Association of putative pathogenicity genes with adherence characteristics and fimbrial genotypes in typical enteroaggregative Escherichia coli from patients with and without diarrhoea in the United Kingdom.

The aim of this study was to compare genotypic characteristics seen in typical EAggEC isolated during a study of intestinal infectious disease from cases and controls, and to identify which genes, or combinations of genes, were most associated with diarrhoeal disease. We also investigated the association of genotype with certain characteristics, such as presence of fimbrial genes and adherence to Hep-2 cells. The aafC gene, encoding the usher for AAFII, was the only gene significantly associated with patients with diarrhoea (P < 0.005), and the aggC gene, which encodes the usher for AAFI, was the only gene significantly associated with the healthy control group (P < 0.002). Putative virulence genes significantly associated with aggregative adherence included aafC, aggR, pet, pic and astA. The shf, pet and astA genes were all more likely to be associated with type II fimbriae than with type I. We conclude that in addition to presence and absence of certain genes, studies of EAggEC pathogenicity should investigate the combinations and associations of putative virulence factors.

Serotypes, intimin subtypes, and antimicrobial resistance patterns of atypical enteropathogenic Escherichia coli isolated in England from 1993 to 1996.

The aim of this study was to characterise the atypical enteropathogenic Escherichia coli (EPEC) strains isolated during a study of intestinal infectious disease in the UK by serotyping, intimin subtyping, and antimicrobial resistance typing. Serotypes, intimin subtypes, and resistance patterns of strains from cases were then compared with those from the control group. A wide range of serotypes, intimin subtypes, and antimicrobial resistance patterns was identified in isolates from both cases and controls, with O70:H11 and O111:H- being the most frequently detected serotypes. The most common intimin types were gamma and gamma(2). Thirty-six percent of the EPEC isolates were resistant to at least one antimicrobial agent. No significant differences in the characteristics of EPEC strains isolated from patients with symptoms of gastrointestinal disease versus those isolated from healthy controls were detected, although strains harbouring the beta-intimin subtype were more commonly isolated from children under 5 years of age (p=0.002). The compilation of data on atypical EPEC strains presented here indicates the need for further study of their virulence and epidemiology in order to assess their significance as human pathogens.

Detection of Clostridium difficile cytotoxin and Clostridium perfringens enterotoxin in cases of diarrhoea in the community.

Faecal specimens from 843 cases of diarrhoea in the community were tested for the presence of Clostridium difficile cytotoxin and Clostridium perfringens enterotoxin. C. difficile cytotoxin was detected in faecal specimens from 0.6 % of cases aged at least 2 years by using a Vero cell assay. Factors associated with detection of C. difficile cytotoxin were antibiotic therapy, age over 60 years and living in a home with other elderly people. Three methods were used for the detection of C. perfringens enterotoxin: a Vero cell assay, a commercial (TechLab) enzyme immunoassay (EIA) and an in-house EIA. The lower level of detection of pure C. perfringens enterotoxin in buffer was 0.01 micro g ml(-1) by the TechLab EIA and 1.0 micro g ml(-1) by the Vero cell assay. C. perfringens enterotoxin was detected by using the TechLab EIA in faecal specimens from 2.5 % of cases. This commercial EIA was less sensitive than the in-house EIA, detecting only 31 % of positive cases, but was specific and could be used for outbreak investigation by routine diagnostic laboratories. Age over 60 years was a factor associated with C. perfringens enterotoxin detection; this age group may be targeted for testing.

Point source outbreaks of Campylobacter jejuni infection--are they more common than we think and what might cause them?

Despite being the commonest bacterial cause of infectious intestinal disease (IID) in England and Wales, outbreaks of campylobacter infection are rarely reported. However, data from the Campylobacter Sentinel Surveillance Scheme suggested that outbreaks might be more common than was previously suspected, since a high proportion of cases reported other illness in the home or in the community at the same time as their illness. To identify factors that might lead to these apparent outbreaks, the exposures of cases of Campylobacter jejuni infection reporting other illness, either in the home or the community, were compared with those for cases not reporting other illness using case-case methodology. Illness in the home was associated with consuming organic meats in the winter, having contact with a pet suffering from diarrhoea or visiting a farm in the 2 weeks before the onset of symptoms. Illness in the community was associated with the consumption of foods in restaurants or drinking unpasteurized milk. Prevention of campylobacter infection requires that better methods of outbreak detection and investigation are developed, which in turn should lead to a better understanding of risk factors.

The infectious intestinal disease study of England: a prospective evaluation of symptoms and health care use after an acute episode.

