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.     

Sorbitol-MacConkey medium for detection of Escherichia coli O157:H7 associated with hemorrhagic colitis.

Escherichia coli serotype O157:H7 is a recently recognized human pathogen associated with hemorrhagic colitis. Unlike most E. coli strains, E. coli O157:H7 does not ferment sorbitol. Therefore, the efficacy of MacConkey agar containing sorbitol (SMAC medium) instead of lactose as a differential medium for the detection of E. coli O157:H7 in stool cultures was determined in comparison with MacConkey agar. The relative frequency of non-sorbitol-fermenting (NSF) organisms other than E. coli O157:H7 in feces was low at 10 to 20% (95% confidence limits), and NSF organisms also occurred mostly in small numbers. In a field trial involving over 1,000 diarrheal stools, E. coli O157:H7 was isolated from 18 stools, all of which were from patients with bloody diarrhea. In every instance, the growth of E. coli O157:H7 on SMAC medium was heavy and occurred in almost pure culture as colorless NSF colonies in contrast to fecal flora, which are mostly sorbitol fermenting and hence appear pink on this medium, whereas on MacConkey agar cultures, the growth of E. coli O157:H7 was indistinguishable from fecal flora. SMAC medium permitted ready recognition of E. coli O157:H7 in stool cultures. Detection of E. coli O157:H7 on SMAC medium had a sensitivity of 100%, a specificity of 85%, and an accuracy of 86%. SMAC medium stool culture is a simple, inexpensive, rapid, and reliable means of detecting E. coli O157:H7, and we recommend routine use of SMAC medium especially for culturing bloody stools.     

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.     

Direct detection of verotoxin genes in stool samples by polymerase chain reaction in hemolytic uremic syndrome patients in France

Objective: To reassess the occurrence of verocytotoxin-producing Escherichia coli (VTEC) in French hemolytic uremic syndrome (HUS) patients.
Method: From March 1991 to January 1995, direct detection of verotoxin genes (VT) by the polymerase chain reaction (PCR) was performed on stool samples from 169 patients suffering from HUS.
Results: Fifty-one were PCR positive (30.1%); one was positive for the VT1 gene and the others for the VT2 gene. VTEC was isolated from only 32 of the 51 PCR-positive samples. E. coli O157:H7 was isolated from five patients. E. coli O111 was isolated from seven patients during an outbreak of HUS. Among the other VT2 E. coli strains, only four were serotypable. Of the 51 PCR-positive stools, 19 were culture negative for VTEC.
Conclusions: This study provides evidence that in France E. coli O157 and other VTEC serotypes are involved in HUS.     

Impact of free verotoxin testing on epidemiology of diarrhea caused by verotoxin-producing Escherichia coli.

During a 10-week period in the summer of 1990, an epidemiologic investigation of the prevalence of verotoxin (VT)-producing Escherichia coli infection was conducted in Calgary, Alberta, Canada. Consecutive stool specimens (n = 3,577) were cultured for E. coli O157:H7, and fecal filtrates were tested for free VTs (FVTs). E. coli O157:H7 was recovered from 22 specimens (0.6%), but VT was detected in 74 specimens (2.1%). Sixty-nine stool specimens positive for FVTs or E. coli O157:H7 were probed for VT genes by colony blot hybridization; 22 of 38 VT gene probe-positive isolates were non-O157:H7 E. coli organisms. Fourteen of 22 strains could not be induced to produce VT in vitro, despite the presence of FVTs in the stool sample, positivity on colony blot hybridization, positive PCR probes with the primers described by Pollard et al. (D. R. Pollard, W. M. Johnson, H. Lior, S. D. Tyler, and K. R. Rozee, J. Clin. Microbiol. 28:540-545, 1990) or Gannon et al. (V. P. Gannon, R. K. King, J. Y. Kim, and E. J. Golsteyn-Thomas, Appl. Environ. Microbiol. 58:3809-3815, 1992) (but not those described by Karch and Meyer [H. Karch and T. Meyer, J. Clin. Microbiol. 27:2751-2757, 1989]), and positive Southern blot analysis of isolates in 10 of 14 strains. The patient survey questionnaire showed that E. coli O157:H7 infection was associated with bloody diarrhea of short duration, whereas infection with other serotypes or persistence of FVT only was associated with longer-duration nonbloody diarrheal illness.(ABSTRACT TRUNCATED AT 250 WORDS)     

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.     

