New immuno-PCR assay for detection of low concentrations of shiga toxin 2 and its variants

Shiga toxin (Stx)-producing Escherichia coli (STEC) strains secrete toxins that are major virulence factors and diagnostic targets, but some STEC strains secrete Stx in amounts that cannot be detected using conventional cell cytotoxicity or immunological assays. Therefore, there is an urgent need for more-sensitive Stx detection methods. We describe the development of an assay that can detect low concentrations of Stx2 and its variants. An immuno-PCR Stx2 assay was developed based on an enzyme immunoassay (EIA) combining antibody capture and DNA amplification to increase the signal. The immuno-PCR assay detected 10 pg/ml of purified Stx2, compared to 1 ng/ml Stx2 detected by commercial EIA. Consequently, immuno-PCR detected Stx2 and its variants in STEC strains that produce the toxins at levels that are nondetectable by using the EIA, as well as the Stx2 in EIA-negative enriched stool cultures from patients. Our data demonstrate that the immuno-PCR developed here is a highly sensitive and specific method for the detection of trace amounts of Stx2 and Stx2 variants. It is therefore suitable for use by clinical microbiological laboratories to improve the toxin detection in clinical samples.     

Evaluation of the duopath verotoxin test for detection of shiga toxins in cultures of human stools

The Duopath Verotoxin test (Merck KgaA, Darmstadt, Germany) is a newly developed immunochromatographic test for the confirmation of Shiga toxin (Stx)-producing Escherichia coli (STEC) strains from food products. This test detects both Stx 1 (Stx1)-positive and Stx2-positive samples individually with the same device. By modification of the original protocol, the present study evaluated its performance and feasibility for clinical application with human stool samples, consisting of 41 frozen samples known to contain STEC isolates (O157:H7 and non-O157 serotypes) and 250 fresh specimens. The test specimens were polymyxin B extracts of colony sweeps taken from overnight sorbitol-MacConkey agar cultures of stools containing STEC isolates and other bacteria. All 41 frozen STEC-positive stool samples were positive by the Duopath Verotoxin test, as were 2 fresh stool samples with culture-confirmed STEC O157 infection. Thus, 100% sensitivity and no false-positive results were obtained when the Premier EHEC assay (Meridian Bioscience, Cincinnati, Ohio) was used as the "gold standard." The Duopath Verotoxin test is simple to perform and easy to interpret, providing a turnaround time of 24 h. Despite its original intended use, the Duopath Verotoxin test has a great potential for clinical application.     

Direct Detection of Legionella Species from Bronchoalveolar Lavage and Open Lung Biopsy Specimens: Comparison of LightCycler PCR, In Situ Hybridization, Direct Fluorescence Antigen Detection, and Culture

We developed a rapid thermocycling, real-time detection (also known as real-time PCR) method for the detection of Legionella species directly from clinical specimens. This method uses the LightCycler (Roche Molecular Biochemicals, Indianapolis, Ind.) and requires approximately 1 to 2 h to perform. Both a Legionella genus PCR assay and Legionella pneumophila species-specific PCR assay were designed. A total of 43 archived specimens from 35 patients were evaluated, including 19 bronchoalveolar lavage (BAL) specimens and 24 formalin-fixed, paraffin-embedded open lung biopsy specimens. Twenty-five of the specimens were culture-positive for Legionella (9 BAL specimens and 16 tissue specimens). BAL specimens were tested by LightCycler PCR (LC-PCR) methods and by a direct fluorescent antibody (DFA) assay, which detects L. pneumophila serogroups 1 to 6 and several other Legionella species. Tissue sections were tested by the two LC-PCR methods, by DFA, by an in situ hybridization (ISH) assay, specifically designed to detect L. pneumophila, and by Warthin-Starry (WS) staining. The results were compared to the "gold standard" method of bacterial culture. With BAL specimens the following assays yielded the indicated sensitivities and specificities, respectively: Legionella genus detection by Legionella genus LC-PCR, 100 and 100%; Legionella genus detection by DFA assay, 33 and 100%; and L. pneumophila detection by L. pneumophila species-specific LC-PCR, 100 and 100%. With open lung biopsy specimens the following assays yielded the indicated sensitivities and specificities, respectively: Legionella genus detection by LC-PCR 68.8 and 100%; Legionella genus detection by DFA assay, 44 and 100%; Legionella genus detection by WS staining, 63 and 100%; L. pneumophila species-specific detection by LC-PCR, 17 and 100%; and L. pneumophila species-specific detection by ISH, 100 and 100%. The analytical sensitivity of both LC-PCR assays was <10 CFU/reaction. LC-PCR is a reliable method for the direct detection of Legionella species from BAL specimens. The Legionella genus LC-PCR assay could be performed initially; if positive, L. pneumophila species-specific LC-PCR could then be performed (if species differentiation is desired). The speed with which the LC-PCR procedure can be performed offers significant advantages over both culture-based methods and conventional PCR techniques. In contrast, for the methods evaluated, culture was the best for detecting multiple Legionella species in lung tissue. WS staining, Legionella genus LC-PCR, and L. pneumophila species-specific ISH were useful as rapid tests with lung tissue.     