The sequelae of Infectious Intestinal Disease (IID) in a population-based sample of cases and matched controls were investigated for a period of 3 months following the initial infection. Incident cases of IID presenting to GPs or occurring in the community and controls were studied at 3 weeks and over a 3-month follow-up period. Cases were six times more likely than controls to have gastrointestinal symptoms, particularly diarrhoea, at 3 weeks. Ten per cent of cases consulted their GP in the 3 months after episode and 2.3% were referred to hospital. GP presentation rates were twice as high in cases. Gastrointestinal symptoms persist after IID, leading to an increased likelihood of GP consultation and hospital referral. Diagnosis of irritable bowel syndrome may be more likely following IID. The burden of IID is likely to be considerable given its high incidence and the frequency of such sequelae.

Vero cytotoxin-producing Escherichia coli in a study of infectious intestinal disease in England.

An investigation of infectious intestinal disease in England included examination of feces for Vero cytotoxin-producing Escherichia coli (VTEC). Using DNA probe hybridization 27 VTEC strains were identified, 12 were from cases, and of these three belonged to serogroup O157. The remaining 15 strains were isolated from controls. The strains were confirmed biochemically as E. coli, they were serotyped and characterized according to their toxin production, the presence of sequences encoding intimin (eae) and enterohemolysin was determined and resistance to antimicrobial agents was determined. Six of the nine cases with non-O157 VTEC were less than 16 years old, only two of the 15 controls were under 16. Infection with more than one micro-organism was also considered.

The study of infectious intestinal disease in England: risk factors for cases of infectious intestinal disease with Campylobacter jejuni infection.

This is a case-control study aimed at identifying risk factors for intestinal infection with Campylobacter jejuni. Cases were defined as subjects with diarrhoea occurring in community cohorts or presenting to General Practitioners (GPs) with Campylobacter jejuni in stools. Controls were selected from GP lists or cohorts, matched by age, sex, and GP practice. Travel abroad and consumption of chicken in a restaurant were statistically significantly associated with being a case. There was no statistically significant risk associated with consumption of chicken other than in restaurants nor with reported domestic kitchen hygiene practices. Consumption of some foods was associated with a lower risk of being a case. Most cases remained unexplained. We suggest that infection with low numbers of micro-organisms, and individual susceptibility may play a greater role in the causation of campylobacter infection than previously thought. It is possible that in mild, sporadic cases infection may result from cross contamination from kitchen hygiene practices usually regarded as acceptable. Chicken may be a less important vehicle of infection for sporadic cases than for outbreaks, although its role as a source of infection in both settings requires further clarification in particular in relation to the effect of domestic hygiene practices. The potential effect of diet in reducing the risk of campylobacteriosis requires exploration.

A study of infectious intestinal disease in England: risk factors associated with group A rotavirus in children.

OBJECTIVE: To identify risk factors for infectious intestinal disease (IID) due to rotavirus group A in children aged under 16 years. METHODS: Case-control study of cases of IID with rotavirus infection presenting to general practitioners (GPs) or occurring in community cohorts, and matched controls. RESULTS: There were 139 matched pairs. In children under 16 years the following risk factors were significantly associated with rotavirus IID: living in rented council housing (adjusted OR = 3.78, P = 0.022), accommodation with more than five rooms (OR = 0.72, P = 0.002), contact with someone ill with IID (OR = 3.45, P < 0.001). Some foods were associated with decreased risk. In infants, bottle feeding with or without breast feeding was associated with increased risk (OR = 9.06, P < 0.05). CONCLUSIONS: Contact with persons with IID, living in rented council housing and accommodation with fewer rooms, were significant risk factors for sporadic rotavirus IID in children whereas breast feeding is protective in infants.

Effect of clarithromycin and omeprazole therapy on the diversity and stability of genotypes of Helicobacter pylori from duodenal ulcer patients.

The genotypes of multiple isolates of Helicobacter pylori from 17 duodenal ulcer patients in the United Kingdom were compared to determine reasons for treatment failure. Isolates were from antrum and corpus biopsies taken before and after dual therapy with clarithromycin and omeprazole. All isolates were tested for antibiotic resistance and characterised by a novel scheme combining polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) analysis of the ureA + ureB and 23S rRNA genes, vacA signal and midregion genotypes, and PCR detection of cagA. Combined genotypes of paired pre- and post-treatment isolates from 8 patients showed an infection with a single strain of H. pylori that had acquired resistance to clarithromycin. In 4 other patients, acquisition of clarithromycin resistance was associated with the presence of different strain types of H. pylori. The remaining 5 patients had clarithromycin-sensitive isolates. Overall, H. pylori from different patients had diverse genotypes, yet most (70%) were colonized by the same predominant and stable strain in both the antrum and corpus. There was no link between the emergence of in vitro clarithromycin resistance and a particular strain genotype for these UK isolates. It was concluded that colonization with a clarithromycin-resistant H. pylori was due to selection of a resistant strain or clonal variant within the infecting population. Present genomic markers had low predictive value for emergence of resistance.

