Diarrhoea in close contacts as a risk factor for childhood haemolytic uraemic syndrome. The CPKDRC co-investigators.

To determine whether the risk factors for childhood haemolytic uraemic syndrome (HUS) are similar to risk factors previously reported for Escherichia coli O 157. H7 gastroenteritis, we conducted a case-control study at eight paediatric hospitals in the summer of 1990. Thirty-four consecutive children with HUS were prospectively enrolled; all had diarrhoea and 88% had laboratory evidence of exposure to verotoxin-producing E. coli (VTEC). The 102 controls were otherwise healthy children with minor acute injuries. Parents of all subjects responded to a questionnaire about each child''s exposure to various foods, methods of food preparation, sources of water, travel, and individuals with diarrhoea. Children with HUS were significantly more likely than controls to have had close contact with an individual with diarrhoea in the 2 weeks before the onset of illness (74 v. 29%, P < 0.00001; odds ratio 7.0, 95% CI 2.7-18.5). The onset of diarrhoea in the contacts occurred a median of 6 days (range, 1- > 14 days) before the onset of diarrhoea in the HUS patients. Exposure to undercooked ground meat was not significantly more common in the patients with HUS (15 v. 8%; P = 0.05). These data provide evidence consistent with person-to-person transmission of VTEC in a substantial proportion of episodes of childhood HUS.     

Characteristic of verotoxigenic strains of Escherichia coli

The aim of the study was the isolation from faecal samples of patients with diarrhoea of verotoxigenic strains of E. coli (VTEC) on the basis of characteristic biochemical properties and production of enterohaemolysin and comparison of isolated verotoxigenic strains with reference strains of VTEC. For isolation of VTEC from 257 stool samples derived from patients with diarrhoea were used selective medium sorbiol--Mac Conkey agar (SMAC) and media supplemented with unwashed and washed in PBS sheep erythrocytes for detection of haemolysins of E. coli. In all haemolytic and sorbitolo-positive or -negative strains isolated from 93 stool samples were examined the activity of beta-glucuronidase using MUG (4-methylumbelliferyl-beta-D-glukuronid) as a substrate for that enzyme. All isolated haemolytic strains as well as reference VTEC were examined on Vero cell line. Verotoxigenic strains from examined samples were investigated by agglutination assay with antiserum to E. coli O157 and then with antisera to eneropathogenic E. coli (EPEC). After that they were examined with ID GN and ATB GN tests. In 93 (36.2%) examined samples there were haemolytic strains of E. coli which fermented or not sorbitol and were MUG-positive or negative. Only in 2 (0.2%) stool samples there were verotoxigenic strains of E. coli which were sorbiol-positive and MG-positive. Both strains belonged to O26 serotype and were derived from samples of two children with diarrhoea. Isolated verotoxigenic strains of E. coli O26 were susceptible on all tested antibiotics.     

Incidence and clinical symptoms of Aeromonas-associated travellers' diarrhoea in Tokyo.

In a survey examining the causes of travellers'' diarrhoea treated in Tokyo between July 1986 and December 1995, Aeromonas species were isolated from 1265 (5.5%) of 23,215 travellers returning from developing countries. Aeromonas species were the fourth most frequent enteropathogen isolated, following enterotoxigenic E. coli (8.5%), Salmonella spp. (7.6%) and Plesiomonas shigelloides (5.6%). Aeromonas species were found in 1191 (5.6%) of 21,257 patients with diarrhoea and in 74 (3.8%) of 1958 healthy individuals without diarrhoea. Mixed infection was observed in 512 (40.5%) cases. No significant difference in the prevalence of Aeromonas by year, season, age distributions, or sex was observed, but a slight difference was noted depending on the country where the travellers visited. Of the 1265 Aeromonas isolates, 893 strains (70.6%) were A. veronii biovar sobria, 330 (26.1%) were A. hydrophila, and 42 (3.3%) were A. caviae. The clinical symptoms of patients from whom Aeromonas species was isolated as the only potential enteric pathogen were almost similar, which were watery diarrhoea (about 60%), abdominal cramps (43%), fever (around 15%), and nausea or vomiting (13%). Although the severity of illness was milder than that of enterotoxigenic E. coli alone, these data suggest that Aeromonas species are important enteric pathogens in travellers'' diarrhoea.     