Isolation of enteric pathogeic agents in Côte d'Ivoire: Escherichia coli O157:H7 and enteroaggregative E. coli

New pathogens including Escherichia coli O157:H7 have emerged and spread world-wide as the most important cause of foodborne infections. We established a prospective study in Abidjan from 1996 to 1999 to determine the prevalence of Shiga-toxin producing E. coli (STEC) in our environment. Two O157 strains were found. One (EA47) O157:H7 was isolated from chicken and the other (EH144) O157:HNM from human diarrhoeal stool specimens. Both O157 strains carried stx2, eae, and UidA genes, but not e-hly one. Four other pathogenic E. coli were isolated, including three enteroaggregative E. coli (EAggEC) and one isolate which expresses a cytolethal distending toxin gene (cdtB). This is the first report of Shiga-toxin producing E. coli (STEC) in C?te d'Ivoire. Given its low prevalence (0.8%), E. Coli does not appear to be a public health problem in C?te d'Ivoire.     

Culture confirmation of Escherichia coli serotype O157:H7 by direct immunofluorescence.

An evaluation of a fluorescein-labeled, polyclonal, affinity-purified goat antibody to Escherichia coli serotype O157:H7 (Kirkegaard & Perry Laboratories Inc., Gaithersburg, Md.) was conducted to determine the efficacy of this research reagent for the rapid direct immunofluorescence identification of E. coli O157:H7 isolated from fecal specimens cultured on sorbitol-MacConkey (SMAC) agar. The E. coli O157:H7 fluorescent-antibody conjugate proved to be 100% sensitive and specific for the rapid identification of non-sorbitol-fermenting E. coli O157:H7 from SMAC agar inoculated with fecal specimens. The addition of SMAC agar to the battery of primary isolation media routinely used for fecal specimens resulted in the identification of 16 patients with E. coli O157:H7 disease from a total of 799 fecal specimens cultured during 1988.     

Hemolytic uremic syndrome in Belgium: incidence and association with verocytotoxin-producing Escherichia coli infection

Objective: To evaluate the incidence of hemolytic uremic syndrome (HUS) in Belgium and to determine the role of verocytotoxin-producing Escherichia coli O 157:H7 and other serotypes (non-O 157 VTEC).
Methods: Twenty-two centers, including the seven university hospitals, registered prospectively all cases of HUS; they collected clinical samples for isolation of VTEC strains and serum for detection of specific O-lipopolysaccharide antibodies.
Results: Forty-seven cases of HUS (including five incomplete cases) were recorded. Three cases were seen in nonresidents. The incidence of complete HUS in Belgian residents was 4.3 cases/100 000 in children <5 years old, 1.8 cases/100 000 when all children <15 years were considered, and 0.42/100 000 when patients of all ages were taken into account. By combining bacteriologic and serologic results, evidence of VTEC infection was obtained in 64% of the patients, mainly but not exclusively in children with prodromal diarrhea. The 13 VTEC isolates belonged to serotypes O157:H7 (nine isolates), O26:H11, O121:H-, O145:H- and O172:H- (one each) and all produced VT2 (+VT2vh-a in three O157 strains) and were positive for the eaeA gene.
Conclusions: The incidence rate found in this study and the high mortality and morbidity linked with this syndrome warrant further registration of pediatric and post-diarrheic adult HUS cases and also examination of stools for both O157 and non-O157 VTEC strains. For effective prevention of this disease, further study of the serotypes and accessory virulence factors associated with HUS is needed.     

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-.     

PCR detection of Escherichia coli O157:H7 directly from stools: evaluation of commercial extraction methods for purifying fecal DNA

Rapid identification of Escherichia coli O157:H7 is important for patient management and for prompt epidemiological investigations. We evaluated one in-house method and three commercially available kits for their ability to extract E. coli O157:H7 DNA directly from stool specimens for PCR. Of the 153 stool specimens tested, 107 were culture positive and 46 were culture negative. The sensitivities and specificities of the in-house enrichment method, IsoQuick kit, NucliSens kit, and QIAamp kit were comparable, as follows: 83 and 98%, 85 and 100%, 74 and 98%, and 86 and 100%, respectively. False-negative PCR results may be due to the presence of either inherent inhibitors or small numbers of organisms. The presence of large amounts of bacteria relative to the amount of the E. coli O157:H7 target may result in the lower sensitivities of the assays. All commercial kits were rapid and easy to use, although DNA extracted with the QIAamp kit did not require further dilution of the DNA template prior to PCR.     

