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Volume 50,
Issue 12,
2001
Volume 50, Issue 12, 2001
- Editorial
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- Review Article
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Detection of Helicobacter pylori in faeces by culture, PCR and enzyme immunoassay
More LessVarious techniques such as culture, PCR and enzyme immunoassay have been used to detect Helicobacter pylori infection in human faecal specimens. Attempts to culture H. pylori have had limited success as the bacterium exists predominantly in a non-culturable (coccoid) form in the faeces. Several PCR protocols, differing from each other in the choice of genomic targets and primers, have been used to detect H. pylori infection. Substances in faeces that inhibit PCR have been removed by various pre-PCR steps such as filtration through a polypropylene membrane, biochemical separation by column chromatography and isolation of H. pylori with immunomagnetic beads, the former two techniques yielding results with a high degree of sensitivity and specificity. An enzyme immunoassay based on the detection of H. pylori antigen in faeces has become a convenient tool for the pre-treatment diagnosis of the infection. The stool antigen assay is convenient, especially for children, as it involves neither surgery nor the discomfort associated with the urea breath test. However, its applicability in monitoring eradication therapy has been controversial, as the assay can detect dead or partially degraded bacteria long after actual eradication, thus giving false positive results.
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- Diagnostic Microbiology
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Comparison of PCR, capture ELISA and immunoblotting for detection of Toxoplasma gondii in infected mice
More LessPCR was compared with capture ELISA and immunoblotting for the detection of Toxoplasma gondii in sera of acutely infected mice. One hundred animals were inoculated intraperitoneally with 5000 trophozoites of RH strain and five of them were killed every 3 h from 3 h to 21 h post infection (p.i.), and every day from day 1 to day 7 p.i.. No assay detected the parasite from 3 h p.i. to 15 h p.i. PCR was the most sensitive assay and detected the T. gondii from 18 h p.i., whereas the other assays detected it only from day 1.
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- Epidemiology And Bacterial Typing
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Characterisation and differentiation of lactobacilli by lectin typing
More LessLactobacillus isolates from healthy Estonian and Swedish children were characterised by a lectin typing technique; 56 isolates from six species (L. acidophilus, L. paracasei, L. plantarum, L. fermentum, L. brevis and L. buchneri) were tested. The typing system was based on an agglutination assay with a panel of six commercially available lectins, which were chosen on the basis of their carbohydrate specificities. The isolates were also subjected to proteolytic degradation before lectin typing to decrease auto-agglutination of whole cells in the assay. The 56 isolates were divided into 15 different lectin types by their lectin agglutination patterns. Proteolytic treatment reduced auto-agglutination for the majority of species, apart from L. acidophilus, which remained predominantly auto-agglutinating (eight of nine strains). The system produced stable and reproducible results under standardised culture conditions. Lactobacilli are important bacteria for use as probiotics and this system may supplement current molecular typing techniques and may help in identification of strains that could be useful in this role.
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Comparison of SmaI-defined genotypes of Campylobacter jejuni examined by KpnI: a population-based study
More LessPulsed-field gel electrophoresis (PFGE) was used to analyse 147 isolates collected in two regions of Québec province (Estrie and Montréal) between March 1998 and Feb. 1999, to determine the utility of molecular strain typing for a population-based collection of Campylobacter jejuni and to compare directly the discriminatory power of SmaI and KpnI restriction digests. With a combination of epidemiological criteria including space and time plus molecular strain typing, 49% of isolates from Estrie and 39% of isolates from Montréal were identified as belonging to a putative cluster. For 41% of the cases, sources were either missing or explicitly unknown; the remaining sources were subject to recall bias. Thus, the evaluation of sporadic cases of campylobacter enteritis by descriptive clinical investigation alone is neither sensitive nor reliable for identifying sources of infection. In the PFGE analysis, KpnI digests provided appreciably greater discriminatory power than SmaI digests. When combining the PFGE analyses with basic epidemiological criteria, 30% of the putative SmaI clusters were inconsistent with the epidemiological criteria compared with 17% of the KpnI clusters. Among the 98 isolates assigned to clusters by SmaI, only 65% gave concordant results with KpnI. In contrast, among the 81 isolates assigned to clusters by KpnI, 92% gave concordant results with SmaI. Finally, clusters that were epidemiologically related to ingestion of raw milk and specific water sources correlated better with the typing results based on KpnI than SmaI. Thus, KpnI is the enzyme of choice for molecular epidemiology studies of C. jejuni. The combination of continuous epidemiological surveillance and molecular strain typing may be useful for identifying new sources and mechanisms of transmission for community-acquired C. jejuni infection and ultimately for developing new approaches to prevention.