An outbreak of gastroenteritis in Osaka,Japan due to Escherichia coli serogroup O166:H15 that had a coding gene for enteroaggregative E. coli heat-stable enterotoxin 1 (EAST1)

In an outbreak of gastroenteritis on 23 July 1996, in Osaka, Japan, 54 of 91 persons who had attended a meeting the previous day became ill. Escherichia coli O166:H15 was isolated from stool specimens of patients (29/33, 88%). Laboratory tests for other bacterial pathogens and viruses were negative. The E. coli 0166 organisms did not adhere to HEp-2 cells in a localized, diffuse, or enteroaggregative manner. The organisms did not express known enterotoxigenic E. coli (ETEC) colonization factors. In polymerase chain reaction tests, the bacteria did not have coding genes for shigatoxin of enterohemorrhagic E. coli (EHEC), heat-labile, or heat-stable enterotoxin of ETEC, attachment and effacement (eaeA) of EPEC, or invasion (invE) of enteroinvasive E. coli (EIEC). Consequently, they could not be assigned to any of the recognized diarrhoeagenic groups of E. coli: EPEC, ETEC, EHEC, EIEC, enteroaggregative E. coli (EAggEC), or diffusely adhering E. coli. However, the organisms possessed the EAggEC heat-stable enterotoxin (EAST1) gene. To our knowledge, this is the first report of an outbreak caused by E. coli that did not have well-characterized virulence genes other than EAST1. The isolates showed the same DNA banding pattern in pulsed-field gel electrophoresis after digestion with the restriction enzymes XbaI or NotI. Three O166:H15 strains isolated from two sporadic cases and another outbreak during 1997-8 were distinct, indicating that multiple clones have spread already. We propose that diarrhoeal specimens should be examined for E. coli possessing the EAST1 gene.     

Asymptomatic carriage of verocytotoxin-producing Escherichia coli O157 in farm workers in Northern Italy

Faecal samples from 350 farm workers on 276 dairy farms and 50 abattoir employees from seven different operations were examined for the presence of Verocytotoxin-producing Escherichia coli 0157 (VTEC O157) by an O157-specific enzyme-linked fluorescent assay followed by immunoconcentration. VTEC O157 was isolated from four (1.1%) of the farm workers. A second stool sample was obtained from the positive farm workers as well as from their household contacts. VTEC O157 was isolated from the wife of one of them. The strains from the same household shared the same Verocytotoxin genes profile, phage type and pulsed-field gel electrophoresis pattern. The VTEC O157-positive subjects had neither intestinal symptoms at the moment of sampling nor a history of bloody diarrhoea or renal failure. Our study seems to confirm the hypothesis that farm residents often develop immunity to VTEC O157 infection, possibly due to recurrent exposure to less virulent strains of VTEC.     

A controlled study of endemic sporadic diarrhoea among adult residents of southern Brazil

The aetiology of sporadic summer diarrhoea and enterotoxin (LT) antibody titres was studied among 43 adult patients in southern Brazil who had an acute, untreated diarrhoeal illness and 43 age- and sex-matched controls from the same area. A potential pathogen was identified in 33 of 34 patients and in 17 of 43 controls (p less than 0.01). 10 Shigella, one invasive Escherichia coli, one Salmonella and one Entamoeba histolytica were identified in 17 patients with inflammatory diarrhoea (faecal polymorphonuclear neutrophil leucocytes (PMN) present). In the other four only enterotoxigenic strains of E. coli were identified. Among 26 patients with non-inflammatory (no faecal PMN) diarrhoea, heat-stable (ST) toxin-producing coliforms were the most common pathogens isolated (27%). Heat-labile (LT) toxin-producing coliforms or serological rises to LT were seen in five patients, Strongyloides larvae in five, Giardia lamblia trophozoites in four and an LT-producing Salmonella in one. No pathogen was found in 10 patients with non-inflammatory diarrhoea. Among 43 controls, 11 LT coliforms, two Shigella and two Strongyloides larvae were found. 40 of 41 controls had detectable serum anti-LT antibody. A single ST-producing E. coli was found in one other control. No pathogen was identified in 26 controls. No rotaviruses or Norwalk-like viral agents were seen by direct or immune electron microscopy of stool filtrates. While LT-producing E. coli are frequently associated with endemic summer childhood diarrhoea in southern Brazil and are recognized causes of travellers' diarrhoea, this study shows that they are often carried asymptomatically by adults living in this region.(ABSTRACT TRUNCATED AT 250 WORDS)     

Sequence heterogeneity of the eae gene and detection of verotoxin-producing Escherichia coli using serotype-specific primers.

