Evaluation of the premier EHEC assay for detection of Shiga toxin-producing Escherichia coli.

An enzyme-linked immunosorbent assay for the detection of Shiga toxins (Premier EHEC assay; Meridian Diagnostics, Inc.) was compared to conventional sorbitol-MacConkey culture for the recovery of enterohemorrhagic Escherichia coli. A total of 74 enteric pathogens, including 8 E. coli O157:H7 isolates, were recovered from 974 stool specimens. Two of these specimens were not tested by Premier assaying due to insufficient sample and are not considered in the data analysis. The Premier EHEC assay detected the 6 evaluable specimens which were culture positive for E. coli O157:H7 and identified an additional 10 specimens as containing Shiga toxin. Seven isolates were recovered from these 10 specimens by an immunoblot assay and were confirmed as toxin producers by a cytotoxin assay. Of these seven, four isolates were serotype O157:H7, one was O26:NM, one was O6:H-, and one was O untypeable:H untypeable. Three specimens contained Shiga toxin by both EHEC immunoassaying and cytotoxin testing; however, no cytotoxin-producing E. coli could be recovered. The sorbitol-MacConkey method had a sensitivity and a specificity of 60 and 100%, respectively, while the Premier EHEC assay had a sensitivity and a specificity of 100 and 99.7%, respectively, for E. coli O157:H7 only. The Premier EHEC assay also detected an additional 20% Shiga toxin-producing E. coli (STEC) that were non-O157:H7. Thus, the Premier EHEC assay is a sensitive and specific method for the detection of all STEC isolates. Routine use would improve the detection of E. coli O157:H7 and allow for determination of the true incidence of STEC other than O157:H7. The presence of blood in the stool and/or the ages of the patients were poor predictors of the presence of STEC. Criteria need to be determined which would allow for the cost-effective incorporation of this assay into the routine screen for enteric pathogens in high-risk individuals, especially children.     

Rapid Immunoassay for detection of Escherichia coli O157 directly from stool specimens.

A new and rapid ( < 1 h) enzyme-linked immunosorbent assay (ELISA) was compared with conventional sorbitol-MacConkey agar (SMAC) culture for the detection of Escherichia coli O157 from stool specimens. Among 34 positive specimens, confirmed by colony-sweeping and immunofluorescence stain methods, 6 did not exhibit visible sorbitol-negative colonies on SMAC. These six specimens would have been considered to be negative if SMAC alone had been used. The ELISA detected 31 of the 34 positive samples, including 5 of the above-mentioned 6 false-negative samples, resulting in a sensitivity and specificity of 91.2 and 99.5%, respectively. Cross-reactivity with other enteric pathogens was not noted by ELISA. The SMAC method had a sensitivity and specificity of 82.4 and 100%, respectively. The ELISA-negative specimens do not require culture confirmation, whereas positive results must be considered to be presumptive until confirmed by culture. The test is accurate and is easy to perform, making it a very efficient method for screening stool specimens for E. coli O157.     

Use of an alkaline phosphatase-labeled synthetic oligonucleotide probe for detection of Campylobacter jejuni and Campylobacter coli.

A commercially available synthetic nucleic acid probe (SNAP) conjugated to alkaline phosphatase was compared with standard culture techniques for detecting Campylobacter species. The SNAP was able to detect either 5 ng of C. jejuni DNA or 10(5) CFU of bacteria. The SNAP could also detect DNA extracted from 10(5) CFU in mock-infected stool samples. The SNAP detected C. jejuni and C. coli but showed no reactivity with C. laridis, C. fetus subsp. fetus, C. fetus subsp. venerealis, C. fennelliae, "C. upsaliensis," C. cinaedii, C. fecalis, C. hyointestinalis, C. mucosalis, or Helicobacter (Campylobacter) pylori. The SNAP also showed no cross-reactivity with other enteric pathogens. When applied to pure cultures, the SNAP detected 55 clinical isolates of C. jejuni and 11 clinical isolates of C. coli, with an accuracy of 100%. When applied directly to clinical specimens, the SNAP detected Campylobacter spp. in 19 of 23 culture-positive stool specimens (sensitivity, 82.6%; specificity, 100%). Pure cultures of the Campylobacter strains isolated from the four probe-negative, culture-positive stool specimens gave positive reactions with the SNAP. While the SNAP had excellent sensitivity and specificity for isolated bacterial colony isolates, the main limitation to the Campylobacter probe detection kit may be the sensitivity limit on direct detection of Campylobacter organisms in stools.     

