Helicobacter pylori isolated from patients with tonsillar cancer or tonsillitis chronica could be of different genotype compared to isolates from gastrointestinal tract.

Helicobacter pylori from patients with different diseases, including so-called autoimmune thyroiditis, chronic tonsillitis and tonsillar cancer, was isolated and cultured. It was identified according to the genotype using labeled hybridization probes complementary to six sequences of cagA and vacA genes. Different types of strains were found in isolates from gastrointestinal tract and patients suffering from thyroiditis. Six out of seven genotyped isolates from patients in our Department of Otorhinolaryngology and Head and Neck Surgery exhibited the same genotype, differing from isolates obtained from other patients; the 7th isolate originated from a patient who had undergone surgery for deviatio septi nasi, at the same time suffering from autoimmune thyroiditis, having confirmed gastric infection by H. pylori from biopsy. This data made it possible to formulate the hypothesis on probable association of specific H. pylori genotype with chronic tonsillitis and tonsillar cancer. We assessed commercial transport media and improved nucleic acid isolation techniques and the RT-PCR-based tests, which allowed us to skip a culture step and to test directly the patients' samples; however, for full confirmation of our hypothesis and explanation of possible mechanisms of the contribution of Helicobacter sp. to the pathogenesis of the disease further data are to be collected and evaluated.

[Detection of CagA protein in strains of Helicobacter pylori isolated from patients with gastritis and gastric ulcer disease]. / Prukaz CagA proteinu u kmenu Helicobacter pylori izolovaných od nemocných s gastritidou a vredovou chorobou zaludku.

UNLABELLED: A total of 156 Helicobacter pylori strains isolated from adult patients (81 men, 75 women) with diagnosis of gastritis (43 strains GAS) and gastric ulcer (113 strains GU) were analyzed for the production of CagA protein by quantitative and qualitative immunoblot methods. 80% of gastric ulcer group strains (84% in men, 73% in women) produced CagA protein, while in the gastritis patients group only 63% of strains were CagA protein positive (59% in men, 65% in women). Quantitative production was estimated by CagA index (mean production of CagA protein in CagA positive strains) in both groups of strains GAS and GU in men and women. Strains of GU group were shown to be higher producers (index CagA 2.42) as compared to GAS group (2.09). Index CagA was higher in strains isolated from men (index CagA 2.54) than is strains from women (index CagA 2.13). CONCLUSION: CagA production has been proved in 75% of analysed Helicobacter pylori strains. CagA positive strains were more frequent in gastric ulcer disease patients, slightly higher in the male than in the female patients. Strains isolated from men, irrespective of diagnosis (GAS and GU), are higher producers of CagA protein.

[Helicobacter pylori infection in a group of symptomatic and asymptomatic children and adolescents in the Czech Republic]. / Infekce Helicobacter pylori u souboru symptomatických a asymptomatických detí a dospívajících v Ceské republice.

BACKGROUND: There exist only inconsistent results of studies on the relationship between Helicobacter pylori (H. pylori) infection and functional dyspepsia and/or recurrent abdominal pain in children and adolescents. The answer could bring a comparison of the epidemiological features of H. pylori infection between children and adolescents with dyspepsia and/or recurrent abdominal pain (symptomatic) and without these symptoms (asymptomatic), living in the Czech republic. METHODS AND RESULTS: Study includes children and adolescents (2-18 years) with upper gastrointestinal symptoms visiting paediatric gastroenterology department between 1994 and 1999. Age, sex, socioeconomic level matched control (asymptomatic) group consisted of children and adolescents visiting the general paediatric service with symptoms not related to the upper gastrointestinal tract. Demographic and socioeconomic factors, including parent(s) educational level, place of residence, living conditions, type of drinking water and pets in their household were evaluated. Serum of the children and adolescents was tested for H. pylori IgG by enzyme linked immunosorbent assay. In addition, the symptomatic children and adolescents underwent endoscopic examination and biopsies from gastric antrum and corpus were taken for the quick urease test, histology/histoscopy, and H. pylori cultivation. H. pylori infection in this group was stated when at least two tests were positive. Altogether 829 children and adolescents were examined, 624 cases were symptomatic and 205 represented controls (asymptomatic). The prevalence of infection was 33% among symptomatic children vs. 7.5% among controls (OR = 6.2, p.001) and was similar among boys and girls (32% vs. 34.5%, respectively). H. pylori prevalence increased with age among symptomatic children (10% for children below 6 years and 37% between 11 to 16 years) (p.001). In contrary, prevalence tended to fall with age among asymptomatic children (11% in children below 6 years vs. 6% in children over 10 years (OR = 2.0, 95% CI = 0.7-6.2). In both groups, an inverse correlation between H. pylori occurrence and mother's educational level was observed. The H. pylori incidence was 10% among asymptomatic children who drank water from municipal water supply or from a well vs. 3% among those who drank bottled water (OR = 4, 95% CI = 1.1 to 18, p.05). CONCLUSIONS: H. pylori infection was more prevalent among symptomatic children and adolescents vs. asymptomatic children and adolescents within the same population. H. pylori incidence increased with age among symptomatic children and adolescents and tended to fall among controls, showing deep differences between the two groups. With exception of the bottled water drinking, presence of any other factor studied represented significant risk for acquiring the H. pylori infection.