The distribution of the Escherichia coli attaching and effacing (eae) gene in strains of verotoxin-producing E. coli (VTEC) isolated from cattle and humans was studied. The majority of strains isolated from humans with bloody diarrhoea or HUS and cattle with severe diarrhoea were eae positive (82 and 83% respectively). In contrast, 59% of VTEC isolated from asymptomatic cattle were eae negative and of the remaining 41% that were eae positive, the majority were serotype O157. H7. The nucleotide sequence of the 3'' end of the eae gene of enteropathogenic E. coli (EPEC) of serotype O55. H7 was found to be almost identical to that of serotype O157. H7. Specific primers are described which detect the eae sequences of VTEC serotypes O157. H7, O157. H-, and EPEC serotypes O55. H7 and O55. H-. The nucleotide sequence of the 3'' end of the eae gene of serotype O111. H8 differed significantly from that of O157. H7. Primers were developed to specifically identify the eae sequences of VTEC serotypes O111. H- and O111. H8. We conclude that whereas the majority of VTEC associated with disease in cattle and humans possess the eae gene, the gene itself may not be necessary to produce haemorrhagic colitis and HUS. Sequence heterogeneity in the 3'' end of eae alleles of VTEC permits specific identification of subsets of these organisms.     

An international outbreak of Vero cytotoxin-producing Escherichia coli O157 infection amongst tourists; a challenge for the European infectious disease surveillance network

In March 1997, an outbreak of Vero cytotoxin-producing Escherichia coli O157 (VTEC) infection occurred amongst holidaymakers returning from Fuerteventura, Canary Islands. For the investigation, a confirmed case was an individual staying in Fuerteventura during March 1997, with either E. coli O157 VTEC isolated in stool, HUS or serological evidence of recent infection; a probable case was an individual with bloody diarrhoea without laboratory confirmation. Local and Europe-wide active case finding was undertaken through national centres, Salm-Net and the European Programme of Intervention Epidemiology, followed by a case-control study. Fourteen confirmed and one probable case were identified from England (7), Finland (5), Wales (1), Sweden (1) and Denmark (1) staying in four hotels. Three of the four hotels were supplied with water from a private well which appeared to be the probable vehicle of transmission. The case-control study showed illness was associated with consumption of raw vegetables (OR 8.4, 95% CI 1-5-48.2) which may have been washed in well water. This investigation shows the importance of international collaboration in the detection and investigation of clusters of enteric infection.     

Verotoxin-producing Escherichia coli infections in Sheffield: cattle as a possible source

During 1986 and 1987, faecal samples from patients with haemorrhagic colitis (HC) or haemolytic-uraemic syndrome (HUS) were examined for evidence of infection by verotoxin-producing Escherichia coli (VTEC). During the 2-year period VTEC infections were found in 31 (78%) of 40 patients initially presenting with HC, and in 5 (63%) of 8 patients initially presenting with HUS. VTEC were found in only 2 (0.9%) of 229 age and sex matched control patients with acute non-bloody diarrhoea. All but one VTEC belonged to E. coli serogroup O 157. During 1987 this serogroup was isolated from 2 (1%) of 207 samples of faeces taken from cattle arriving at a Sheffield abattoir, indicating a possible source of these infections for man. We are unaware of previous reports of isolation of this organism from cattle in England.     

Serum antibodies to secreted proteins in patients infected with Escherichia coli O157 and other VTEC.