Evaluation of performance and potential clinical impact of ProSpecT Shiga toxin Escherichia coli microplate assay for detection of Shiga Toxin-producing E. coli in stool samples

Shiga toxin-producing Escherichia coli bacteria (STEC) are emerging pathogens capable of producing sporadic and epidemic diarrhea, hemorrhagic colitis, and potentially life-threatening hemolytic-uremic syndrome. Although the presence of E. coli O157 can be readily detected in stool by sorbitol-MacConkey agar culture (SMAC), STEC non-O157 serotypes cannot. In contrast to culture, testing for the presence of Shiga toxins 1 and 2 in stool detects both O157 and non-O157 STEC serotypes capable of causing disease. Over two consecutive summers, we evaluated the performance of the ProSpecT Shiga toxin E. coli Microplate assay (Alexon-Trend, Ramsey, Minn.), an enzyme immunoassay for the detection of Shiga toxins 1 and 2, on all stools submitted for culture of enteric pathogens, and the potential clinical impact of Shiga toxin detection. Twenty-nine stool specimens were STEC positive by ProSpecT assay. Twenty-seven of 29 STEC-positive isolates were confirmed by SMAC and serotyping or by a second enzyme immunoassay and PCR (positive predictive value, 93%). Thirteen of 27 confirmed Shiga toxin-producing strains were serotype O157. The remaining 14 strains represented 8 other serotypes. The ProSpecT assay was 100% sensitive and specific for detection of E. coli O157 in stool (7 of 7) compared to SMAC. In addition, the ProSpecT assay detected twice as many STEC as SMAC. Fifty-two percent of confirmed STEC-positive stools were nonbloody. Thus, in our population, screening strategies that test only visibly bloody stools for STEC would miss a majority of cases. Eleven (41%) STEC-positive patients were hospitalized, and eight (30%) developed severe disease (two developed hemolytic-uremic syndrome, and six developed hemorrhagic colitis). Prior to detection of STEC infection, seven (26%) and eight patients (30%) underwent unnecessary diagnostic procedures or received potentially deleterious empirical treatment, respectively. We propose that establishing a specific diagnosis of STEC may have prevented these potentially harmful interventions. We conclude that the ProSpecT assay is sensitive and specific for the detection of Shiga toxins 1 and 2 in stool and has potentially significant clinical impact for the individual patient and public health. Shiga toxin assays should be considered for routine use in settings where prevalence of STEC disease warrants testing.     

Performance of the ImmunoCard STAT! E. coli O157:H7 test for detection of Escherichia coli O157:H7 in stools

ImmunoCard STAT! E. coli O157:H7 (Meridian Diagnostics, Inc., Cincinnati, Ohio) is a novel rapid (10-min) test for the presence of Escherichia coli O157:H7 in stools. The test may be performed either directly on stool specimens or on an overnight broth culture of stool. In a multicenter prospective study, 14 of 14 specimens positive by culture for E. coli O157:H7 were positive by the ImmunoCard STAT! O157:H7 test, and there were no false positives from 263 culture-negative specimens. In a retrospective study, the test was positive in 339 (81%) of 417 stored culture-positive specimens and the specificity was 95% (98 of 103 specimens). No false positives were associated with alternate stool pathogens. The ImmunoCard STAT! O157:H7 test has high sensitivity and specificity.     