[Helicobacter pylori and diabetes mellitus]. / Helicobacter pylori a diabetes mellitus.

BACKGROUND: Helicobacter (H.) pylori participates significantly on the pathogeny of chronic gastritis, duodenal a gastric ulcer, carcinoma and lymphoma of the stomach. Attention was attracted also by some extragastric diseases, including diabetes mellitus, where the elevated antibodies levels against H. pylori was found. The aim of the work was to determine the seroprevalence in relation to the sex and type of disease in a cohort of diabetics and in a control group of blood donors. METHODS AND RESULTS: In 195 diabetic patients (type I and II) and 216 blood donors levels of IgG antibodies were determined by ELISA method. Seroprevalence in the group of diabetic patients was 27%, in males 26% and in women 28% (n.s.). No differences related to sex or diabetes type were found. Significant differences in seroprevalence were found among the group of diabetic patients and blood donors (27% vs. 51%, p < 0.001), male diabetics of both types and blood donors (26% vs. 62%, p < 0.001). No differences were found between female diabetic patients and controls (28%, 27% vs. 40%, n.s.). CONCLUSIONS: Our study has shown a lower seroprevalence of H. pylori in diabetic patients of type I and II in comparison with the healthy population. Such finding differs from the generally accepted experience of the higher sensitivity of these patients to infection. The practical significance of the observation remains unsolved.

[Helicobacter pylori infections: familial clustering and transmission]. / Infekce Helicobacter pylori: shlukování a prenos v rodinách.

BACKGROUND: The importance of Helicobacter pylori for the development of gastritis and gastro-duodenal ulceration is generally accepted. The objective of the presented work was to find out whether its transmission is involved in families of children examined on account of upper dyspeptic syndrome. Aggregation and transmission of Helicobacter pylori infection were not described so far in the Czech Republic. METHODS AND RESULTS: The authors examined IgG antibodies (ELISA) against Helicobacter pylori in 411 subjects incl. 102 children with non-ulcerative or ulcerative dyspepsia and in 115 children of similar age but without gastrointestinal complaints and in 194 family members of children with dyspepsia. The specificity of the ELISA test is 97.1% and the sensitivity 92.1% (discrimination analysis). Children with dyspepsia had more frequently a positive finding of IgG antibodies against H. pylori (p = 0.0001), as compared with the control group of children of the same age but without dyspepsia. A statistically significant source of infection with H. pylori for dyspeptic children was the mother (p = 0.0006) and both parents (p = 0.0069), who were also a source of infection for other family members (p = 0.0003 and 0.0084). The most frequent positive IgG finding was in fathers (76.7%), but the father was not a statistically significant source of infection for the examined children (p = 0.6120) nor for other members of the family (p = 0.2330). IgG positive mothers had 85.2% positive husbands, IgG negative mothers had 68.4% positive husbands (p = 0.1601). The frequency of IgG antibodies in the mother and father (in couples) was thus statistically independent. However, there was a significant correlation (p = 0.01) of (P/N) IgG antibodies in couples--i.e. father and mother of the examined children. Siblings of IgG positive children had a significantly higher mean value of antibodies (p = 0.05) and were older. CONCLUSIONS: The presented facts provide evidence that in the investigated group the Helicobacter pylori infection is more frequent in children with the upper dyspeptic syndrome than in children of similar age without gastrointestinal complaints. In families of children with dyspepsia the infection aggregates and is transmitted. The mother and both parents are involved in the transmission to children. Nevertheless it is probable that there is also mutual transmission in children and from adults to children outside the family and in another environment.

