Biomedical subjects
Publications and source records attributed to H Goossens.
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Four strains of Campylobacter jejuni isolated from children with inflammatory diarrhoea were assayed in the rabbit ileal loop model of infectious diarrhoea. All caused inflammatory reactions with severe macroscopic and microscopic damage in infected rabbit ileal tissue similar to that observed in the patients by endoscopy and histological analysis of colonic biopsies. Haemoglobin and other proteins were observed in loop fluids, consistent with leakage of serum from damaged mucosa. Loop fluids also contained significant bicarbonate concentrations, indicative of an active secretory component similar to that in control loops inoculated with cholera toxin. However, although three of the four clinical strains produced small amounts of a protein immunologically related to cholera toxin in vitro, none such was detected in either tissues or fluids of infected ileal loops. We propose instead that host-derived mediators of secretion may be important in pathogenesis. A mutant strain of C. jejuni with impaired motility, obtained from the National Collection of Type Cultures, did not induce tissue damage or fluid secretion in rabbit ileal loops.
Infection of rabbit ileal loops with inflammatory Campylobacter jejuni strains caused elevation of cyclic AMP, prostaglandin E2, and leukotriene B4 levels in tissue and fluids. Incubation of cultured Caco-2 cells with loop fluids caused elevated cellular cyclic AMP levels, an effect which was inhibited by antiserum against prostaglandin E2.
DNA polymorphisms of Arcobacter butzleri outbreak-related strains and Arcobacter reference strains were determined by use of the polymerase chain reaction with primers aimed at repetitive sequences. The epidemiological relationship among 14 outbreak-related strains was substantiated, as they showed virtually no genomic variations. Their DNA amplification patterns were, however, clearly different from those of all Arcobacter reference strains studied; each reference strain was characterized by a unique DNA fingerprint.
By comparing nucleic acid sequences determined for one of the most variable areas of 23S rRNA genes of 23 Campylobacter strains, we were able to identify regions specific for thermophilic Campylobacter strains. Oligonucleotide primers corresponding to these unique regions were synthesized and used in the polymerase chain reaction. One primer pair selectively detected all thermophilic Campylobacter species, while four other primer pairs allowed discrimination among the thermophilic species Campylobacter coli, Campylobacter jejuni subsp. jejuni, Campylobacter lari, and Campylobacter upsaliensis. All primer sets were tested successfully on a large number of clinical isolates.
The application of polymerase chain reaction (PCR) fingerprinting assays enables discrimination between species and strains of microorganisms. PCR primers aiming at arbitrary sequences in combination with primers directed against the repetitive extragenic palindrome (REP) or enterobacterial repetitive intergenic consensus (ERIC) motifs generate isolate-specific DNA banding patterns. Analysis of these PCR fingerprints obtained for 33 isolates of Campylobacter jejuni, 30 isolates of Campylobacter coli, and 8 isolates of Campylobacter lari revealed that besides generation of isolate-specific fragments, species-specific DNA fragments of identical size were synthesized. It appeared that these DNA fragments could be used as species-specific probes, since they are unique for the pattern which they are deriving from. The probes do not cross-react with amplified DNA originating from a large panel of nonrelated microorganisms. Moreover, these probes displayed species specificity, as they reacted with a single restriction fragment on Southern blots containing DNA from C. jejuni, C. coli, and C. lari and other Campylobacter species. This combination of PCR fingerprinting and probe hybridization results in a highly specific identification assay and provides an example of specific test development without the prior need for DNA sequence information. The principle of the procedure holds great promise for the rapid isolation of DNA probes which, in combination with a general PCR assay, may lead to efficient typing and detection procedures for a multitude of medically important nonviral microorganisms.
The systemic immune response to Helicobacter pylori was studied in 247 infected adult patients before antimicrobial therapy and at different intervals following therapy. Endoscopy with simultaneous collection of biopsies was performed in all patients immediately before treatment, 4 to 6 weeks after the end of therapy and 6 to 12 months later. A 14C-urea breath test was performed 3 to 6 months after the end of treatment. Biopsy specimens were cultured and examined histologically using Giemsa stain. Sera were tested for Helicobacter pylori IgG antibodies with a commercial enzyme immunoassay using species-specific antigens. Overall, Helicobacter pylori was eradicated in 120 patients while the other 127 remained infected with the organism. The follow-up period ranged from 4 weeks to 33 months (mean 10.2 months). Pretreatment IgG levels did not differ significantly between the two groups of patients. Six weeks after the end of treatment a slight but definite decrease in the IgG antibody levels was seen irrespective of treatment success. In the 127 patients who remained Helicobacter pylori-positive, the level of IgG antibodies remained stable or increased with time. A continuous fall in antibody levels was observed following bacterial eradication in the other 120 patients, but the difference in antibody levels between treatment responders and nonresponders became significant only more than six months after the end of treatment (p = 0.001). Serological testing may be useful for monitoring the outcome of long-term treatment of Helicobacter pylori infection and obviate the need for endoscopy.