Duplex real-time SYBR green PCR assays for detection of 17 species of food- or waterborne pathogens in stools

A duplex real-time SYBR Green LightCycler PCR (LC-PCR) assay with DNA extraction using the QIAamp DNA Stool Mini kit was evaluated with regard to detection of 8 of 17 species of food- or waterborne pathogens in five stool specimens in 2 h or less. The protocol used the same LC-PCR with 20 pairs of specific primers. The products formed were identified based on a melting point temperature (T(m)) curve analysis. The 17 species of food- or waterborne pathogens examined were enteroinvasive Escherichia coli, enteropathogenic E. coli, enterohemorrhagic E. coli, enterotoxigenic E. coli, enteroaggregative E. coli, Salmonella spp., Shigella spp., Yersinia enterocolitica, Yersinia pseudotuberculosis, Campylobacter jejuni, Vibrio cholerae, Vibrio parahaemolyticus, Vibrio vulnificus, Aeromonas spp., Staphylococcus aureus, Clostridium perfringens, and Bacillus cereus. No interference with the LC-PCR assay was observed when stool specimens were artificially inoculated with each bacterial species. The detection levels were approximately 10(5) food- or waterborne pathogenic bacteria per g of stool. The protocol for processing stool specimens for less than 10(4) food- or waterborne pathogenic bacteria per g of stool requires an overnight enrichment step to achieve adequate sensitivity. However, the rapid amplification and reliable detection of specific genes of greater than 10(5) food- or waterborne pathogenic bacteria per g in samples should facilitate the diagnosis and management of food- or waterborne outbreaks.     

Multicenter Evaluation of the BD Max Enteric Bacterial Panel PCR Assay for Rapid Detection of Salmonella spp., Shigella spp., Campylobacter spp. (C. jejuni and C. coli), and Shiga Toxin 1 and 2 Genes

Diarrhea due to enteric bacterial pathogens causes significant morbidity and mortality in the United States and worldwide. However, bacterial pathogens may be infrequently identified. Currently, culture and enzyme immunoassays (EIAs) are the primary methods used by clinical laboratories to detect enteric bacterial pathogens. We conducted a multicenter evaluation of the BD Max enteric bacterial panel (EBP) PCR assay in comparison to culture for the detection of Salmonella spp., Shigella spp., Campylobacter jejuni, and Campylobacter coli and an EIA for Shiga toxins 1 and 2. A total of 4,242 preserved or unpreserved stool specimens, including 3,457 specimens collected prospectively and 785 frozen, retrospective samples, were evaluated. Compared to culture or EIA, the positive percent agreement (PPA) and negative percent agreement (NPA) values for the BD Max EBP assay for all specimens combined were as follows: 97.1% and 99.2% for Salmonella spp., 99.1% and 99.7% for Shigella spp., 97.2% and 98.4% for C. jejuni and C. coli, and 97.4% and 99.3% for Shiga toxins, respectively. Discrepant results for prospective samples were resolved with alternate PCR assays and bidirectional sequencing of amplicons. Following discrepant analysis, PPA and NPA values were as follows: 97.3% and 99.8% for Salmonella spp., 99.2% and 100% for Shigella spp., 97.5% and 99.0% for C. jejuni and C. coli, and 100% and 99.7% for Shiga toxins, respectively. No differences in detection were observed for samples preserved in Cary-Blair medium and unpreserved samples. In this large, multicenter study, the BD Max EBP assay showed superior sensitivity compared to conventional methods and excellent specificity for the detection of enteric bacterial pathogens in stool specimens.     

Comparison of Escherichia coli O157:H7 antigen detection in stool and broth cultures to that in sorbitol-MacConkey agar stool cultures

We evaluated the Meridian IC-STAT direct fecal and broth culture antigen detection methods with samples from children infected with Escherichia coli O157:H7 and correlated the antigen detection results with the culture results. Stools of 16 children who had recently had stool cultures positive for this pathogen (population A) and 102 children with diarrhea of unknown cause (population B) were tested with the IC-STAT device (direct testing). Fecal broth cultures were also tested with this device (broth testing). The results were correlated to a standard of the combined yield from direct culture of stools on sorbitol-MacConkey (SMAC) agar and culture of broth on SMAC agar. Eleven (69%) of the population A stool specimens yielded E. coli O157:H7 when plated directly on SMAC agar. Two more specimens yielded this pathogen when the broth culture was similarly plated. Of these 13 stool specimens, 8 and 13 were positive by direct and broth testing (respective sensitivities, 62 and 100%). Compared to the sensitivity of a simultaneously performed SMAC agar culture, the sensitivity of direct testing was 73%. Three (3%) of the population B stool specimens contained E. coli O157:H7 on SMAC agar culture; one and three of these stool specimens were positive by direct and broth testing, respectively. The direct and broth IC-STAT tests were 100% specific with samples from children from population B. Direct IC-STAT testing of stools is rapid, easily performed, and specific but is insufficiently sensitive to exclude the possibility of infection with E. coli O157:H7. Performing the IC-STAT test with a broth culture increases its sensitivity. However, attempts to recover E. coli O157:H7 by culture should not be abandoned but, rather, should be increased when the IC-STAT test result is positive.     

