction fragment profile analysis and by DNA/DNA hybridisation. Both methods gave broadly similar results and showed that each serotype tended to contain either a single plasmid species, or a limited range of species, and that structural similarities could traverse serotype boundaries. None of the plasmid-bearing strains showed any significant level of resistance to a range of antibiotics.

Vaccine containing iron-regulated proteins of Pasteurella haemolytica A2 enhances protection against experimental pasteurellosis in lambs.

A vaccine containing sodium salicylate extract (SSE) of Pasteurella haemolytica A2 cells grown in a medium chemically depleted of available iron by the addition of alpha alpha dipyridyl to induce iron-regulated proteins (IRPs) conferred protection to specific pathogen-free (SPF) lambs exposed to an aerosol of P. haemolytica A2. The disease score in these lambs was significantly lower (p less than 0.005) than those in unvaccinated lambs or in lambs immunized with SSE prepared from cells grown in iron-replete medium. Immunoblotting of sera from these SPF lambs against whole cell antigens of P. haemolytica A2 grown under iron-restricted conditions demonstrated that antibodies to IRPs were present only in the sera of animals immunized with SSE-IRP. The antibody profile of sera from the SSE-IRP group was similar to that obtained with serum from a lamb which had recovered from P. haemolytica A2 disease produced experimentally. Negligible levels of cytotoxin-neutralizing and bactericidal antibodies were detectable in the SSE-IRP group and therefore appear not to be involved in the protection observed in this experiment.