A study of infectious intestinal disease in England: microbiological findings in cases and controls.

A study was undertaken to identify the microorganisms and toxins in stool specimens associated with infectious intestinal disease (IID) among cases in the community and presenting to general practitioners (GPs) and in asymptomatic controls. Population based cohorts were recruited from practice lists in 70 practices and followed for 26 weeks (cohort component). Seven hundred and sixty-one cases of IID identified from the cohorts, 2893 cases who presented to GPs in 34 of the practices (GP component), and age/sex matched control subjects (555 and 2264, respectively) submitted stool specimens by post for comprehensive microbiological examination. Campylobacter spp (12.2% of stools tested), rotavirus group A (7.7%), and small round structured virus (SRSV) (6.5%) were the organisms most commonly detected in the GP component. SRSV was identified in 7.0% of cases in the community cohort. No target microorganisms or toxins were identified in 45.1% and 63.1% of cases in the two components. Aeromonas spp, Yersinia spp, and some enterovirulent groups of Escherichia coli were detected as frequently in controls as in cases. The higher frequency of detection of campylobacter, salmonella, and rotavirus among cases who presented to GPs than among those in the community suggests that those pathogens cause more severe illness. No enteropathogens were detected from a large proportion of cases although comprehensive standard methods were used to seek them.

A study of infectious intestinal disease in England: plan and methods of data collection.

The Committee on the Microbiological Safety of Food, set up in 1989 by the Department of Health in response to national epidemics of foodborne infection, considered the available evidence and commissioned a study of infectious intestinal disease (IID) in England. Seventy practices (with 489,500) patients overall) recruited from the Medical Research Council's General Practice Research Framework between August 1993 and January 1995 collected data for one year. The practice populations were representative of practices in England by area and urban/rural location, but with fewer small and affluent practices. There were five main components. i) A population cohort of 9776 (40% of those eligible) were enrolled to estimate the incidence and aetiology of IID in the community, and a large proportion were followed up. A median of 10% of patients on practice age-sex registers had moved away or died. ii) A nested case control component based on cases ascertained in the cohort was used to identify risk factors for IID in the community. iii) In a case control component used to identify risk factors and to estimate the incidence and aetiology of IID presenting in 34 general practices 70% of the 4026 cases returned risk factor questionnaires, 75% submitted stools, and matched controls were found for 75% of cases. iv) An enumeration component was used to estimate the incidence of IID presenting to general practitioners (GPs) in 36 practices and the proportion of specimens sent routinely for microbiological examination. v) In a socioeconomic costs component used to estimate the burden of illness of IID in the community and presenting to GPs 63% of those who returned a risk factor questionnaire also returned a socioeconomic questionnaire and were representative by age, sex, and social class. Despite variable enrolment and compliance the study sample had sufficient power for the multivariable analysis. The characteristics associated with low enrollment and compliance must be considered in the interpretation of the main study results.

Study of infectious intestinal disease in England: rates in the community, presenting to general practice, and reported to national surveillance. The Infectious Intestinal Disease Study Executive.

OBJECTIVE: To establish the incidence and aetiology of infectious intestinal disease in the community and presenting to general practitioners. Comparison with incidence and aetiology of cases reaching national laboratory based surveillance. DESIGN: Population based community cohort incidence study, general practice based incidence studies, and case linkage to national laboratory surveillance. SETTING: 70 general practices throughout England. PARTICIPANTS: 459 975 patients served by the practices. Community surveillance of 9776 randomly selected patients. MAIN OUTCOME MEASURES: Incidence of infectious intestinal disease in community and reported to general practice. RESULTS: 781 cases were identified in the community cohort, giving an incidence of 19.4/100 person years (95% confidence interval 18.1 to 20.8). 8770 cases presented to general practice (3.3/100 person years (2.94 to 3.75)). One case was reported to national surveillance for every 1.4 laboratory identifications, 6.2 stools sent for laboratory investigation, 23 cases presenting to general practice, and 136 community cases. The ratio of cases in the community to cases reaching national surveillance was lower for bacterial pathogens (salmonella 3.2:1, campylobacter 7.6:1) than for viruses (rotavirus 35:1, small round structured viruses 1562:1). There were many cases for which no organism was identified. CONCLUSIONS: Infectious intestinal disease occurs in 1 in 5 people each year, of whom 1 in 6 presents to a general practitioner. The proportion of cases not recorded by national laboratory surveillance is large and varies widely by microorganism. Ways of supplementing the national laboratory surveillance system for infectious intestinal diseases should be considered.

