Shiga toxin-producing Escherichia coli: incidence and clinical features in a setting with complete screening of patients with suspected infective diarrhoea.

OBJECTIVES: Shiga toxin-producing Escherichia coli (STEC) causes diarrhoeal disease, bloody diarrhoea, and haemolytic uraemic syndrome. The aim of this study was to describe the incidence of STEC and the clinical features of STEC patients from a well-defined Danish population in which all fecal samples of patients with suspected infective gastroenteritis were analysed for STEC. METHODS: In this population-based cohort study, all stool samples referred to two clinical microbiology laboratories were screened for STEC by culture and/or PCR. Epidemiological (n=170) and clinical (n=209) characteristics were analysed using data from local and national registries. RESULTS: Overall, 75,132 samples from 30,073 patients were screened resulting in 217 unique STEC-isolates. The epidemiological analysis showed an incidence of 10.1 cases per 100,000 person-years, which was more than twofold higher than the incidence in the rest of Denmark (3.4 cases per 100,000 person-years, p <0.001). Three groups were associated with a higher incidence: age <5 years (n=28, p <0.001), age ≥65 years (n=38, p 0.045), and foreign ethnicity (n=27, p 0.003). In the clinical analysis, patients with STEC harbouring only the Shiga toxin 1 gene (stx1-only isolates) showed a lower frequency of acute (n=11, p <0.05) and bloody diarrhoea (n=5, p <0.05) and a higher frequency of gastrointestinal symptoms for ≥3 months (n=8, p <0.05) than the other STEC patients. CONCLUSIONS: We report a more than twofold higher incidence in the project area compared with the rest of Denmark, indicating that patients remain undiagnosed when selective STEC screening is used. We found an association between patients with stx1-only isolates and long-term gastrointestinal symptoms.

A new pathogenicity island carrying an allelic variant of the Subtilase cytotoxin is common among Shiga toxin producing Escherichia coli of human and ovine origin.

Subtilase (SubAB) is a cytotoxin elaborated by some Shiga Toxin (Stx)-producing Escherichia coli (STEC) strains usually lacking the locus of enterocyte effacement (LEE). Two variants of SubAB coding genes have been described: subAB(1) , located on the plasmid of the STEC O113 98NK2 strain, and subAB(2) , located on a pathogenicity island (PAI) together with the tia gene, encoding an invasion determinant described in enterotoxigenic E. coli. In the present study, we determined the entire nucleotide sequence of the PAI containing the subAB(2) operon, termed Subtilase-Encoding PAI (SE-PAI), and identified its integration site in the pheV tRNA locus. In addition, a PCR strategy for discriminating the two subAB allelic variants was developed and used to investigate their presence in E. coli strains belonging to different pathotypes and in a large collection of LEE-negative STEC of human and ovine origin. The results confirmed that subAB genes are carried predominantly by STEC and showed their presence in 72% and 86% of the LEE-negative strains from human cases of diarrhoea and from healthy sheep respectively. Most of the subAB-positive strains (98%) identified possessed the subAB(2) allelic variant and were also positive for tia, suggesting the presence of SE-PAI. Altogether, our observations indicate that subAB(2) is the prevalent SubAB-coding operon in LEE-negative STEC circulating in European countries, and that sheep may represent an important reservoir for human infections with these strains. Further studies are needed to assess the role of tia and/or other genes carried by SE-PAI in the colonization of the host intestinal mucosa.

A verocytotoxin-producing E. coli outbreak with a surprisingly high risk of haemolytic uraemic syndrome, Denmark, September-October 2012.

Denmark faced an outbreak of verocytotoxin-producing E. coli (VTEC) O157:H7 infections in autumn 2012. Thirteen cases were diagnosed of which eight had haemolytic uraemic syndrome (HUS). Epidemiological investigations suggested ground beef as the vehicle of the outbreak. The outbreak strain had a rare toxin gene subtype profile: eae, vtx1a and vtx2a, and a high proportion of HUS (62%) among cases, a finding previously linked with the outbreak subtype profile. Toxin subtyping can be useful to identify high risk VTEC strains.