Multicenter evaluation of a sequence-based protocol for subtyping shiga toxins and standardizing stx nomenclature

When Shiga toxin-producing Escherichia coli (STEC) strains emerged as agents of human disease, two types of toxin were identified: Shiga toxin type 1 (Stx1) (almost identical to Shiga toxin produced by Shigella dysenteriae type 1) and the immunologically distinct type 2 (Stx2). Subsequently, numerous STEC strains have been characterized that express toxins with variations in amino acid sequence, some of which confer unique biological properties. These variants were grouped within the Stx1 or Stx2 type and often assigned names to indicate that they were not identical in sequence or phenotype to the main Stx1 or Stx2 type. A lack of specificity or consistency in toxin nomenclature has led to much confusion in the characterization of STEC strains. Because serious outcomes of infection have been attributed to certain Stx subtypes and less so with others, we sought to better define the toxin subtypes within the main Stx1 and Stx2 types. We compared the levels of relatedness of 285 valid sequence variants of Stx1 and Stx2 and identified common sequences characteristic of each of three Stx/Stx1 and seven Stx2 subtypes. A novel, simple PCR subtyping method was developed, independently tested on a battery of 48 prototypic STEC strains, and improved at six clinical and research centers to test the reproducibility, sensitivity, and specificity of the PCR. Using a consistent schema for nomenclature of the Stx toxins and stx genes by phylogenetic sequence-based relatedness of the holotoxin proteins, we developed a typing approach that should obviate the need to bioassay each newly described toxin and that predicts important biological characteristics.     

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.     

Real-time fluorescence PCR assays for detection and characterization of heat-labile I and heat-stable I enterotoxin genes from enterotoxigenic Escherichia coli

To facilitate the diagnosis of enterotoxigenic Escherichia coli (ETEC) infections in humans, we developed and evaluated real-time fluorescence PCR assays for the Roche LightCycler (LC) against the enterotoxin genes commonly present in strains associated with human illness. Separate LC-PCR assays with identical cycling conditions were designed for the type I heat-labile enterotoxin (LT I) and the type I heat-stable enterotoxin (ST I) genes, using the LC hybridization probe format. A duplex assay for ST I with two sets of amplification primers and three hybridization probes was required to detect the major nucleotide sequence variants of ST I, ST Ia and ST Ib. LC-PCR findings from the testing of 161 E. coli isolates of human origin (138 ETEC and 23 non-ETEC) were compared with those obtained by block cycler PCR analysis. The sensitivities and specificities of the LC-PCR assays were each 100% for the LT I and ST I genes. The LC-PCR and block cycler PCR assays were also compared for their abilities to detect LT I and ST I genes in spiked stool specimens with different methods of sample preparation. Findings from these experiments revealed that the limits of detection for the LC-PCR assays were the same or substantially lower than those observed for the block cycler PCR assay. Melting curve analysis of the amplified LT I and ST I genes revealed sequence variation within each gene, which for the ST I genes correlated with the presence of ST Ia and ST Ib. The rapidity, sensitivity, and specificity of the LC-PCR assays make them attractive alternatives to block cycler PCR assays for the detection and characterization of ETEC.     

Loop-mediated isothermal amplification assays for detecting shiga toxin-producing Escherichia coli in ground beef and human stools