Relevance of antibiotic sensitivities in predicting failure of omeprazole, clarithromycin, and tinidazole to eradicate Helicobacter pylori.

Omeprazole 20mg once (od) or twice daily (bd), clarithromycin 250mg bd, and tinidazole 500 mg bd for 7 days (OCT) is an effective regimen against Helicobacter pylori, but the effect of 5-nitroimidazole resistance is unclear. We aimed to evaluate this using the disc diffusion technique and E-test to assess 5-nitroimidazole resistance. H. pylori was cultured from antral biopsies of infected patients as determined by 13C-urea breath test (13C-UBT), histology, and/or rapid urease test. Patients were prescribed OCT, and H. pylori eradication was assessed by 13C-UBT at least 4 weeks after completion of therapy. Antibiotic sensitivities to metronidazole and clarithromycin were evaluated by the disc diffusion method and by minimum inhibitory concentration (MIC) using the E-test. One hundred and forty-one H. pylori-infected patients were enrolled into the study and the organism was successfully cultured in 119 patients (84%). The overall eradication rate was 125/141 (89%). OCT was successful in 62/69 (90%) patients harboring fully sensitive strains of H. pylori compared with 42/45 (93%) of patients with strains that were resistant to metronidazole alone (P = 0.74, Fisher's exact test). MIC was assessed in 22 samples. Using a cut-off point of > 32 microg/ml to define metronidazole resistance, eradication rates were higher against sensitive (9/12; 75%) compared with resistant (3/10; 30%) strains (P = 0.08, Fisher's exact test). 5-Nitroimidazole resistance assessed by the disc diffusion technique is not helpful in predicting OCT failure, but the E-test may be of value.

Use of an amplified-fragment length polymorphism technique to fingerprint and differentiate isolates of Helicobacter pylori.

Amplified-fragment length polymorphism (AFLP) analysis is the name given to a genotypic technique in which adapter oligonucleotides are ligated to restriction enzyme fragments and then used as target sites for primers in a PCR amplification process. The amplified fragments are electrophoretically separated to give strain-specific band profiles. We have developed a single-enzyme approach that did not require costly equipment or reagents for the fingerprinting of strains of Helicobacter pylori. The method was assessed with 46 isolates of H. pylori from 28 patients, and the results were compared with those from other genotypic tests. The AFLP profiles derived from HindIII fragments differentiated strains of H. pylori from unrelated individuals and confirmed the common origin of strains in some family members. AFLP analysis was also applied to investigate persistent infection following antibiotic therapy. Overall, the modified technique was relatively rapid and technically simple yet gave reproducible and discriminatory results. AFLP analysis samples variation throughout the genome and is a valuable addition to the existing genotypic fingerprinting methods for H. pylori.

Relevance of antibiotic sensitivities in predicting failure of omeprazole, clarithromycin, and tinidazole to eradicate Helicobacter pylori.

Omeprazole 20 mg once (o.d.) or twice daily (b.d.), clarithromycin 250 mg b.d., and tinidazole 500 mg b.d. for 7 days (OCT) is an effective regimen against Helicobacter pylori, but the effect of 5-nitroimidazole resistance is unclear. We aimed to evaluate this using the disc diffusion technique (Mast Diagnostics, Bootle, UK) and E-test (Cambridge Diagnostics Services, Cambridge, UK) to assess 5-nitroimidazole resistance. H. pylori was cultured from antral biopsies of infected patients, as determined by 13C-urea breath test (13C-UBT), histology, and/or rapid urease test. Patients were prescribed OCT and H. pylori eradication was assessed by 13C-UBT at least 4 weeks after completion of therapy. Antibiotic sensitivities to metronidazole and clarithromycin were evaluated by the disc diffusion method and by the measurement of minimum inhibitory concentration (MIC) using the E-test. One hundred and forty-one H. pylori-infected patients were enrolled in the study and the organism was successfully cultured from 119 patients (84%). The overall eradication rate was 125/141 (89%). OCT was successful in 62/69 (90%) patients harboring fully sensitive strains of H. pylori, compared with 42/45 (93%) of patients with strains that were resistant to metronidazole alone (P = 0.74, Fisher's exact test). MIC was assessed in 22 samples. Using a cut-off point of >32 microg/ml to define metronidazole resistance eradication rates were higher against sensitive (9/12; 75%) than resistant (3/10; 30%) strains (P = 0.08, Fisher's exact test). 5-Nitroimidazole resistance assessed by the disc diffusion technique is not helpful in predicting OCT failure, but the E-test may be of value.