Escherichia coli clonal group A causing bacteraemia of urinary tract origin.

Escherichia coli clonal group A (CgA) causes disease in humans. This is the first study investigating the prevalence of CgA among E. coli from non-urine, extraintestinal infections in a northern European country. E. coli blood (n = 196) and paired urine (n = 195) isolates from the same patients with bacteraemia of urinary tract origin were analysed. The isolates were collected from January 2003 through May 2005 at four hospitals in Copenhagen, Denmark. Pulsed-field gel electrophoresis (PFGE) patterns, antimicrobial resistance and patient characteristics were determined for all CgA isolates; presence of virulence-associated genes (VAGs) and serotypes were determined for the blood CgA isolates. Thirty blood isolates (15%) belonged to CgA. CgA blood isolates were associated with female patients and sulfamethoxazole-trimethoprim resistance and they harboured a distinctive VAG profile. The blood and urine isolates from each pair were found to be related in 26 of 27 CgA blood/urine pairs, confirming a urinary tract origin of infection. Furthermore, a relationship between the PFGE patterns of CgA blood/urine isolates and CgA isolates from UTI patients in general practice and a CgA isolate from a community-dwelling human reported previously, was found, suggesting a community origin of CgA. The finding of CgA strains in 15% of the E. coli bloodstream infections with a urinary tract origin in Denmark suggests that CgA constitutes an important clonal lineage among extraintestinal pathogenic E. coli. A reservoir of this pathogenic E. coli group in the community causing not only UTI but also more severe infections such as bacteraemia has implications for public health.

Clonality and virulence traits of Escherichia coli associated with haemorrhagic septicaemia in turkeys.

Fifty-five clinical isolates of avian pathogenic Escherichia coli (APEC) from seven outbreaks of acute haemorrhagic septicaemia in turkeys were characterized by serotyping, plasmid profiling including restriction analysis with HindIII, ribotyping with EcoRI and HindIII, multilocus sequence typing (MLST) and virulence profiling. A clonal relationship was demonstrated for each outbreak according to serotype, plasmid profiling, ribotyping, and MLST. In addition, isolates demonstrated highly similar virulence profiles, as all isolates were positive for F11 pili and possessed genes encoding aerobactin (iucD), increased serum survival (iss), temperature-sensitive haemagglutinin (tsh) and colicin V plasmid operon genes (cva/cvi). However, only 20% of the isolates produced colicin V and 42% exhibited serum resistance. All strains with O group O111 and a single O18ac strain (demonstrating non-clonal DNA profiles) were positive for enteroaggregative heat-stabile toxin (EAST1), while isolates of a single outbreak all possessed the enteroaggregative toxin gene (astA). All isolates were negative for genes encoding verocytotoxins (vtx/stx), iron-repressible protein (irp2), P-fimbria (papC), invasion plasmid antigen (ipaH), attaching and effacing gene (eae), enterohaemolysin (ehxA), and enterotoxins LT, STIa (ST(p)) and STIb (ST(h)). In conclusion, highly similar virulence profiles were demonstrated for isolates of E. coli associated with a single well-defined lesion type of colibacillosis in turkeys; acute haemorrhagic septicaemia. The isolates obtained, however, demonstrated a different phylogenetic background, underlining the importance of using well-defined strain collections for characterization of APEC pathotypes.

Characteristics of the enteroaggregative Shiga toxin/verotoxin-producing Escherichia coli O104:H4 strain causing the outbreak of haemolytic uraemic syndrome in Germany, May to June 2011.

The Escherichia coli strain causing a large outbreak of haemolytic uraemic syndrome and bloody diarrhoea in Germany in May and June 2011 possesses an unusual combination of pathogenic features typical of enteroaggregative E. coli together with the capacity to produce Shiga toxin. Through rapid national and international exchange of information and strains the known occurrence in humans was quickly assessed.We describe simple diagnostic screening tools to detect the outbreak strain in clinical specimens and a novel real-time PCR for its detection in foods.