Comparison of a direct fecal Shiga-like toxin assay and sorbitol-MacConkey agar culture for laboratory diagnosis of enterohemorrhagic Escherichia coli infection.

A direct fecal Shiga-like toxin assay (DSLTA) was used to prospectively screen 9,449 unselected stool samples, received at the British Columbia Provincial Health Laboratories and the Metropolitan Laboratories of Vancouver, for Shiga-like toxin I and Shiga-like toxin II. The results were compared with results of routine stool culture on sorbitol-MacConkey agar (SMAC) for Escherichia coli O157:H7. Of 80 specimens positive by either method, 59 (74%) and 74 (93%) were positive by SMAC and DSLTA, respectively; 53 (66%) were positive by both methods, 21 (26%) were positive by DSLTA only, and 6 (7%) were positive by SMAC only. On further screening, Shiga-like toxin-producing E. coli were detected in 8 (38%) of the 21 stools positive by DSLTA only, including serotypes O157:H7 (1 stool), O26:K60 (5 stools), O128:K67 (1 stool), and O103:H2 (1 stool). For the remaining 13 stools in which no SLTEC was found but DSLTA was positive, clinical information revealed that 11 of 12 patients had diarrheal illnesses, and 4 of these 11 had bloody diarrhea or hemolytic-uremic syndrome. Stools positive only by SMAC were collected earlier in the illness than stools positive by DSLTA, suggesting that free fecal toxin levels may be too low to detect at this time. Overall we found that DSLTA detected 19% more positive specimens than SMAC and that Shiga-like toxin-producing E. coli serotypes other than E. coli O157:H7 are causing disease in the province of British Columbia, Canada.     

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.     

多重实时荧光定量PCR检测肠出血性大肠杆菌O157:H7

目的 利用多重荧光定量PCR技术,建立一种快速、准确、特异检测肠出血性大肠杆菌O157:H7的定量方法.方法 选取肠出血性大肠杆菌O157:H7编码脂多糖基因(rfbE)和编码鞭毛抗原基因(fliC)作为检测的靶基因,设计引物和TaqMan-MGB探针,探针的5'端分别用FAM和HEX进行荧光标记,3'端标记MGB.优化PCR扩增体系,对多重实时荧光定量PCR方法的特异性、灵敏度、重复性评价,同时进行一定数量临床样本鉴定,与常规方法进行比较.结果 本研究所建立的多重实时荧光定量PCR方法可准确、特异地检测和鉴定肠出血性大肠杆菌O157:H7,能够有效甄别肠出血性大肠杆菌O157:H7与非H7菌株,其他菌株均无阳性结果;该方法的灵敏度可达到10 CFU/ml;定量检测的批间和批内变异系数均小于5%;对66例临床样本进行评价,结果显示15例肠出血性大肠杆菌O157:H7阳性,2例为肠出血性大肠杆菌O157:非H7阳性,其中16例与常规培养法结果符合,符合率达到98.49%.结论 本研究建立的检测肠出血性大肠杆菌O157:H7多重实时荧光定量PCR方法快速,结果准确、可靠,操作简便,为肠出血性大肠杆菌O157:H7的临床诊断、现场流行病学调查和食品安全监测提供了新的鉴定方法.     

DNA fingerprinting of Escherichia coli O157:H7 strains by pulsed-field gel electrophoresis.

Pulsed-field gel electrophoresis of genomic DNA was carried out on Escherichia coli O157:H7 strains from different geographic locations to determine its value in an epidemiological survey of O157 infections. Pulsed-field gel electrophoresis of XbaI-digested DNA fragments clearly separated E. coli O157:H7 strains from nontoxigenic E. coli O157:H19, O157:H43, and O157:H45 strains and from Shiga-like-toxin-producing E. coli strains of other serogroups. However, among the E. coli O157:H7 strains, the restriction patterns either were identical or differed only by a few fragment bands. In some cases, it was therefore impossible to distinguish among epidemiologically unrelated strains. Hybridization experiments with a DNA probe complementary to Shiga-like toxin II sequences revealed that the Shiga-like toxin II genes were located on DNA fragments of different lengths. Our data show that for a single highly conserved clone, such as E. coli O157:H7, other typing techniques may need to be performed in addition to DNA fingerprinting in epidemiological surveys.     

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.     