Simultaneous detection of six diarrhea-causing bacterial pathogens with an in-house PCR-luminex assay

Diarrhea can be caused by a range of pathogens, including several bacteria. Conventional diagnostic methods, such as culture, biochemical tests, and enzyme-linked immunosorbent assay (ELISA), are laborious. We developed a 7-plex PCR-Luminex assay to simultaneously screen for several of the major diarrhea-causing bacteria directly in fecal specimens, including pathogenic Aeromonas, Campylobacter jejuni, Campylobacter coli, Salmonella, Shigella, enteroinvasive Escherichia coli (EIEC), Vibrio, and Yersinia. We included an extrinsic control to verify extraction and amplification. The assay was first validated with reference strains or isolates and exhibited a limit of detection of 10(3) to 10(5) CFU/g of stool for each pathogen as well as quantitative detection up to 10(9) CFU/g. A total of 205 clinical fecal specimens from individuals with diarrhea, previously cultured for enteric pathogens and tested for Campylobacter by ELISA, were evaluated. Using these predicate methods as standards, sensitivities and specificities of the PCR-Luminex assay were 89% and 94% for Aeromonas, 89% and 93% for Campylobacter, 96% and 95% for Salmonella, 94% and 94% for Shigella, 92% and 97% for Vibrio, and 100% and 100% for Yersinia, respectively. All discrepant results were further examined by singleplex real-time PCR assays targeting different gene regions, which revealed 89% (55/62 results) concordance with the PCR-Luminex assay. The fluorescent signals obtained with this approach exhibited a statistically significant correlation with the cycle threshold (C(T)) values from the cognate real-time PCR assays (P < 0.05). This multiplex PCR-Luminex assay enables sensitive, specific, and quantitative detection of the major bacterial causes of gastroenteritis.     

Production and characterization of a monoclonal antibody specific for enterohemorrhagic Escherichia coli of serotypes O157:H7 and O26:H11

A monoclonal antibody (MAb 4E8C12) specific for Escherichia coli O157:H7 and O26:H11 was produced by immunizing BALB/c mice with a rough strain of E. coli O157:H7. The antibody reacted strongly by a direct enzyme-linked immunosorbent assay with each of 36 strains of E. coli O157:H7. No cross-reactivity was observed with strains of Salmonella spp., Yersinia enterocolitica, Shigella dysenteriae, Proteus spp., Escherichia hermanii, Klebsiella pneumoniae, Campylobacter jejuni, Serratia marcescens, Citrobacter spp., Enterobacter cloacae, Hafnia alvei, Aeromonas hydrophila, and all except five strains of E. coli other than serotype O157:H7 (including strains of serotype O157 but not H7). The E. coli strains (all of serotype O26:H11) that reacted with the antibody were enterohemorrhagic E. coli (EHEC) that were isolated from patients with hemolytic uremic syndrome or hemorrhagic colitis and produced verotoxin similar to that of E. coli O157:H7. MAb 4E8C12 belongs to the subclass immunoglobulin G2a and has a kappa light chain. Tricine-sodium dodecyl sulfate-polyacrylamide gel electrophoresis of outer membrane proteins of E. coli of different serotypes followed by Western immunoblot analysis revealed that MAb 4E8C12 reacted specifically with two proteins of EHEC strains of serotypes O157:H7 and O26:H11 with apparent molecular weights of 5,000 to 6,000. These proteins appeared to be markers specific for EHEC strains of serotypes O157:H7 and O26:H11. This MAb, because of its specificity, may be a useful reagent of an immunoassay for the rapid detection of these types of EHEC isolates in clinical and food specimens.     