Certain strains of verotoxigenic Escherichia coli (VTEC), and in particular those belonging to serogroup O157, cause attaching and effacing (AE) lesions of the host gut mucosa during pathogenesis. The mechanisms involved with bacterial attachment and the destruction of microvilli are determined by a cluster of genes within the LEE region, which also encode five secreted proteins. Sera from patients with antibodies to the lipopolysaccharide (LPS) of E. coli O157 and other VTEC were tested for antibodies to these secreted proteins. Twenty-one of 34 (62%) sera with antibodies to the lipopolysaccharide (LPS) of E. coli O157 also contained antibodies to one or more of the secreted proteins. Five of 12 sera containing antibodies to the LPS of a range of other VTEC serogroups also contained antibodies to 1 or more of the 5 secreted proteins, as did 16 of 70 (23%) sera from patients with haemolytic uraemic syndrome (HUS), haemorrhagic colitis (HC) or diarrhoea, but without bacteriological evidence of infection with VTEC and which did not contain antibodies to VTEC serogroups O5, O115, O145, O153 or O157. The detection of serum antibodies to secreted proteins may provide additional information for interpreting the results of established lipopolysaccharide-based VTEC serology.     

Vero cytotoxin-producing Escherichia coli, particularly serogroup O157, associated with human infections in England and Wales: 1992-4.

Investigations were performed by the Laboratory of Enteric Pathogens on Vero cytotoxin-producing Escherichia coli (VTEC) in England and Wales from 1992-4. Bacterial isolates, faeces and sera obtained from patients with diarrhoea, bloody diarrhoea and haemolytic uraemic syndrome were examined. Using serotyping, Vero cytotoxin gene probing and serodiagnostic tests for E. coli O157, evidence of infection was detected in 543, 434 and 491 individuals in 1992, 1993 and 1994 respectively; VTEC of serogroup O157 were isolated from 470, 385 and 411 cases. The O157 VTEC strains belonged to at least 19 different phage types (PT) although 84% belonged to PT2, PT49, PT8, PT1 or PT4. Antibodies to E. coli O157 lipopolysaccharide were detected in 13% of the cases. The average annual rate of infection with O157 VTEC was 0.83/100000 and 12% of the 1458 individuals with evidence of infection with VTEC or E. coli O157 developed haemolytic uraemic syndrome. There were at least 18 general outbreaks and many family outbreaks.     

Aetiology of diarrhoea in a birth cohort of children aged 0-2 year(s) in rural Mirzapur, Bangladesh

The incidence of aetiology-specific diarrhoea and the pathogenicity of infectious agents in a birth cohort (n=252) in rural Bangladesh were determined. Stool specimens or rectal swabs were collected from diarrhoeal cases over two years and routinely on a monthly basis. Stool samples from children with diarrhoea were compared with stool samples from children without diarrhoea to calculate rates of isolation and pathogenicity of agents. In total, 1750 stool specimens from diarrhoea patients and 5679 stool specimens from children without diarrhoea were tested. An infectious agent was identified in 58% of the stool specimens from diarrhoea patients and 21.6% of the stool specimens from children without diarrhoea. The most commonly-isolated pathogens from all specimens were enterotoxigenic Escherichia coli (ETEC), enteroadherent E. coli, Shigella, Campylobacter jejuni, Giardia, and rotavirus. ETEC (ST and LT-ST toxin), enterotoxigenic Bacteroides fragilis, Shigella, and rotavirus were associated more with disease than with asymptomatic infections. Aetiology-specific infections were associated with acute episodes. The isolated enteropathogens were essentially the same as those found in other tropical rural settings. Enterotoxigenic B. fragilis was also identified as a pathogen. Ongoing vaccine efforts focusing on Shigella, rotavirus, and ETEC would be useful.     

A community outbreak of haemolytic-uraemic syndrome in children occurring in a large area of northern Italy over a period of several months.

From March to October 1993, 15 cases of haemolytic-uraemic syndrome (HUS) in children were detected in a large area of northern Italy, where only 8 cases had occurred in the previous 5 years. Analysis of stool and serum specimens obtained from 14 cases showed evidence of Verotoxin-producing Escherichia coli (VTEC) infection in 13. Serum antibodies to the E. coli O157 lipopolysaccharide (LPS) were found in 8 patients and to the O111 LPS in 2. An O86 VTEC was isolated from another patient. Fourteen children needed dialysis, and 1 died. No obvious epidemiologic link was observed among cases, most of whom lived in small townships. A case-control study did not show an association between HUS and food or exposure to cattle, but suggested an association with contact with chicken coops (OR = 6.5, 95% C.I. 1.2-34.9). However, VTEC were not isolated from stool samples obtained from the chicken coops involved. The risk factors for VTEC infection related to living in rural settlements, including the exposure to live poultry, should be considered in outbreak investigations.     