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PCR ribotyping of clinically important Clostridium difficile strains from Hungary
More LessIsolates of Clostridium difficile from different hospital wards at the University Hospital of Szeged in Hungary were typed by PCR amplification of rRNA intergenic spacer regions (PCR ribotyping). A total of 15 different ribotypes was detected among the 65 isolates tested. The predominant type, PCR ribotype 087, accounted for 39% of all isolates, in contrast with an international typing study where ribotype 001 was the most common. Two non-toxigenic C. difficile strains were found to exhibit the same pattern, which was distinct from those of all the ribotypes described previously, suggesting that this is a new type.
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Prevalence of cytolethal distending toxin (cdt) genes and CDT production in Campylobacter spp. isolated from Danish broilers
More LessThe pathogenesis of campylobacter infection in man is largely unknown, although cytolethal distending toxin (CDT) has been incriminated as a virulence factor. However, little is known about the cdt genes in Campylobacter spp. isolated from broiler chickens. A total of 350 cloacal swabs was collected and tested by conventional culture and PCR. Of the 114 Campylobacter isolates obtained, 101 (88.6%) were identified as C. jejuni and 13 (11.4%) as C. coli by conventional methods. cdt genes were detected by PCR in all the isolates except one C. jejuni isolate. Cytotoxic effects were produced in a Vero cell line, by 100 of the C. jejuni isolates. In contrast, 10 C. coli isolates produced much lower levels of toxin and 3 produced no detectable toxin. These results confirm the common occurrence of campylobacter infection in chickens and indicate that cdt genes are commonly present in both C. jejuni and C. coli isolates from broilers, but that there are distinct differences in CDT production in these two closely related species.
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Seasonal variations in nasopharyngeal carriage of respiratory pathogens in healthy Italian children attending day-care centres or schools
The aim of this study was to investigate seasonal variations in the prevalence of the nasopharyngeal carriage of respiratory pathogens and identify factors affecting colonisation patterns in healthy children. The nasopharyngeal carriage of Streptococcus pneumoniae, Haemophilus influenzae and Moraxella catarrhalis during two seasons (autumn and spring) was evaluated in 1580 healthy children aged 1–7 years by means of a cohort study conducted in day-care centres and schools in eight Italian cities. A questionnaire was used to obtain the epidemiological data. In all, 309 children (19.5%) carried one or more respiratory pathogens in the autumn, and 375 children (23.7%) in the spring. This variation was due to H. influenzae alone or in combination (autumn: S. pneumoniae 60, 3.8%; H. influenzae 206, 13.0%, M. catarrhalis 71, 4.5%; spring: S. pneumoniae 75, 4.7%; H. influenzae 288, 18.2%, M. catarrhalis 82, 5.2%). Colonisation with two or more pathogens increased from 9.1% in the spring to 17.3% in the autumn. Seasonal variations occur in the prevalence of the nasopharyngeal carriage of respiratory pathogens in healthy children attending day-care centres or schools in Italy. However, although statistically significant, the difference was slight and had limited clinical relevance. Therefore, seasonal influence on the nasopharyngeal carriage of respiratory pathogens in healthy children was negligible.