Detection,isolation, and molecular subtyping of Escherichia coli O157:H7 and Campylobacter jejuni associated with a large waterborne outbreak

The largest reported outbreak of waterborne Escherichia coli O157:H7 in the United States occurred in upstate New York following a county fair in August 1999. Culture methods were used to isolate E. coli O157:H7 from specimens from 128 of 775 patients with suspected infections. Campylobacter jejuni was also isolated from stools of 44 persons who developed diarrheal illness after attending this fair. There was one case of a confirmed coinfection with E. coli O157:H7 and C. jejuni. Molecular detection of stx(1) and stx(2) Shiga toxin genes, immunomagnetic separation (IMS), and selective culture enrichment were utilized to detect and isolate E. coli O157:H7 from an unchlorinated well and its distribution points, a dry well, and a nearby septic tank. PCR for stx(1) and stx(2) was shown to provide a useful screen for toxin-producing E. coli O157:H7, and IMS subculture improved recovery. Pulsed-field gel electrophoresis (PFGE) was used to compare patient and environmental E. coli O157:H7 isolates. Among patient isolates, 117 of 128 (91.5%) were type 1 or 1a (three or fewer bands different). Among the water distribution system isolates, 13 of 19 (68%) were type 1 or 1a. Additionally, PFGE of C. jejuni isolates revealed that 29 of 35 (83%) had indistinguishable PFGE patterns. The PFGE results implicated the water distribution system as the main source of the E. coli O157:H7 outbreak. This investigation demonstrates the potential for outbreaks involving more than one pathogen and the importance of analyzing isolates from multiple patients and environmental samples to develop a better understanding of bacterial transmission during an outbreak.     

Evaluation of multiplex PCRs for diagnosis of infection with diarrheagenic Escherichia coli and Shigella spp

We have developed two multiplex PCR assays that detect typical and atypical enteropathogenic Escherichia coli (EPEC) isolates, enteroaggregative E. coli (EAEC) isolates, enterotoxigenic E. coli (ETEC) isolates, enteroinvasive E. coli (EIEC) isolates, Shiga toxin-producing E. coli (STEC) isolates, and Shigella spp. The targets selected for each group were eae and bfpA for EPEC isolates, the target of probe CVD432 for EAEC isolates, the genes encoding heat-labile and heat-stable toxins for ETEC isolates, stx(1) and stx(2) for STEC isolates, and ipaH for EIEC isolates and Shigella spp. These PCRs were specific and sensitive for rapid detection of target isolates in stools. Among 150 stool specimens from the acute diarrhea tested, 9 samples (6%) had atypical EPEC, 9 (6%) had typical EPEC, 7 (4.7%) had EAEC, 3 (2%) had EIEC, 3 (2%) had Shigella spp., and 1 (0.7%) had an O26 STEC strain; we also detected mixed infections, 2 (1.3%) with EAEC and Shigella spp., 1 (0.7%) with atypical and typical EPEC strains, and another with atypical EPEC and EAEC strains. One of the multiplex PCRs directly applied to 36 stool specimens correctly identified 100% of EPEC and EAEC isolates.     

Etiology of childhood diarrhea in Korea.

To assess the role of recently recognized enteropathogens in childhood diarrhea in Korea, 231 children with diarrhea admitted to and 104 children without diarrhea seen at the well-baby clinic or the outpatient department of Hanyang University Hospital in Seoul, Korea, were evaluated during a 14-month period. Stools were cultured for bacterial pathogens, including enterotoxigenic (heat-labile and heat-stable enterotoxin-producing) and enteroadherent organisms. Only those stools obtained from patients with diarrhea were examined for rotavirus. All Escherichia coli isolates were screened for Shiga-like toxin (SLT) I, SLT-II, enterohemorrhagic E. coli fimbriae, and enteroinvasiveness by colony hybridization. One or more pathogens were identified in 75.8% of the children with diarrhea. Rotavirus was the most frequently identified pathogen, accounting for 47% of the cases. Other major enteropathogens were enterotoxigenic E. coli (22%), Clostridium difficile (16%), enteroadherent E. coli (15%), and enteropathogenic E. coli (6%). Shigella spp., Campylobacter jejuni, Salmonella spp., SLT-I-and enterohemorrhagic-E. coli-fimbria-probe-positive E. coli serotype O26:H11 and enteroinvasive E. coli were isolated from only a few patients. Aeromonas hydrophila and E. coli O157 were not isolated. Compared with those of the controls, the isolation rates of heat-stable-enterotoxin-producing E. coli (P less than 0.05), C. difficile (P less than 0.025), and enteroadherent E. coli (P less than 0.05) were significantly higher in the patients with diarrhea. The greatest number of rotavirus, enterotoxigenic E. coli, and C. difficile cases were identified during the cool, dry months of October and November.     