Do patients need to fast for a 13C-urea breath test?

OBJECTIVE: The 13C-urea breath test (13C-UBT) is a useful non-invasive method of diagnosing Helicobacter pylori infection. One of its limitations, however, is that patients have to fast for 4 h before testing. We have compared the accuracy of a non-fasting 13C-UBT (NF13C-UBT) with a fasting 13C-UBT (F13C-UBT) test and against a gold standard. DESIGN: An unblinded prospective crossover study. METHODS: H. pylori status was assessed by histology, culture and rapid urease test. Patients were defined as H. pylori positive if two or more tests gave a positive result and negative if all tests were negative. H. pylori status was indeterminate if only one test gave a positive result. Following endoscopy patients had a F13C-UBT and then a further NF13C-UBT up to 14 days later after eating two slices of toast with jam or honey and tea or coffee. RESULTS: Of the 222 patients recruited to the study, 123 were gold standard H. pylori positive and 94 were negative with five patients having indeterminate status. Compared to this gold standard the NF13C-UBT had a 98% sensitivity and 96% specificity and the F13C-UBT had a 96% sensitivity and 97% specificity. The NF13C-UBT and F13C-UBT agreed in 217/222 (98%) cases. CONCLUSION: Relaxation of the fasting state does not reduce the accuracy of the 13C-UBT, making this test more convenient for patients.

Randomised double blind controlled study of recurrence of gastric ulcer after treatment for eradication of Helicobacter pylori infection.

OBJECTIVE: To determine whether eradication of Helicobacter pylori infection reduces recurrence of benign gastric ulceration. DESIGN: Randomised, double blind, controlled study. Patients were randomised in a 1:2 ratio to either omeprazole 40 mg once daily for eight weeks or the same treatment plus amoxycillin 750 mg twice daily for weeks 7 and 8. A 12 month untreated follow up ensued. SETTING: Teaching and district general hospitals between 1991 and 1994. SUBJECTS: 107 patients with benign gastric ulcer associated with H pylori. MAIN OUTCOME MEASURES: Endoscopically confirmed relapse with gastric ulcer (analysed with life table methods), H pylori eradication, and healing of gastric ulcers (Mantel-Haenszel test). RESULTS: 172 patients were enrolled. Malignancy was diagnosed in 19; 24 were not infected with H pylori; four withdrew because of adverse events; and 18 failed to attend for start of treatment, leaving 107 patients eligible for analysis (35 omeprazole alone; 72 omeprazole plus amoxycillin). In the omeprazole/amoxycillin group 93% (67/72; 95% confidence interval 84% to 98%) of gastric ulcers healed and 83% (29/35; 66% to 94%) in the omeprazole group (P = 0.103). Eradication of H pylori was 58% (42/72; 46% to 70%) and 6% (2/35; 1% to 19%) (P < 0.001) and relapse after treatment was 22% (16/72) and 49% (17/35) (life table analysis, P < 0.001), in the two groups, respectively. The recurrence rates were 7% (3/44) after successful H pylori eradication and 48% (30/63) in those who continued to be infected (P < 0.001). CONCLUSIONS: Eradication of H pylori reduces relapse with gastric ulcer over one year. Eradication rates achieved with this regimen, however, are too low for it to be recommended for routine use.

Failed treatment of Helicobacter pylori infection associated with resistance to clarithromycin.

BACKGROUND: Resistance of Helicobacter pylori to clarithromycin is uncommon. Initial studies have suggested that primary resistance has a significant adverse effect on bacteriological cure rates and acquired resistance develops frequently with failure of treatment following regimens containing clarithromycin. MATERIALS AND METHODS: H. pylori isolates were obtained from patients with duodenal ulcer treated with clarithromycin and omeprazole and examined for susceptibility to clarithromycin using the E-test method. A 13C urea breath test was used to confirm infection with H. pylori and successful treatment. RESULTS: H. pylori infection was successfully treated in 101 patients, all with susceptible isolates, and persisted in 30 patients of whom 4 had resistant isolates. Of 16 patients with bacteriological cure failure who had susceptible isolates pretreatment, 11 (68.8%) had resistant isolates post-treatment. CONCLUSIONS: This study confirms previous reports that acquired resistance of H. pylori to clarithromycin develops frequently (68.8-96%) in individuals with failed dual therapy regimens. Primary resistance, although uncommon (3%), was 100% predictive of treatment failure with the regimen used in this study. Effective treatment of H. pylori infection should help to prevent the development of resistance to clarithromycin.