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- Bacterial Pathogenicity
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Interaction of Yersinia enterocolitica and Y. pseudotuberculosis with platelets
More LessYersinia enterocolitica is a bacterium capable of growth at 4°C in donated blood and has been responsible for many deaths following transfusion. Interaction of Y. enterocolitica with blood cells is of interest in understanding the mechanisms of survival and growth in blood. The closely related organism Y. pseudotuberculosis is known to invade platelets and cause platelet aggregation by a mechanism that involves expression of the chromosomal inv gene. Yersinia isolates were made to express green fluorescent protein (GFP) and their interaction with platelets was studied by flow cytometry. Y. enterocolitica did not cause platelet aggregation or activation, not even when grown at 22°C to maximise inv expression. Attachment of Y. enterocolitica O:9 to platelets occurred with virulence plasmid-bearing (pYV+) strains grown at 37°C but not with pYV− strains nor with strains grown at 22°C. Y. pseudotuberculosis containing inv did cause platelet activation and aggregation when grown at 22°C, as has been shown before, but also showed enhanced attachment to platelets when grown at 37°C. Electron microscopy studies confirmed that inv-expressing Y. pseudotuberculosis invaded platelets but Y. enterocolitica attached only to the outer surface of platelets. Interaction of Y. enterocolitica O:9 with platelets provided a modest protection against bacterial killing by human serum. Interaction of Y. enterocolitica O:9 with platelets does not lead to platelet invasion or activation, and is mediated through plasmid-coded factors, not inv.
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Characterisation of p-nitrophenylglycerol-resistant Proteus mirabilis super-swarming mutants
More Lessp-Nitrophenylglycerol (PNPG) inhibits the co-ordinately regulated activities of swarming behaviour and virulence factor expression in Proteus mirabilis. The inhibitory action of PNPG was investigated by the isolation of Tn5 insertion mutants that could swarm, albeit with much reduced ability, in the presence of PNPG. The mutants exhibited a super-swarming phenotype in the absence of PNPG; i.e., they migrated further in a given time than did the wild-type cells. Cloning and sequence analysis of the mutants indicated that Tn5 was inserted into the rsbA gene, which may encode a membrane sensor histidine kinase of the bacterial two-component signalling system. In the absence of PNPG, the mutants exhibited several swarming-related phenotypes that were different from those of the wild type; they initiated swarming earlier and had a less conspicuous consolidation phase, they differentiated earlier and maintained a differentiated state for longer, they started to express virulence factors earlier and maintained high expression levels of these factors for longer, and they had higher cell invasion ability than the wild type. These mutant phenotypes could be complemented by a plasmid-borne copy of rsbA. Together, these data suggest that RsbA may act as a repressor of swarming and virulence factor expression. In the presence of PNPG, these rsbA-mutated mutants could still swarm, differentiate and express virulence factors, whereas the wild type could not, suggesting that PNPG may target RsbA or RsbA-regulated pathways to exert its inhibitory effect. Together, these data reveal a novel mechanism through which bacteria may negatively regulate swarming differentiation and virulence factor expression and identify a potential target of PNPG action.
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Survival and distribution of cell-free SEF 21 of Salmonella enterica serovar Enteritidis in the stomach and various compartments of the rat gastrointestinal tract in vivo
Rats were dosed for 6 days with purified SEF 21 fimbriae of Salmonella enterica serovar Enteritidis 10360. The levels of fimbriae in gut contents associated with tissues and in the faeces were quantified by direct non-competitive ELISA. SEF 21 was distributed throughout the gut. The majority was found in the large intestine where it was primarily in the luminal contents. In contrast, a high proportion of SEF 21 detected in the ileum, the main site of salmonella colonisation and invasion, was tissue-bound. Thus, purified SEF 21 survived intestinal passage and associated with the stomach and gastrointestinal tract in a pattern similar to that found with whole Salmonella cells.