A longitudinal study of Vero cytotoxin producing Escherichia coli in cattle calves in Sri Lanka.

Two cohorts of 10 and 16 calves were followed at weekly or fortnightly intervals from 4-28 and 1-9 weeks respectively to determine whether natural infection by Vero cytotoxin (VT) producing Escherichia coli (VTEC) occurred. Ninety-one of 171 (53%) faecal specimens were VTEC positive and 20-80% of animals at any given time excreted VTEC. Of 104 VTEC strains studied further, 6 different serogroups (O 22.H16; O 25.H5; O 49.H-; O 86.H26; O 88.H25; O 153.H12) and an untypable strain (O? .H21) were identified. All strains belonging to the same serotype had identical profiles of reactivity with DNA probes to toxins VT1 or 2, LTI or II and a probe (CVD419) derived from a plasmid carried by enterohaemorrhagic Escherichia coli O 157.H7. Four of these serotypes were found in the faecal flora of the calves, taken as a group, throughout the 4-month study period. Sixty percent of the strains hybridized with the probe for VT1, 4% with the probe for VT2, and 36% with both probes. Faecal VTEC were significantly associated with overt diarrhoeal illness in animals < 10 weeks of age, but no characteristic profile of markers (serotype or hybridization pattern) in E. coli isolates was associated with diarrhoea. A serological response to VT1 was detected in some animals, but faecal VT1 VTEC excretion persisted in spite of seroconversion. VT1 seroconversion was not associated with diarrhoea. A serological response to VT2 was not detected even in those animals excreting VT2 VTEC in the faeces.     

Children hospitalized with acute gastroenteritis. II. No relation between clinical symptoms and causative agents isolated from feces

OBJECTIVE: To determine if it is possible to predict a bacterial cause in children with gastroenteritis on the basis of clinical features and if the number of stool cultures can be reduced. DESIGN: Retrospective. METHOD: The results of the stool cultures of 227 children admitted with acute gastroenteritis to the department of Paediatrics of Sint Joseph Hospital, Veldhoven, the Netherlands, in the period 1992-1996 were related by review of medical records to clinical symptoms and blood tests. The diagnostic values of the various parameters were calculated. RESULTS: Rotavirus was identified in 40% of the faeces of the children, other viruses in 10% and bacteria in 8%. In 95% the bacterial pathogen was identified in the first stool sample. The diagnostic value of the investigated parameters was low. CONCLUSION: It is not possible on the basis of clinical parameters to predict a bacterial gastroenteritis. The number of stool samples may, however, be reduced if the stools of each child with acute diarrhoea are first investigated for rotavirus. Only if this test is negative are further investigations required. The number of bacterial cultures can be limited to one stool sample.     

Virulence factors of enteric Escherichia coli in young Aboriginal children in north-west Australia.

Enterotoxigenic Escherichia coli (ETEC) were the most frequently identified enteric pathogens associated with diarrhoea in 0-5 year old Aboriginal children in tropical north-west Australia with an incidence similar to those from other tropical regions. Heat-stable toxin-producing (ST+) strains were associated with diarrhoea throughout the year but heat-labile toxin-producing (LT+) strains were more important in the monsoonal summer season. ST+ strains were commonest in children with diarrhoea between 6 and 18 months of age while LT+ strains were associated with diarrhoea in children aged 18-24 months. Vero-toxigenic E. coli (VTEC) which produced VT1, but not VT2, and enteroadherent (EAF+) E. coli were significant causes of diarrhoea, mainly in children below 18 months but without a seasonal pattern.     

A prospective study of exposure to verotoxin-producing Escherichia coli among Canadian children with haemolytic uraemic syndrome. The CPKDRC co-investigators.