From 1984 to 1989, stool samples from 2811 gastroenteritis cases were examined for the presence of Campylobacter jejuni and C. Coli, Salmonella, Shigella and Yersinia species. Isolation rates were: Campylobacter jejuni and C. Coli, 5.3%, Salmonella spp., 14.8%, Shigella spp., 4.6% and Yersinia enterocolitica, 1.1%. Age group distribution analysis shows a higher Campylobacter isolation rate in children under one year of age. Seasonal distribution revealed a peak incidence in winter as in other Mediterranean countries. Predominant biotypes were C. jejuni I (51%), C. jejuni II (21.5%) and C. coli I (18.8%). Antimicrobial susceptibility testing did not reveal resistance to erythromycin. Thirty of the strains harboured plasmids with 7 different profiles.
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A collection of 44 Campylobacter isolates (37 C. jejuni and seven C. coli) from children with colitis (21 strains) or watery diarrhoea (23 strains) was analysed for toxin production, association with HeLa cells, and invasion of differentiated Caco-2 cell cultures. There was no obvious association of clinical symptoms with species, biotype or enterotoxin production. All colitis strains and most of the isolates from watery diarrhoea were cytotoxic for Chinese hamster ovary cells. Measurements of bacterial association indices with HeLa cells varied with time, and were considered to be unreliable for discriminating between isolates from the two diagnostic groups. Statistically significant differences were observed between the two groups (all colitis strains and 65% of strains from non-inflammatory diarrhoea) with respect to invasion of both HeLa and Caco-2 cell monolayers. However, among the strains from non-inflammatory diarrhoea that did invade, numbers of internalised bacteria were similar to the range observed for colitis strains. Of the colitis strains, 86% were able to transcytose through polarised Caco-2 monolayers grown on filters, compared with 48% of isolates from non-inflammatory disease. We propose the use of Caco-2 cells as a model for studying invasion of intestinal epithelia by C. jejuni and C. coli.
We evaluated a commercially available second-generation anti-H. pylori immunoglobulin G enzyme immunoassay (EIA) (Cobas Core Anti-Helicobacter pylori EIA; Roche S. A., Basel, Switzerland) for serodiagnosis of H. pylori infection. The results of the assay were assessed in relation to the results of bacterial culture, urease testing, and histological Giemsa stain of gastric biopsy specimens from 1,134 patients with a variety of symptoms relating to the upper gastrointestinal tract. H. pylori was detected in biopsy specimens from 660 (58.2%) patients: 6 had a normal mucosa, 123 had chronic gastritis only, and 531 were found to have chronic active gastritis by histology; endoscopy showed duodenal and gastric ulcers in 137 and 64 patients of the last two groups, respectively. The test was evaluated with different age and ethnic groups. The prevalence, sensitivity, specificity, and positive and negative predictive values were, respectively, (i) for Belgian patients between 18 and 40 years old, 34, 93, 95, 91, and 96%; (ii) for Belgian patients more than 40 years old, 53, 96, 91, 93, and 95%; and (iii) the Mediterranean patients more than 17 years old, 87, 94, 70, 95, and 64%. All sera showing discordant immunoassay results compared with the results of histology and culture of biopsy specimens, as well as those with borderline immunoassay results, were tested further by immunoblotting. Among the EIA results considered false negative, we demonstrated an absence of seroconversion in 14 of 19 patients tested by immunoblotting. Among the EIA results considered false positive, immunoblotting showed the presence of specific antibodies in 28 of 37 patients tested. Among the borderline results obtained in the first assay with 22 patients' sera, a second assay showed positive results in 10 patients (8 were positive by immunoblotting) and negative reactions in 10 patients (9 were negative by immunoblotting), whereas 2 remained borderline. These data indicate that sera showing borderline immunoassay results must be tested again. In conclusion, this commercially available second-generation EIA, which is easy and quick to perform, was found highly reliable for the serodiagnosis of H. pylori infection.