Survey of Culture,GoldenGate Assay,Universal Biosensor Assay,and 16S rRNA Gene Sequencing as Alternative Methods of Bacterial Pathogen Detection

Cultivation-based assays combined with PCR or enzyme-linked immunosorbent assay (ELISA)-based methods for finding virulence factors are standard methods for detecting bacterial pathogens in stools; however, with emerging molecular technologies, new methods have become available. The aim of this study was to compare four distinct detection technologies for the identification of pathogens in stools from children under 5 years of age in The Gambia, Mali, Kenya, and Bangladesh. The children were identified, using currently accepted clinical protocols, as either controls or cases with moderate to severe diarrhea. A total of 3,610 stool samples were tested by established clinical culture techniques: 3,179 DNA samples by the Universal Biosensor assay (Ibis Biosciences, Inc.), 1,466 DNA samples by the GoldenGate assay (Illumina), and 1,006 DNA samples by sequencing of 16S rRNA genes. Each method detected different proportions of samples testing positive for each of seven enteric pathogens, enteroaggregative Escherichia coli (EAEC), enterotoxigenic E. coli (ETEC), enteropathogenic E. coli (EPEC), Shigella spp., Campylobacter jejuni, Salmonella enterica, and Aeromonas spp. The comparisons among detection methods included the frequency of positive stool samples and kappa values for making pairwise comparisons. Overall, the standard culture methods detected Shigella spp., EPEC, ETEC, and EAEC in smaller proportions of the samples than either of the methods based on detection of the virulence genes from DNA in whole stools. The GoldenGate method revealed the greatest agreement with the other methods. The agreement among methods was higher in cases than in controls. The new molecular technologies have a high potential for highly sensitive identification of bacterial diarrheal pathogens.     

A method for detecting Shiga toxin and Shiga-like toxin-I in pure and mixed culture

Shiga toxin and Shiga-like toxins (SLTs, syn. Verotoxins) are currently detected by tissue culture assays that are expensive, time-consuming and require specialised facilities and experienced personnel. We have developed a rapid method to detect Shiga toxin and SLT-I (Verotoxin 1) based on their binding to globotriosyl ceramide (Gb3). Bound toxin was then detected by an enzyme-linked immunosorbent assay (ELISA) with monoclonal antibodies. The direct detection of cytotoxins from pure culture plates and from a mixed bacterial culture was studied. Using polymyxin extraction (0.1 g/L, 30 min, 37 degrees C) and Gb3-based ELISA we detected toxin from reference strains Shigella dysenteriae 1 strain 60R (Shiga toxin) and Escherichia coli O26:H11 strain H30 (SLT-I), and from clinical isolates of E. coli O157:H7 and O26:H11 (both SLT-I) from 11 patients with diarrhoea, haemorrhagic colitis or haemolytic uraemic syndrome. Toxin production by these strains was confirmed by a radiolabelled HeLa cell assay and the structural genes were detected by DNA hybridisation. The Gb3-based ELISA could detect SLT-I in extracts of a mixed culture even when the toxin-positive strains represented only 1% of the mixture. No cross-reactivity was found with bacteria that produce other cytotoxins, such as other E. coli and Shigella, Salmonella, Aeromonas and Campylobacter spp.     