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A Portuguese isolate of Borrelia lusitaniae induces disease in C3H/HeN mice
A low-passage, Portuguese isolate of Borrelia lusitaniae, strain PotiB2, was inoculated into C3H/HeN mice and disease was monitored by histopathology at 8 weeks after spirochaete challenge. Ear, heart, bladder, femoro-tibial joint, brain and spinal cord were examined. B. lusitaniae strain PotiB2 (6 of 10 mice) and B. burgdorferi sensu stricto strain N40 (9 of 10 mice) induced similar lesions in the bladder of infected mice characterised as a multifocal, lymphoid, interstitial cystitis. Moreover, both B. lusitaniae PotiB2 and B. burdorferi N40 induced lesions in the heart of infected mice. The lesions induced by B. lusitaniae PotiB2 (2 of 10 mice) were characterised as a severe, necrotising endarteritis of the aorta, with a minimal, mixed inflammatory infiltrate (neutrophils, macrophages and lymphoid cells) extending into the adjacent myocardium. In contrast, B. burgdorferi N40 induced a periarteritis of the pulmonary artery (7 of 10 mice), with no involvement of the endothelium and more extensive inflammation and subsequent necrosis of the adjacent myocardium. This infiltrate was composed entirely of mononuclear cells, predominantly mature lymphocytes and plasma cells. No lesions were noted in the joints or central nervous system with inoculation of strains N40 or PotiB2, and co-inoculation of either strain with Ixodes ricinus salivary gland lysate did not affect the resulting pathology. Serology, examined 8 weeks after inoculation, indicated a different reactivity in mice infected with B. lusitaniae PotiB2 compared with B. burgdorferi N40. Immunoblot analysis demonstrated that mice with lesions resulting from infection with B. lusitaniae PotiB2 reacted only to the flagellin protein (41 kDa) or to flagellin and OspC, whereas mice infected with B. burgdorferi N40 reacted with multiple high and low mol. wt proteins, including flagellin, p93, p39, OspA, OspB and OspC. These results indicate that B. lusitaniae PotiB2 induced pathology similar to B. burgdorferi N40 when inoculated into susceptible mice. Moreover, these results establish the first animal model of disease with B. lusitaniae. This mouse model can be used to characterise the immunopathogenesis of B. lusitaniae infection and to delineate the proteins responsible for disease induction in susceptible mice.
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Isolation and characterisation of a novel spirochaete from severe virulent ovine foot rot
More LessA novel spirochaete was isolated from a case of severe virulent ovine foot rot (SVOFR) by immunomagnetic separation with beads coated with polyclonal anti-treponemal antisera and prolonged anaerobic broth culture. The as yet unnamed treponeme differs considerably from the only other spirochaete isolated from ovine foot rot as regards morphology, enzymic profile and 16S rDNA sequence. On the basis of 16S rDNA, it was most closely related to another unnamed spirochaete isolated from cases of bovine digital dermatitis in the USA, raising the possibility of cross-species transmission. Further information is required to establish this novel ovine spirochaete as the cause of SVOFR.
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- Mycology
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Comparative performance of Fungichrom I, Candifast and API 20C Aux systems in the identification of clinically significant yeasts
More LessTo compare the performance of current chromogenic yeast identification methods, three commercial systems (API 20C Aux, Fungichrom I and Candifast) were evaluated in parallel, along with conventional tests to identify yeasts commonly isolated in this clinical microbiology laboratory. In all, 116 clinical isolates, (68 Candida albicans, 12 C. parapsilosis, 12 C. glabrata and 24 other yeasts) were tested. Germ-tube production, microscopical morphology and other conventional methods were used as standards to definitively identify yeast isolates. The percentage of isolates identified correctly varied between 82.7% and 95.6%. Overall, the performance obtained with Fungichrom I was highest with 95.6% identification (111 of 116 isolates). The performance of API 20C Aux was higher with 87% (101 of 116 isolates) than that of Candifast with 82.7% (96 of 116). The Fungichrom I method was found to be rapid, as 90% of strains were identified after incubation for 24 h at 30°C. Both of the chromogenic yeast identification systems provided a simple, accurate alternative to API 20C Aux and conventional assimilation methods for the rapid identification of most commonly encountered isolates of Candida spp. Fungichrom seemed to be the most appropriate system for use in a clinical microbiology laboratory, due to its good performance with regard to sensitivity, ease of use and reading, rapidity and the cost per test.
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Volumes and issues
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Volume 72 (2022 - 2023)
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Volume 71 (2022)
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Volume 70 (2021)
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Volume 69 (2020)
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Volume 68 (2019)
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