Isolation and preliminary evaluation of a low-molecular-mass antigen preparation for improved detection of Helicobacter pylori immunoglobulin G antibodies.

Previously, immunoglobulin G (IgG) antibodies to five antigens with a relative molecular mass of between 15 and 30 kDa from Helicobacter pylori were found to be significantly more frequent in H. pylori-infected patients than in noninfected patients. In this study, these specific low-molecular-mass (LMW) antigens were separated by ultrafiltration of whole-cell sonicates. The LMW antigen preparation was evaluated by enzyme-linked immunosorbent assay with serum samples from 76 children with abdominal symptoms and 151 adults with dyspeptic symptoms. H. pylori was cultured or seen in 40 (53%) children and 83 (55%) adults. Increased antibody levels to H. pylori were found in serum from 35 (46%) children and 88 (58%) adults. Values for sensitivity, specificity, and predictive value of positive and negative results of the test were higher with LMW antigens than with the heat-stable antigen previously described. The low specificity and predictive value of a positive result were due to seropositive results for 21 persons with a negative culture for H. pylori and negative microscopy results for Helicobacter-like organisms in biopsies from gastric mucosa. Histologically, chronic gastritis was demonstrated in 43% of these persons, and 19% had peptic ulcer, indicating that they have or have had H. pylori infection. Specific antibodies to H. pylori were confirmed in all 21 patients by the Western immunoblot technique. Use of the LMW antigen improved the IgG antibody detection in patients with H. pylori infection, even though the results reflect the difficulties in establishing a true gold standard for diagnosis of H. pylori infection.

[Serologic detection of antibodies to Helicobacter pylori in children and adolescents using ELISA]. / Sérologický prukaz protilátek proti Helicobacter pylori u deti a dospívajících metodou ELISA.

Serum antibodies anti-Helicobacter pylori (H.p.) which are specific against antigenic determinants of the bacterial body, were determined in 183 non-ulcer dyspeptic children and adolescents, aged 7- to 20 years. 108 patients (59%) of the same group underwent fibroendoscopy for direct detection of H.p. in bioptic specimens of the stomach and duodenum microbiological methods. Discrimination analysis revealed a overall positive correlation between the results of the two methods: P < 0.01. For detection of specific IgG antibodies against H.p., evaluation by the P/N ratio was used, i.e. the absorbance of the examined serum (P) was compared with the mean absorbance of three negative serum controls (N). The specificity of the ELISA test in this examination was 96.1%, the sensitivity of 90.3%. The mentioned ELISA method for detection of specific IgG antibodies against H.p. is comparable with direct microbiological prove of H.p. presence in mucus specimens in our group examined. It is a very important non-invasive method useful for primary detection of H.p. infection.

[Determination of antibodies to Campylobacter pylori in children and adolescents with gastroduodenal disorders]. / Stanovení protilátek proti Campylobacter pylori u detí a dorostu s gastroduodenálními obtízemi.

Serological examination of antibodies against Campylobacter pylori was made in 195 boys and girls aged 9-20 years. In all on account of gastrointestinal complaints endoscopy was indicated. The results of microbiological and histological finding), in particular in the antrum and corpus of the stomach). Positive titres antrum and body of the stomach were compared with results of the complement fixation test and the ELISA reaction. Antibodies against C. pylori were detected in 64 of the examined subjects. The titres assessed by the complement fixation and ELISA test were in good agreement. Patients with positive sera had significantly more often a positive endoscopic finding (bacteria, chronic gastritis) and bacteriological finding), in particular in the antrum and corpus of the stomach). Positive titres of the complement fixation reaction persisted frequently for 15 months after the first examination. From the results ensues that serological examination of antibodies against C. pylori can be considered a sparing examination method which in patients with gastrointestinal complaints should precede endoscopic examination and moreover it can be used to follow up the course of the disease and success of therapy.

[Can epigastric pain and non-ulcerative dyspepsia in children and adolescents be Campylobacter pylori infection?]. / Mohou byt bolesti v epigastriu a neulcerózní dyspepsie u detí a dospívajících infekcním onemocnením Campylobacter pylori?