Lymphoid follicle-dense mucosa at the terminal rectum is the principal site of colonization of enterohemorrhagic Escherichia coli O157:H7 in the bovine host

Escherichia coli O157:H7 causes bloody diarrhea and potentially fatal systemic sequelae in humans. Cattle are most frequently identified as the primary source of infection, and E. coli O157:H7 generally colonizes the gastrointestinal tracts of cattle without causing disease. In this study, persistence and tropism were assessed for four different E. coli O157:H7 strains. Experimentally infected calves shed the organism for at least 14 days prior to necropsy. For the majority of these animals, as well as for a naturally colonized animal obtained from a commercial beef farm, the highest numbers of E. coli O157:H7 were found in the feces, with negative or significantly lower levels detected in lumen contents taken from the gastrointestinal tract. Detailed examination demonstrated that in these individuals the majority of tissue-associated bacteria were adherent to mucosal epithelium within a defined region extending up to 5 cm proximally from the recto-anal junction. The tissue targeted by E. coli O157:H7 was characterized by a high density of lymphoid follicles. Microcolonies of the bacterium were readily detected on the epithelium of this region by immunofluorescence microscopy. As a consequence of this specific distribution, E. coli O157:H7 was present predominantly on the surface of the fecal stool. In contrast, other E. coli serotypes were present at consistent levels throughout the large intestine and were equally distributed in the stool. This is a novel tropism that may enhance dissemination both between animals and from animals to humans. The accessibility of this site may facilitate simple intervention strategies.     

Evaluation of an immunoassay for direct detection of Escherichia coli O157 in stool specimens.

An enzyme-linked immunosorbent assay (ELISA) produced by LMD Laboratories, Inc., Carlsbad, Calif., was compared with culture for the detection of Escherichia coli O157. Nine of 185 stool specimens evaluated had positive results by the LMD E. coli O157 ELISA and grew E. coli O157 on culture; 174 had negative by LMD E. coli O157 ELISA results and did not grow E. coli O157 on culture. Of 174 specimens negative by LMD E. coli O157 ELISA, 117 specimens grew other enteric pathogens: Campylobacter spp. (46 isolates), Salmonella spp. (43 isolates), Yersinia spp. (20 isolates), and Shigella spp. (8 isolates). There were two indeterminant results by the LMD E. coli O157 ELISA. One stool specimen did not grow other enteric pathogens on culture, and one grew a Campylobacter sp. on culture. Both had negative LMD E. coli O157 ELISA results upon repeat testing. The LMD E. coli O157 ELISA is an accurate, easy-to-read screening method for the detection of E. coli O157 in fecal specimens.     

Profile of Escherichia coli O157:H7 pathogen responsible for hamburger-borne outbreak of hemorrhagic colitis and hemolytic uremic syndrome in Washington.

We analyzed Escherichia coli O157:H7 isolates from stool samples of five patients who had bloody diarrhea and were infected during a large food-borne outbreak of hemorrhagic colitis in Washington state. The isolates were assessed for Shiga-like toxin profile, adherence and plasmid traits, mouse virulence, capsule, and enterohemolysin production. The profiles of the five isolates were indistinguishable from each other and similar to that of E. coli O157:H7 strain EDL933, an organism responsible for a similar hamburger-associated food poisoning episode in 1982.     

Direct detection of verotoxin-producing Escherichia coli in stool samples by PCR.

A method for the rapid detection of verotoxin-producing Escherichia coli in stool samples by PCR was evaluated. Verotoxin-1 and verotoxin-2 genes in DNA extracted directly from stool samples were amplified with oligonucleotide primers. Stools spiked with control organisms, E. coli C600 (H19B) (verotoxin-1) or E. coli C600 (933W) (verotoxin-2), demonstrated that verotoxin-1-containing organisms could be detected at 10(2) CFU per 0.1 g of stool and verotoxin-2-containing organisms could be detected at 10(7) CFU per 0.1 g of stool. Testing of stool samples from patients with diarrhea showed a high concordance between PCR positivity and the presence of verotoxin-producing E. coli, determined by isolation of serotype O157:H7 on sorbitol-MacConkey medium (34 of 35 stool samples) or by colony blots with gene probes (19 of 21 stool samples). Conversely, only 1 of 20 (5.0%) stool samples that were O157:H7 culture negative and colony blot negative and that contained free verotoxin only was positive by PCR. As well, only 4 of 145 (2.8%) stool samples that were negative for serotype O157:H7 or free verotoxin were PCR positive. PCR of DNA extracted directly from stool samples provides a rapid method for the detection of stool samples containing verotoxin-producing E. coli compared with colony blot testing.