Outbreaks of gastroenteritis linked to lettuce, Denmark, January 2010.

At least 11 linked outbreaks of gastroenteritis with a total of 260 cases have occurred in Denmark in mid January 2010. Investigations showed that the outbreaks were caused by norovirus of several genotypes and by enterotoxigenic Escherichia coli. Lettuce of the lollo bionda type grown in France was found to be the vehicle.

Oral health status of handicapped primary school pupils in Dar es Salaam, Tanzania.

BACKGROUND: There is hardly any information regarding oral health status of handicapped primary school pupils in Tanzania. Determination of their oral health status could help in planning sustainable intervention programmes for this disadvantaged group. OBJECTIVES: To determine caries and periodontal status and treatment needs of handicapped primary school pupils in Dar es Salaam, Tanzania. DESIGN: A descriptive cross-sectional study. SETTING: Uhuru Mchanganyiko and Buguruni special schools, Dar es Salaam. RESULTS: The sample consisted of 179 (55.8%) males and 142 (44.2%) females aged between 7 and 22 years. Majority (71%) were deaf followed by blind (17.8%) and mentally retarded (8.7%). Six (1.9%) pupils were both deaf and blind, while one (0.3%) pupil was blind and mentally retarded. Forty one (12.8%) pupils had at least one decayed deciduous tooth, with the mean (dmfs) ranging from 0.25 to 3.24. The deaf had the highest mean decayed surfaces, followed by the mentally retarded and the blind. There was only one (0.3%) pupil who had a filled deciduous tooth. Thirty three (10.3%) pupils had decayed permanent teeth and 31 (9.7%) had missing permanent teeth. None of the decayed permanent teeth were restored. The blind had the lowest mean deciduous surfaces (DS) scores of between 0 and 1.0. In the mentally retarded group the mean DS ranged from 0.25 to 1.75. About 73.5% of the studied group had bleeding of the gums, with the blind having the highest mean bleeding index scores (p < 0.001) and about 82.8% of the pupils had calculus, with highest mean scores mainly among the blind (p = 0.008). CONCLUSION: The caries prevalence among handicapped primary school pupils was quite low. However, there was relatively high level of gingival bleeding and calculus. Regarding treatment needs, 23% required dental fillings mainly of one and two surface restorations and 82% required scaling and polishing. Despite these treatment needs these pupils had not received any dental attention.

Attaching and effacing Escherichia coli isolates from Danish children: clinical significance and microbiological characteristics.

This study describes the prevalence, clinical manifestations and microbiological characteristics of attaching and effacing Escherichia coli isolates, i.e., enteropathogenic E. coli (EPEC) belonging to the classical EPEC serotypes, non-EPEC attaching and effacing E. coli (A/EEC) and verocytotoxin-producing E. coli (VTEC), isolated in a case-control study of Danish children aged <5 years. Among 424 children with diarrhoea and 866 healthy controls, EPEC and VTEC were more prevalent in cases (2.4% and 2.6%, respectively) than in controls (0.7% and 0.7%, respectively). There was a high frequency of A/EEC isolates (n = 121), but these were equally prevalent in cases (11.3%) and controls (12.5%), and comprised a heterogeneous distribution of O:H serotypes. The intimin (eae) subtypes in A/EEC isolates showed an even distribution; the eae-gamma subtype predominated in classical EPEC cases. The virulence genes encoding the bundle-forming pilus (bfpA) and enteroaggregative heat-stable enterotoxin (astA) were rare among all isolates, and seemed to be of limited pathogenic importance in this population. Virulence characterisation of A/EEC isolates did not reveal any significant differences between cases and controls. Colonisation of children with A/EEC was associated with contact with sheep or goats (OR 2.2). The role of A/EEC, not being VTEC or belonging to the classical EPEC serotypes, requires further clarification, but serotyping is useful in discriminating between EPEC and A/EEC strains.

A method for fast and simple detection of major diarrhoeagenic Escherichia coli in the routine diagnostic laboratory.

