Molecular epidemiology of human Campylobacter jejuni shows association between seasonal and international patterns of disease.

We sought to explain seasonality and other aspects of Campylobacter jejuni epidemiology by integrating population genetic and epidemiological analysis in a large 3-year longitudinal, two-centre, population-based study. Epidemiological information was collected for 1505 isolates, which were multilocus sequence-typed. Analyses compared pathogen population structure between areas, over time, and between clinical presentations. Pooled analysis was performed with published international datasets. Subtype association with virulence was not observed. UK sites had nearly identical C. jejuni populations. A clade formed by ST45 and ST283 clonal complexes showed a summer peak. This clade was common in a Finnish dataset but not in New Zealand and Australian collections, countries with less marked seasonality. The UK, New Zealand and Australian collections were otherwise similar. These findings map to known in-vitro differences of this clade. This identifies a target for studies to elucidate the drivers of the summer peak in human C. jejuni infection.

Serology and urea breath test in the diagnosis of H. pylori infection.

Helicobacter pylori is a chronic infection that has the potential for causing and initiating serious gastric disease. Specific treatment can be successful in eradication of the infection but is currently complex which hampers essential patient compliance. Therefore, the accurate detection of H. pylori and, importantly, the post-treatment check for cure is vital in the effective management of this infection. This is especially true in cases of asymptomatic individuals. Serology is now a simple ELISA with a high degree of accuracy and has been shown to be useful as a screening tool prior to endoscopy in selected cases. The urea breath test, either using C13 or C14, is a sensitive test easily applied and is the test of choice for post-treatment check for cure. It is also the gold standard for the validation of serology in different populations.

Serology and Urea Breath Test in the Diagnosis of H. pylori Infection.

The selectivity of Helicobacter pylori for the antral mucosa of the stomach and gastric metaplasia in the duodenum combined with the inaccessibility of those sites has hindered the investigation of this infection. Consequently, the study of the epidemiology and treatment of H. pylori has relied heavily on indirect methods of detecting infection. Therefore, the accuracy of findings from such studies, particularly those with small populations, was highly dependent on the sensitivity and specificity of the detection systems employed. The calculation of sensitivity, specificity, and positive and negative values of a test requires a "gold standard.". The choice of gold standard, against which to validate the test, is important as it will have its own margins of error. Ideally, the gold standard and its margins of error should be identified when quoting sensitivity and specificity rates in validation of any new diagnostic test. For example, if a test has a sensitivity of 95% against a gold standard that has a true sensitivity of 85%, then the true sensitivity of the first test for its target would be 0.95 × 0.85 = 0.8, i.e., 80 and not 95%. In the case of H. pylori, the most commonly used gold standard in early work on indirect diagnostic methods was culture and/or histology. These two methods possibly have relatively the lowest sensitivity of any test for the presence of H. pylori. This is owing to the potential sampling error in removing biopsy material from an uninfected site. This, combined with variable culture efficiency and identification in histological sections, will lead to false negatives (1). This would be a particular problem in patients with any changes to the gastric mucosal epithelium from typical antral epithelium, such as intestinal metaplasia, because the changes would remove the niche availability to H. pylori and reduce the infective load.The problems can be minimized, for example, by staining histological sections with the modified Giemsa stain, making the recognition of H. pylori easier, even when the organism is present in small numbers. The combination of culture and histology is preferable to using either of the two independently, and certainly preferable to using endoscopic appearance alone, to estimate the sensitivity and specificity of a test. Many workers only count a case as H. pylori positive when two out of three indirect tests are positive.

Estimation of prevalence of Helicobacter pylori infection in an asymptomatic elderly population comparing [14C] urea breath test and serology.

A non-invasive serological assay devised in this laboratory had a sensitivity and specificity of 100% as determined by culture and confirmed by histology in a group of 47 patients who had undergone endoscopy. The correlation between serology and the non-invasive [14C] breath test was very good. Only one of 24 culture positive patients was, while all 23 culture negative patients were, breath test negative. In a group of 46 healthy elderly persons, however, significant anomalies between serology and breath test were observed. Only 83% of the breath test negative persons were seronegative, while only 68% of the breath test positive persons were seropositive. These results can be explained in terms of age related atrophic gastritis and immune incompetence, causing reduced colonisation and decreased antibody production, respectively. These investigations suggest that non-invasive tests for H pylori infection may not be reliable in the elderly.

The demonstration of nickel in the urease of Helicobacter pylori by atomic absorption spectroscopy.

Fast protein liquid chromatography and SDS-PAGE have been used to isolate and purify Helicobacter pylori urease. A nickel component of the urease was detected in the purified proteins by atomic absorption spectroscopy. The nickel was present only in the 61 kDa polypeptide and in the ratio of between five and six atoms to one molecule of urease, suggesting a hexameric structure. These results are discussed in relation to other bacterial ureases and urease activity at low pH.

Antigenicity of fractions of Helicobacter pylori prepared by fast protein liquid chromatography and urease captured by monoclonal antibodies.

The antigenicity of Helicobacter pylori protein fractions separated by fast protein liquid chromatography size exclusion was investigated by EIA with sera from patients of well defined Helicobacter pylori status. The antigenic material of Helicobacter pylori was confined to fractions 8 and 14 to 21. Urease containing fractions (14/15) and flagella containing fractions (17/18) were identified. Fraction 8 non-specifically bound human immunoglobulin as demonstrated by the binding of Helicobacter pylori negative sera. The remaining fractions 14 to 21 when used individually as EIA antigens were 91-100% specific, however fractions 16 to 19 showed a reduced sensitivity (78%) compared with the acid extract (95%). The urease fractions were 91% sensitive. Purified urease antigen captured by antiurease monoclonal antibodies was 83% sensitive and 93.3% specific.

Investigation of the structure and localization of the urease of Helicobacter pylori using monoclonal antibodies.

The urease of Helicobacter pylori (formerly Campylobacter pylori) has been partly purified by fast protein liquid chromatography. This material contained 10 nm doughnut-like structures when examined by electron microscopy and comprised three major polypeptides (61 kDa, 56 kDa and 28 kDa). Only two of these polypeptides (61 kDa and 28 kDa) were observed in urease-containing material isolated by preparative non-denatured PAGE. Monoclonal antibodies (mAbs) were produced which were directed against two of these polypeptides (56 kDa and 28 kDa). Only mAbs directed against the 28 kDa polypeptide inhibited or captured urease activity. These results suggest that the 56 kDa polypeptide is not essential for enzyme activity. Anti-urease mAbs were used in an indirect immunogold technique to localize the enzyme at the ultrastructural level. In both prefixed bacteria and ultrathin cryosectioned bacteria the enzyme was located on the cell surface and in material apparently shed from that surface.

Antigenic conservation of the ureases of spiral- and helical-shaped bacteria colonising the stomachs of man and animals.

A monoclonal antibody, CP11, has been produced which is directed against the ureas of Campylobacter pylori. This antibody has been used to look for antigenic cross-reactivity, in other ureolytic and non-ureolytic campylobacters, by immunohistological techniques. It has also been used to investigate the helical-shaped organisms found in the stomach of the human, monkey and cat (CS1) and the ileum of the rat (ST1). Interestingly the antibody cross-reacted with the gastric helical organisms from the human, monkey and cat but not with the rat helical organism. No cross-reactivity was observed with C. mustelae or the other ureolytic campylobacters, C. nitrofigilis and the urease positive thermophilic campylobacters. These results are discussed in relation to the phylotaxonomy of these organisms.