The authors examined 263 children and adolescents aged 5-20 years who suffered from so-called non-ulcerative dyspepsia or epigastric pain, because of the suspected presence of Campylobacter pylori. In 31.9% of the examined subjects direct microbiological methods revealed its presence in the gastroduodenal mucosa. The confidence limit of positive results of Campylobacter pylori in the population is between 27.58% and 38.84%, the probability being 95%. The authors proved a mutual correlation between endoscopic, histological, serological findings and detection of Campylobacter pylori by microbiological methods. The statistical significance of the correlation of campylobacter pylori and chronic gastritis B (mostly inactive) provides further support for the hypothesis of the aetiological role of Campylobacter pylori in the development of chronic gastritis B in children and adolescents. From the investigation it does not ensue, however, that colonization with Campylobacter pylori is associated with certain clinical symptoms in all instances. However, in the differential diagnosis of so-called non-ulcerative dyspepsia and epigastric pain in children and adolescents we must include infection with Campylobacter pylori among their possible causes.

[Cytotoxic changes in cell cultures induced by newly isolated strains of Campylobacter pylori]. / Cytotoxické zmeny vyvolané cerstve izolovanymi kmeny Campylobacter pylori v bunecných kulturách.

Suspensions of primocultures of two strains of Camplyobacter pylori, isolated from bioptic materials collected from patients with nodular hyperplasia of the pyloric antrum and with gastric ulcers were inoculated to 20-hour confluent or almost confluent cell cultures line VERO, McCoy and MA 104 in test tubes and Petri dishes. Both strains of C. pylori adhered markedly to cell lines VERO and McCoy and after 72 hours led to marked morphological changes of the cells. The changes were characterized in VERO cells by the presence of polygonal to star-shaped cells with processes, a reduced cytoplasm and only in the final stages by the formation of circular cells separating from the medium. Some cells were vacuolized. Cells of the McCoy line responded to the infection with C. pylori by a similar morphological picture, changes in the cell periphery were, as compared with the VERO line, more marked in the early stages. Cells of line MA 104 did not respond to infection with C. pylori by any morphological changes, not even after six days. The intensity of morphological changes was more marked in cell cultures line VERO infected with a strain isolated from a patient with a gastric ulcer, as compared with changes caused by a strain of C. pylori isolated from a patient with nodular hyperplasia of the pyloric antrum. The possibility to induce morphological changes of some stable lines supports the idea of the pathogenic action of Campylobacter pylori on the gastric mucosa.

[New aspects of intolerance of the aqueous iodinated x-ray contrast medium, adipiodone--fatal interaction of adipiodone-Ultrabil with the immunomodulators Corynebacterium parvum (CP) and Shigella dysenteriae endotoxin (LPS)]. / Nový aspekt intolerance vodné jódované rtg kontrastní látky typu adipiodonu--fatální interakce adipiodonu-Ultrabilu s imunomodulátory Corynebacterium parvum (CP) a endotoxinem (LPS) Shigella dysenteriae.

The authors assume a new type of interaction of the aqueous X-ray contrast substance type adipiodone (Ultrabil 50% Spofa) with immunomodulators of Corynebacterium parvum (CP) and the endotoxin of Shigella dysenteriae (LPS). The fatal type of interaction developed regularly as a result of the concurrent i.v. administration of the X-ray contrast substance adipiodone with the endotoxin of Shigella dys. (LPS) to laboratory animals (mice) which had five days previously a single dose of Corynebacterium parvum (CP). The effect of the interaction was evaluated using LD50 of adipiodone (Ultrabil Spofa) which in interaction with the immunomodulators of CP and LPS was identical as the dose used in diagnostic practice. The authors considered the possible development of "oxygen stress" as a consequence interacting processes and complement activation.

Relation of enterotoxin production and lipolytic activity in Staphylococcus aureus strains isolated from acute diarrhoeal diseases to clinical course of illness.