A multiplex PCR was developed for the detection of the following genes characteristic of diarrhoeagenic Escherichia coli (DEC): verocytotoxins 1 (vtx1) and 2 (vtx2), characteristic of verocytotoxin-producing E. coli (VTEC); intimin (eae), found in enteropathogenic E. coli (EPEC), attaching and effacing E. coli and VTEC; heat-stable enterotoxin (estA) and heat-labile enterotoxin (eltA), characteristic of enterotoxigenic E. coli (ETEC); and invasive plasmid antigen (ipaH), characteristic of enteroinvasive E. coli (EIEC) and Shigella spp. The method allowed the simultaneous identification of all six genes in one reaction, and included a 16S rDNA internal PCR control. When applied to pure cultures from a reference strain collection, all virulence genes in 124 different DEC strains and 15 Shigella spp. were identified correctly, and there were no cross-reactions with 13 non-E. coli species. The detection limit of the method was 10(2)-10(3) DEC CFU/PCR in the presence of 10(6) non-target cells. When the multiplex PCR was tested with colonies from plate cultures of clinical stool samples, it was a faster, more sensitive, less expensive and less laborious diagnostic procedure than DNA hybridisation. When used with DNA purified from spiked stool samples (by two different commercial kits), the method had a detection limit of 10(6) CFU/mL stool sample.

Patients' satisfaction with dental care provided by public dental clinics in Dar es Salaam, Tanzania.

BACKGROUND: In Tanzania, patient satisfaction with dental services has received only minor attention. OBJECTIVE: To assess patients' satisfaction with public dental health services in Dar es Salaam. DESIGN: A cross-sectional study. SETTING: Five public dental clinics randomly selected from a list of all the nine public dental clinics in Dar es Salaam. SUBJECTS: Five hundred and sixteen consecutive patients, 193 males and 323 females aged between 12 and 77 years who, during the study period between July and November, 2002 were attending five dental clinics were randomly selected. RESULTS: No significant differences in patients' satisfaction level (overall or for the specific studied items) were found in all the five public dental clinics. The answers to the specific sub-items, apart from time spent with doctor, were around the average with very few responses with a high ranking. CONCLUSIONS: Findings indicate a moderate level of patient satisfaction with dental care offered in public dental clinics in Dar es Salaam. Areas identified as needing improvement included; technical quality of care, interpersonal aspects and communication.

Standardized pulsed-field gel electrophoresis of Shiga toxin-producing Escherichia coli: the PulseNet Europe Feasibility Study.

PulseNet USA, the American molecular subtyping network for foodborne infections, has since 1996 been highly successful combating infections caused by Shiga toxin-producing Escherichia coli (STEC) O157, Salmonella, Listeria, and Shigella spp. The PulseNet Europe feasibility study was initiated to ascertain the interest of public health and veterinary reference laboratories to establish a similar network, and to determine if it was possible to perform standardized pulsed-field gel electrophoresis (PFGE) typing of Salmonella, Listeria, and STEC on a large scale in Europe. The results of the STEC part of that study are presented here. Twenty-seven veterinary and public health laboratories participated in the study. The participants subtyped eight E. coli strains by PFGE using the restriction enzyme XbaI according to the PulseNet or a similar protocol, with strict adherence to the electrophoretic conditions stated in the former and submitted an image of their gel for centralized anonymous analysis. The quality of the gels was first graded visually as "good," "intermediate," and "unsatisfactory." The number of gels graded this way was 11, 14, and 2, respectively. All "good" and "intermediate" gels were also analysed and compared by computerized analysis to a reference gel. For gels graded "good," on average 5.6, 7.4, and 8 patterns out of 8 per gel were identified with a similarity of 100%, >95%, and >90%, respectively. The corresponding numbers for gels graded "intermediate" were 1.7, 4.9, and 7.4, respectively. The problems causing the grading to be "intermediate" was overloaded lanes, overexposed images, not optimally focused images and incomplete digestion, all problems that led to misinterpretation of the number of restriction fragments in the gel. These problems may be corrected by simple adjustments to the subtyping procedure. Thus, there seems to be little need for training of the participants in PulseNet Europe.