Commercially available complement fixation test reagents (Institute Virion Ltd., Rüschlikon, Zurich, Switzerland) available in package format were evaluated for the serodiagnosis of Helicobacter pylori infection. The assay was compared with bacterial culture and histological Giemsa stain of gastric biopsy specimens obtained from 930 patients of different ages and from different ethnic groups, with a variety of upper gastrointestinal tract symptoms. The prevalence, sensitivity, specificity, and positive and negative predictive values, respectively, were 35, 71, 90, 80, and 85% for Belgian patients aged 40 years or younger, 50, 81, 93, 92, and 83% for Belgian patients older than 40 years, and 83, 83, 79, 95, and 48% for Mediterranean patients. Using 645 serum specimens from 226 patients, we also evaluated the complement fixation test for its ability to monitor the eradication of H. pylori following antimicrobial therapy. Overall, H. pylori was eradicated from 122 patients while 104 patients remained infected with the organism. A significant decrease in antibody levels was observed 3 to 6 months after the end of therapy in the group of patients from whom H. pylori was eradicated.
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For 3 years a filtration system for the isolation of "new" campylobacter was included in the culture protocol of 15,185 stool specimens. "C upsaliensis" was isolated in 99 patients, C jejuni subsp doylei in 4, and C hyointestinalis in 2. "C upsaliensis" was the only organism isolated in 83 patients. Clinical information was available for 77 out of these 83 patients. 92% of the patients had diarrhoea; vomiting and fever were rare (14% and 7%, respectively); the onset was mostly sudden; and the symptoms usually lasted for less than a week. Gross or occult blood was present in a quarter of cases and neutrophils were detected in faecal smears in about a fifth. "C upsaliensis" may be an unrecognised and frequent cause of diarrhoea in man, and selective isolation media should be combined with non-selective isolation systems.
Companion animals ("pets") are occasionally carriers of organisms pathogenic for man. In the present, study fecal samples of clinically inapparent animals with direct contact to 204 patients, suffering from campylobacter enteritis, were investigated for C. jejuni or C. coli (CJC). CJC positive animals were seen in the environment of only five patients (= 2.4%). By comparison of biotypes and serotypes of thermostable and thermolabile antigens from human and animal isolates no clear epidemiological relationship could be deduced. Using chromosomal DNA of the strains, genetic identity of the isolates was studied for HaeIII-restriction fragment length polymorphism (RFLP), applying a biotinylated commercial CJ probe. The probe was found to be specific for most CJ strains and revealed a pattern of one to four bands. In contrast to biotyping no identity of patient strains and animal isolates was seen in three cases; one case with different biotypes had identical RFLP patterns; one patient CJ strain did not show any pattern with the CJ probe. Serotypes were identical for a larger number of animal strains but differed in HaeIII RFLP and vice versa. Comparing the results from the different technological approaches it seems impossible to give a clear statement on the epidemiology of campylobacter infections or carrier state by biotyping alone. It is concluded that DNA RFLP patterns are a useful additional tool, but for epidemiological analysis a set of different methods should be used.
During a 3-year period, "Campylobacter upsaliensis" was isolated from 99 patients. Phenotypic characterization and numerical analysis of protein electrophoregrams showed evidence that "C. upsaliensis" is a distinct Campylobacter species with unique characteristics. The MBCs of 13 antibiotics were determined. In general, these organisms were highly susceptible to drugs that were present in the selective isolation media, making none of the available selective media suitable for the isolation of "C. upsaliensis." Ten strains were found to be resistant to erythromycin (MBCs, greater than or equal to 12.50 mg/liter). Plasmid DNA was detectable in 89 of the 99 strains; 16 plasmid profiles could be identified. Plasmid pattern 16, containing four plasmids of 52, 32, 5.5, and 2.6 megadaltons, represented 60.7% of the plasmid-containing strains. None of the "C. upsaliensis" strains could be agglutinated with antisera against heat-labile antigens from C. jejuni, C. coli, or C. laridis. "C. upsaliensis" was found to be susceptible to serum killing and was readily phagocytized by human polymorphonuclear cells.
In a randomised, double-blind study the efficacy and toxicity of oral fluconazole 50 mg daily and ketoconazole 200 mg daily were compared for the treatment of oropharyngeal candidiasis in patients with acquired immunodeficiency syndrome (AIDS) and AIDS-related complex (ARC). 20 episodes (18 patients) were treated with fluconazole and 20 episodes (19 patients) with ketoconazole. Pretreatment clinical features and laboratory test results were similar in both groups. 17 episodes (85%) in the fluconazole group and 16 (80%) in the ketoconazole group could be evaluated. There was clinical cure at the end of therapy in all fluconazole-treated and 12 of 16 (75%) ketoconazole-treated episodes. Cultures were negative at the end of therapy in 87% of the fluconazole group and 69% of the ketoconazole group. 1 patients stopped taking fluconazole because of severe nausea. 1 of 18 fluconazole-treated and 4 of 19 ketoconazole-treated patients had transient rises in alanine or aspartate aminotransferase. Fluconazole seemed more effective than ketoconazole in the treatment of oral thrush among AIDS and ARC patients.