Phylogeny, clinical associations, and diagnostic utility of the pilin subunit gene (sfpA) of sorbitol-fermenting, enterohemorrhagic Escherichia coli O157:H-

The plasmid-borne sfpA gene encodes the pilin subunit in sorbitol-fermenting (SF) enterohemorrhagic Escherichia coli (EHEC) O157:H-. We investigated the distribution of sfpA among 600 E. coli isolates comprising the complete E. coli standard reference (ECOR) and diarrheagenic E. coli (DEC) strain collections and clinical isolates associated with enteric disease. sfpA was detected in DEC3F SF EHEC O157:H- strain 493/89, each of 107 SF EHEC O157:H- clinical isolates, and 14 Shiga toxin-negative SF E. coli O157:H- strains which contained eae, which encodes gamma-intimin, and fliC, which encodes the H7 antigen. sfpA was absent from all other strains, including the ECOR strain collection, all non-SF EHEC O157:H7 strains, and all E. coli O55:H7 strains (E. coli O55:H7 is the postulated ancestor of Shiga toxin-producing E. coli [STEC] O157). These results suggest that there was a single acquisition of the sfpA gene in the nonmotile SF E. coli O157 branch, presumably after the eae-encoding pathogenicity island (the locus of enterocyte effacement) was acquired and motility was lost. We then applied the sfpA PCR in combination with rfbO157, stx, and eae PCRs to screen 636 stool samples from patients with diarrhea or hemolytic-uremic syndrome for SF STEC O157:H-. In 27 cases, the simultaneous presence of the sfpA, eae, and rfbO157 amplicons indicated the presence of SF E. coli O157:H- strains, and the result was subsequently confirmed by isolation. All but two of these strains possessed stx2. None of the other stool samples was positive by the sfpA PCR; 59 of these stool samples contained EHEC O157:H7. The sfpA gene can be recommended as a target for screening for SF E. coli O157:H-.     

Laboratory investigation of hemorrhagic colitis outbreaks associated with a rare Escherichia coli serotype.

Two outbreaks of hemorrhagic colitis, a newly recognized syndrome characterized by bloody diarrhea, severe abdominal pain, and little or no fever, occurred in 1982. No previously recognized pathogens were recovered from stool specimens from persons in either outbreak. However, a rare E. coli serotype, O157:H7, was isolated from 9 of 20 cases and from no controls. It was also recovered from a meat patty from the implicated lot eaten by persons in one outbreak. No recovery of this organism was made from stools collected 7 or more days after onset of illness; whereas 9 of 12 culture-positive stools had been collected within 4 days of onset of illness. The isolate was not invasive or toxigenic by standard tests, and all strains has a unique biotype. Plasmid profile analysis indicates that all outbreak-associated E. coli O157:H7 isolates are closely related. These results suggest that E. coli O157:H7 was the causative agent of illness in the two outbreaks.     

Laboratory investigation of outbreak of hemorrhagic colitis caused by Escherichia coli O157:H7.

A severe outbreak of hemorrhagic colitis occurred in London, Ontario, during the month of September 1985. A total of 55 residents and 18 employees of a nursing home developed diarrhea, and 17 residents (age range, 78 to 99 years) died. Specimens from 38 patients, 37 employees and contacts, and 10 autopsies were investigated for all enteric pathogens. Specimens were also planted on MacConkey-sorbitol agar. Fecal extracts were tested on Vero cells for cytotoxin (FVT). Escherichia coli isolates were serotyped and tested for verotoxin and beta-glucuronidase production. Of the 38 symptomatic patients, 26 were positive for FVT, verotoxin-producing E. coli (VTEC), or both. Of the 105 specimens that were examined from these 38 patients, FVT and VTEC were both positive in 30 specimens, FVT only was positive in 13 specimens, and VTEC only was positive in 4 specimens. None of the 27 specimens from 10 autopsies was positive for FVT or VTEC. No other enteric pathogen was found in any of the cases. All asymptomatic individuals were negative for both FVT and VTEC. Of 19 VTEC strains that were isolated, 18 belonged to serotype O157:H7. These 18 strains and 2 more strains that were obtained from sporadic cases that had occurred within the 2 previous months were found to give similar biochemical reactions in a 36-test identification system. All isolates of serotype O157:H7 were beta-glucuronidase negative and susceptible to the antimicrobial agents that are used to treat E. coli infections. Testing for FVT and VTEC was found to be the most sensitive and specific technique for the laboratory diagnosis of this disease. Negative sorbitol, positive raffinose, and negative beta-glucuronidase tests appeared to be consistent markers for aiding in the detection of E. coli O157:H7.     