PCR detection of seven virulence and toxin genes of Campylobacter jejuni and Campylobacter coli isolates from Danish pigs and cattle and cytolethal distending toxin production of the isolates.

AIMS: To study the prevalence of seven virulence and toxin genes, and cytolethal distending toxin (CDT) production of Campylobacter jejuni and C. coli isolates from Danish pigs and cattle. METHODS AND RESULTS: The presence of the cadF, ceuE, virB11, flaA, cdtA, cdtB, cdtC and the cdt gene cluster among 40 C. jejuni and C. coli isolates was detected by polymerase chain reaction. The CDT production of the isolates was determined on Vero, colon 205 and chicken embryo cells. The cadF, flaA, ceuE and cdtB genes were detected from 100% of the isolates. The cdtA and cdtC genes were found in 95.0 and 90.0% of the isolates, respectively. The cdt gene cluster was detected in 82.5% isolates. Only 7.5% of the isolates were positive for virB11. Ninety-five per cent of the isolates produced CDT in Vero and colon 205 cell assays, and 90% of the isolates produced CDT in chicken embryo cell assays. CONCLUSIONS: High prevalence of the cadF, ceuE, flaA and cdtB genes was found. Data of the prevalence of cdt genes was consistent with the CDT titres produced by the isolates. Campylobacter coli from pigs produced high CDT titres. SIGNIFICANCE AND IMPACT OF THE STUDY: The high prevalence of seven virulence and toxin genes demonstrated that these putative pathogenic determinants are widespread among Campylobacter isolates from pigs and cattle. Campylobacter coli isolates from pigs produced much higher CDT titres compared with C. coli isolates from other sources suggesting that C. coli may be particularly adapted to or associated with this species.

The effect of choosing different units of analysis when estimating risk of presence of dental caries in the primary dentition.

BACKGROUND: Data on caries are usually collected with the tooth surface or the tooth as the unit, but subsequently analysed by aggregating the data at the level of the individual. AIMS: To evaluate how different units of analysis may affect the result of the statistical analyses in a study of the association between deciduous dental caries and a set of risk factors. METHOD: 293 children (mean age: 7.5 yrs) from two primary schools in Dar es Salaam, Tanzania were examined. Thus the study design was cross-sectional. Logistic regression analysis was used to determine the association between caries experience and nutritional status, socioeconomic background, and microbiological and salivary characteristics using the subject, the tooth, and the tooth surface as unit of analysis, allowing for the fact that teeth and surfaces within the same individual must be considered non-independent. RESULTS: When aggregated data were used, point estimates were larger in some instances. The precision of the estimates increased considerably when the tooth as compared to the individual was used as the unit of analysis. No or limited gain in precision was obtained when the tooth surface as compared to the tooth was used as unit of analysis. CONCLUSIONS: The choice of unit of analysis may strongly influence the result of the statistical analyses and thus the conclusion of an investigation.

Determinants of utilization of dental services among 20- to 34-year-old Danes.

Our aim was to identify determinants of utilization of dental services among 20- to 34-year-old Danes as outlined in a conceptual framework. Our sample consisted of a sample of 464 individuals who had been interviewed by 10 experienced interviewers from The Danish National Institute of Social Research. Our questionnaire comprised a battery of questions based on our conceptual model. The analysis was done in a hierarchical manner using a framework with three steps: Predisposing factors --> Enabling factors --> Need factors --> Utilization. Associations were evaluated with multivariate logistic regression analysis and expressed as odds ratios. The following factors were significantly associated with irregular use of dental services: age, sex, exercise habits, cost of dental treatment, dental anxiety, and perceived condition of teeth. Our findings suggest that a strategy aiming to increase young people's dental attendance should focus on the transition period that is, when the youngsters leave the public dental health care system and have to make use of the private system. The strategy should also take into account that young men are more likely to become non-users. A concerted effort could be dedicated to the non-negligible group of individuals with dental anxiety and dental phobia.