Haemolytic uraemic syndrome (HUS) is a leading cause of acute renal failure in childhood. Although infection with Escherichia coli O 157. H7 has been associated with HUS in North America and Europe, only a limited number of studies have examined the role of other verotoxin-producing E. coli (VTEC) serotypes in this condition. To address this issue, we conducted a comprehensive, prospective microbiological study of patients treated for HUS at eight Canadian hospitals in the summer of 1990. Of the 34 consecutive patients with HUS enrolled over 4 months, E. coli O 157. H7 was isolated from the stools of 26, and other E. coli serotypes were isolated from four patients. In four subjects no pathogenic E. coli serotypes were identified on stool culture. Using oligonucleotide probes specific for VT-1 and VT-2, verotoxin genes were detected in the stool isolates of all patients with E. coli O 157. H7, and from two with other E. coli serotypes. Two other patients had at least a fourfold rise in anti-verotoxin antibodies. Strong evidence of exposure to a verotoxin was present in 30/34 (88%). Patients with E. coli O 157. H7 infection were more likely to develop an antibody response to VT-2 than to VT-1 (22/22 vs 12/22; P = 0.002). These results further strengthen the association of HUS with verotoxin-producing E. coli in North America, and confirm that E. coli serotypes other than O 157.H7 are isolated in a small proportion of summertime HUS episodes.     

Investigation of human infections with verocytotoxin-producing strains of Escherichia coli (VTEC) belonging to serogroup O118 with evidence for zoonotic transmission

Twenty verocytotoxigenic Escherichia coli (VTEC) O118 strains isolated between 1996 and 1998 from human patients in Germany were analysed for their serotypes, their virulence markers and their epidemiological relatedness. Three strains were typed as O118:H12, these carried only the VT2d-Ount variant gene and were not associated with diarrhoea or haemolytic uraemic syndrome (HUS). Seventeen strains were serotyped as O118:H16 or O118:non-motile (NM). These carried all the genes for VTI, eae and EHEC-haemolysin. The O118:H16/NM strains were from diarrhoea (13 cases) and HUS (2 cases). Sixteen of the patients were young infants and most infections were associated with a rural environment. Evidence for zoonotic transmission from cattle to humans was found in two cases. The epidemiological relationship between the human and bovine O118:H16/NM isolates was indicated by homogeneous plasmid patterns and by very similar XbaI restriction patterns obtained by pulsed-field gel electrophoresis. VTEC O118:H16/NM are emerging pathogens in Germany and should be classified as new enterohaemorrhagic E. coli (EHEC) types.     

The role of Escherichia coli O 157 infections in the classical (enteropathic) haemolytic uraemic syndrome: results of a Central European, multicentre study.

To assess the importance of infection by Verotoxin (VT) producing Escherichia coli (VTEC) in children with HUS in Central Europe, stool and/or serum samples obtained from 147 patients from 28 paediatric centres were prospectively examined for the presence of VTEC and the kinetics of faecal VT titres (FVT), and for VT neutralization titres and antibodies against E. coli O 157 lipopolysaccharide, respectively. Ninety-two percent of the patients had classic (enteropathic) HUS (E+ HUS). Evidence of VTEC infection was obtained in 86% of them. VTEC/FVT were identified in 55/118 E+ cases (47%). A prominent feature was the frequent isolation of sorbitol-fermenting, VT2-producing E. coli O 157.H-.VT1 (C600/H19) was neutralized by 9%, and VT2 (C600/933W) by 99% of the initial serum samples from E+ patients, compared to 3% (VT1) and 100% (VT2) from age-related controls. Fourfold titre rises against VT1 and/or VT2 were observed in 13/70 (19%), and significantly elevated O 157 LPS IgM and/or IgA antibodies in 106/128 (83%) of the E+ patients. The ubiquitous VT2 neutralizing principle in the serum of HUS patients as of healthy controls warrants further investigations.     

Investigation and management of sporadic gastrointestinal infections with potentially Vero cytotoxin producing Escherichia coli in Scotland

Recognition of the potential of Escherichia coli O157 and other Vero cytotoxin producing E. coli (VTEC) organisms to cause serious disease led to the recommendation that all diarrhoeal stool specimens be examined for E. coli O157. National guidelines exist for the testing and exclusion of cases and contacts of VTEC infection. A survey was conducted to discover the extent to which these recommendations are followed in Scotland by asking about current practices for public health management of identified cases and laboratory investigation of E. coli infection. About two thirds of Scottish health boards followed national guidelines for testing and exclusion of cases and contacts of VTEC O157 infection. Most laboratories tested all diarrhoeal stools for E. coli O157 but detection methods varied and a minority tested selected stools for non-O157 E. coli serogroups. Standardisation of policies for laboratory testing of VTEC infection would improve national surveillance. Adherence to evidence based guidelines would standardise public health management of VTEC infections in Scotland.