Prevalence, characterisation and clinical profiles of Shiga toxin-producing Escherichia coli in The Netherlands.

Detection of Shiga toxin-producing Escherichia coli (STEC) in The Netherlands is traditionally limited to serogroup O157. To assess the relative importance of STEC, including non-O157 serogroups, stool samples submitted nationwide for investigation of enteric pathogens or diarrhoea were screened with real-time PCR for the presence of the Shiga toxin genes. Patients were selected if their stool contained blood upon macroscopic examination, if they had a history of bloody diarrhoea, were diagnosed with haemolytic uraemic syndrome, or were aged <6 years (irrespective of the bloody aspect of the stool). PCR-positive stools were forwarded to a central laboratory for STEC isolation and typing. In total, 4069 stools were examined, with 68 (1.7%) positive PCR results. The highest prevalence was for stools containing macroscopic blood (3.5%), followed by stools from patients with a history of bloody diarrhoea (2.4%). Among young children, the prevalence (1.0%) was not significantly higher than among random, non-bloody, stool samples from diarrhoeal patients (1.4%). STEC strains were isolated from 25 (38%) PCR-positive stools. Eleven O-serogroups were detected, including five STEC O157 strains. As serogroup O157 represented only 20% of the STEC isolates, laboratories should be encouraged to use techniques enabling them to detect non-O157 serogroups, in parallel with culture, for isolation and subsequent characterisation of STEC strains for public health surveillance and detection of outbreaks.     

Evaluation of Luminex xTAG Gastrointestinal Pathogen Analyte-Specific Reagents for High-Throughput,Simultaneous Detection of Bacteria,Viruses, and Parasites of Clinical and Public Health Importance

Acute diarrheal disease (ADD) can be caused by a range of pathogens, including bacteria, viruses, and parasites. Conventional diagnostic methods, such as culture, microscopy, biochemical assays, and enzyme-linked immunosorbent assays (ELISA), are laborious and time-consuming and lack sensitivity. Combined, the array of tests performed on a single specimen can increase the turnaround time (TAT) significantly. We validated a 19plex laboratory-developed gastrointestinal pathogen panel (GPP) using Luminex xTAG analyte-specific reagents (ASRs) to simultaneously screen directly in fecal specimens for diarrhea-causing pathogens, including bacteria (Campylobacter jejuni, Salmonella spp., Shigella spp., enterotoxigenic Escherichia coli [ETEC], Shiga toxin-producing E. coli [STEC], E. coli O157:H7, Vibrio cholerae, Yersinia enterocolitica, and toxigenic Clostridium difficile), parasites (Giardia lamblia, Cryptosporidium spp., and Entamoeba histolytica), and viruses (norovirus GI and GII, adenovirus 40/41, and rotavirus A). Performance characteristics of GPP ASRs were determined using 48 reference isolates and 254 clinical specimens. Stool specimens from individuals with diarrhea were tested for pathogens using conventional and molecular methods. Using the predictive methods as standards, the sensitivities of the GPP ASRs were 100% for adenovirus 40/41, norovirus, rotavirus A, Vibrio cholerae, Yersinia enterocolitica, Entamoeba histolytica, Cryptosporidium spp., and E. coli O157:H7; 95% for Giardia lamblia; 94% for ETEC and STEC; 93% for Shigella spp.; 92% for Salmonella spp.; 91% for C. difficile A/B toxins; and 90% for Campylobacter jejuni. The overall comparative performance of the GPP ASRs with conventional methods in clinical samples was 94.5% (range, 90% to 97%), with 99% (99.0% to 99.9%) specificity. Implementation of the GPP ASRs enables our public health laboratory to offer highly sensitive and specific screening and identification of the major ADD-causing pathogens.