Clinical isolates of non-O157 Shiga toxin-producing Escherichia coli: serotypes, virulence characteristics, and molecular profiles of strains of the same serotype.

All human Shiga toxin-producing Escherichia coli (STEC) non-O157 strains (n = 56) isolated in Finland from 1990 to August 2000 were characterized for the O:H serotype, stx(1) and stx(2) genes, production of enterohemolysin, and sensitivity to 12 antimicrobial agents. Strains of the same serotype were genotyped by pulsed-field gel electrophoresis (PFGE) after XbaI restriction of total DNA. The 56 non-O157 isolates belonged to 29 serotypes. Two of the serotypes (O102:H7 and OX181:H49) have not previously been described as being associated with STEC infections in humans or isolated from animals. Thirty-four strains (61%) within seven serotypes (O103:H2 [14 isolates], O26:H11 [6 isolates], O145:H28 [4 isolates], O145:HNM [3 isolates], O15:HNM [3 isolates], OX174:H21 [2 isolates], and O Rough:HNM [2 isolates]) were represented by more than one isolate. Of these strains, O103:H2 isolates were divided into seven, O26:H11 isolates were divided into four, and the rest within a serotype were divided into two genotypes in PFGE. In PCR, 31 (55%) of the 56 strains were positive for the stx(2) gene only and 24 strains (43%) were positive for stx(1) only. One strain (O43:H2) carried both stx(1) and stx(2). Forty-two strains (75%) produced enterohemolysin, and 39 strains (70%) possessed the eae gene. Of the latter 39 strains, 36 (92%) were enterohemolytic, whereas only 6 (35%) of the 17 isolates lacking the eae gene were enterohemolytic (P < 0.001). The majority of the strains (44 strains, 79%) were sensitive to all 12 antimicrobials tested. Of the 56 strains, 20 (36%) were associated with small family outbreaks in nine families and 14 (25%) were associated with recent travel abroad.

Eruption pattern of autotransplanted premolars visualized by radiographic color-coding.

Eruption patterns and root growth were visualized with the use of a new technique, radiographic color-coding, for comparison of the development of autotransplanted premolars with contralateral control teeth. Rates of eruption and root growth were studied. The eruption pattern and rate was assessed relative to the first molar. Maximum rates were found to occur between 30 and 60 days after transplantation. There were no significant differences between transplants and their contralaterals. Two distinct categories of eruption patterns were demonstrated. One group showed a tendency toward an initial rate of transplant eruption that was somewhat faster than that of the contralaterals. The other group showed initially retarded eruption. Possible explanations were discussed. Because no significant differences between the transplants and the contralaterals were observed, it was concluded that autotransplantation is a sound treatment option for substitution of missing teeth, at least from a tooth development point of view.

Determinants of child mortality in a rural Ugandan community.

OBJECTIVE: To estimate the rate of and risk factors associated with child mortality in rural eastern Uganda. DESIGN: A community based cross-sectional study using the preceding birth technique--a robust method of obtaining information of survival of the previous child. SETTING: A rural district in Eastern Uganda. PARTICIPANTS: In total, 2888 multigravidae were interviewed in April and May 1999. MAIN OUTCOME MEASURE: Number of deaths among children born alive. RESULTS: The under-two child mortality rate was 108 per 1000 livebirths. The annual child mortality was 82 per 1000 child-years of risk. Child mortality was associated with low parental education, being born to adolescent mothers or mothers aged 35 or more. Unconditional logistic regression showed that children born to uneducated parents had a doubled risk of not celebrating their second birthday. It was three times more likely for a child to die in the neonatal period than in the first year of life. Child mortality risk decreased by 4% and 6% for every year of education attained by mothers and fathers, respectively. Parity, residence and marital status were not associated with excess risk of child mortality. Seasonal mortality followed the El Nino rainfall pattern. Finally, there were geographical differences in child mortality although this was not statistically significant. CONCLUSION: Monitoring trends in child mortality at district level can be done using the preceding birth technique in antenatal settings. Maternal education, an important predictor of child survival should be included in routine data collection at clinics.