Two sets of cases of acute diarrhoeal disease caused by plasma-coagulase-positive Staphylococcus aureus strains were studied. Testing the isolates for staphylococcal enterotoxin (SE) production and for lipolytic activity on egg-yolk agar in relation to the clinical course of the illness showed that only part of the cases could be attributed to the effect of SE and that lipolytic activity apparently also played some pathogenetic role. In one of the sets (205 cases) SE production was found in only 29% of the strains isolated; the clinical course in the corresponding patients was typical of staphylococcal enterotoxicosis. Pronounced lipolytic activity without SE production was demonstrated in 30% of strains; here the clinical course was much milder and protracted. Where both factors were produced (21% of cases), the clinical pattern of enterotoxicosis predominated but was somewhat modified. Two outbreaks in one of which SE production and in the other only clear-cut lipolytic activity were found also differed mutually in the clinical respect. The first displayed a picture of typical staphylococcal enterotoxicosis, the second comprised mild diarrhoeal cases with an incubation period of 6-9 hours, diffuse abdominal pain and no fever. Accordingly, the above observations showed a certain positive correlation between the presence of some staphylococcal exoproducts (SE, lipolytic-activity factor) and the clinical course of the disease. This was particularly striking in infants up to 2 years of age. Lipolytic activity seemed to be associated with mild diarrhoeal staphylococcal disease, although the co-participation of (an)other, so far undetermined factor(s), could not be precluded.

Purification and some properties of exotoxin from Corynebacterium ulcerans strain ATCC 9015.

Corynebacterium ulcerans strain ATCC 9015 develops during growth in environmental media an exotoxin which is lethal for guinea-pigs. The exotoxin has been purified by ion-exchange chromatography on DEAE-Sephadex A-50, affinity chromatography using immunosorbent techniques, and gel-filtration through Sephadex G-75. The exotoxin is of a protein nature and consists perhaps of two subunits. The molecular weights of the exotoxin and its subunits as estimated by SDS-PAGE were 40 000, 25 000, and 15 000, respectively. The toxic properties of exotoxin were destroyed upon heating (60 degrees C, 30 min), treatment with trypsin (10 micrograms/ml, 37 degrees C, 30 min), and by the extremes of pH (pH 4.0 or 10;0 24 hrs). Homologous antibodies readily neutralized the exotoxin while commercial diphtheria antitoxin had no effects. No immunological cross-reactivity was revealed between exotoxin of C. ulcerans and other bacterial toxins such as diphtheria toxin, enterotoxin from V. cholerae, and exotoxin A of P. aeruginosa.

Proof for the lack of diphtheria toxin in culture filtrates of Corynebacterium ulcerans strain ATCC 9015.

Extensive efforts to identify the diphtheria toxin in culture filtrates of C. ulcerans strain ATCC 9015 was unsuccessful. The culture filtrates did contain a component (called D-antigen) which formed a precipitin line with commercial diphtheria antitoxin. This component was purified by immunosorbent techniques using immobilized preparations of diphtheria antitoxin. It was shown, that D-antigen has a molecular weight of 84000, is not toxic for guinea-pigs, and has nothing in common with purified diphtheria toxin when tested in gel-diffusion tests. The lack of diphtheria toxin in form of its fragments in culture filtrates concentrated 20-fold was proved by the highly sensitive and specific viroimmunoassay.

Enterotoxigenic E. coli in humans.

E. coli strains isolated from persons having diarrhoeal disease were tested for the production of TS enterotoxin. The production of TSE was demonstrated in 2.5% in a series of 80 strains isolated from children under one year of age, having acute diarrhoea. TSE was produced by 8.4% of E. coli strains out of 59 strains isolated from patients over one year of age. Among these strains, an interesting E. coli strain was isolated from the patient T. J., which produced TSE for more than 15 months. The production of TLE was tested though not proved in all strains by experiment on an isolated intestinal loop of an adult rabbit. The test on suckling mice so far appears to be the most suitable test for the demonstration of TSE. The results were considered positive when the index (the ratio of the weight of the whole intestine to the weight of the rest of the body) was higher than 0.08 while indices up to 0.078 were considered negative. E. coli strains with indices of intermediate values and strains with temporary production of TSE, occurring particularly in very small children, deserve special attention. The height of the indices was not influenced by a 30-minute exposure at 60 degrees C, but a decrease in the values of the indices was observed after boiling for a period of 15 min. The occurrence of E. coli strains producing TSE is evidently small in humans in European countries but, without doubt, they are important in the aetiology of diarrhoeal diseases.