Shiga toxin-producing Escherichia coli (STEC), encompassing E. coli O157 and non-O157 STEC, is a significant cause of food-borne illnesses and deaths in the United States and worldwide. Shiga toxins (encoded by stx) and intimin (encoded by eae) are important virulence factors for STEC strains linked to severe human illnesses such as hemorrhagic colitis and hemolytic-uremic syndrome. In this study, the stx(1), stx(2), and eae genes were chosen as targets to design loop-mediated isothermal amplification (LAMP) assays for the rapid, specific, sensitive, and quantitative detection of STEC strains. The assay performances in pure culture and spiked ground beef and human stools were evaluated and compared with those of quantitative PCR (qPCR). No false-positive or false-negative results were observed among 90 bacterial strains used to evaluate assay specificity. The limits of detection for seven STEC strains of various serogroups (O26, O45, O103, O111, O121, O145, and O157) were approximately 1 to 20 CFU/reaction in pure culture and 10(3) to 10(4) CFU/g in spiked ground beef, which were comparable to the results of qPCR. Standard curves generated suggested good linear relationships between STEC cell numbers and LAMP turbidity signals. When applied in ground beef samples spiked with two low levels (1 to 2 and 10 to 20 CFU/25 g) of STEC cultures, the LAMP assays achieved accurate detection after 6 to 8 h enrichment. The assays also consistently detected STEC in human stool specimens spiked with 10(3) or 10(4) CFU/0.5 g stool after 4 h enrichment, while qPCR required 4 to 6 h. In conclusion, the LAMP assays developed in this study may facilitate rapid and reliable identification of STEC contaminations in high-risk food commodities and also facilitate prompt diagnosis of STEC infections in clinical laboratories.     

Rapid and Sensitive Detection of Shiga Toxin-Producing Escherichia coli from Nonenriched Stool Specimens by Real-Time PCR in Comparison to Enzyme Immunoassay and Culture

Shiga toxin (Stx)-producing Escherichia coli (STEC) bacteria are a frequent cause of food-borne gastroenteritis, hemorrhagic colitis, and hemolytic uremic syndrome. Because antimicrobial agents are generally contraindicated in patients infected with STEC, a sensitive and specific diagnostic test with rapid turnaround is essential. Current culture methods may fail to detect non-O157 STEC. We evaluated a Stx gene real-time PCR assay using hybridization probes and the LightCycler instrument with 204 prospectively collected stool specimens, which were also tested for Stx by enzyme immunoassay (EIA) (ProSpecT STEC; Remel, Lenexa, KS) and by culturing on chromogenic agar (Chromagar O157; BD BBL, Sparks, MD). In addition, 85 archived stool specimens previously tested for Stx (by EIA) and/or E. coli O157:H7 (by culture) were tested by PCR. Sample preparation for PCR included mixing the stool in sterile water and extraction of nucleic acid using the MagNA Pure LC instrument (Roche Diagnostics). The PCR assay had 100% sensitivity and specificity compared to EIA and culture for specimens collected prospectively (4 of 204 specimens were positive) and compared to culture and/or EIA for archival specimens (42 of 85 specimens were positive). Both the EIA and PCR produced positive results from a specimen containing an O103 serotype STEC in the prospective specimens, and the PCR test detected three positive specimens that contained nonviable STEC in the archived specimens. The PCR assay demonstrated 100% sensitivity and specificity compared to EIA and/or culture and more rapid turnaround than either EIA or culture.Shiga toxin (Stx)-producing Escherichia coli (STEC) is a frequent cause of food-borne outbreaks of diarrhea (15). Disease caused by STEC is characterized by abdominal pain and bloody diarrhea, and 5 to 15% of those individuals infected with serotype O157:H7 develop hemolytic uremic syndrome (HUS), a potentially life-threatening condition consisting of hemolytic anemia, thrombocytopenia, and kidney failure caused primarily by Stx (8). STEC may carry genes for one or both types of Stx, Stx1 and Stx2 (17).Although STEC strains are a diverse group of pathogens, up to the present, the most common serotype in the United States has been O157:H7. A common association is that of E. coli O157:H7 contaminating ground beef (3, 7), but recent large outbreaks have involved a variety of other foods, including leafy greens (6, 29). The diversity of potentially contaminated food means that patients may acquire STEC infection from many foodstuffs, far beyond the stereotypical risk of undercooked ground beef. The common denominator of tainted food products seems to be direct or indirect contamination from bovine feces. To best detect infected patients and potential outbreaks, clinical laboratories must have tools to quickly and accurately detect STEC in stool specimens. Culture on sorbitol MacConkey agar is an inexpensive, effective, and widely used method based on lack of sorbitol fermentation by E. coli O157:H7. Several drawbacks limit the utility of culture, including slow turnaround, false-negative results in antibiotic-treated patients, and false-negative results due to emerging serotypes of non-O157 STEC that ferment sorbitol (1, 14, 16, 29). Alternatively, a method that is increasingly utilized is detection of Stx antigen from stool, either directly or after broth enrichment. Our experience concurs with enzyme immunoassay (EIA) product insert data that optimal sensitivity and specificity are achieved only when a broth enrichment step is employed; this results in slow turnaround.Here, we describe a real-time PCR assay that can detect STEC using nucleic acid extracts of stool specimens. We evaluated the performance of this assay using both archived stool specimens and prospectively collected specimens and compared the results to those of culture and Stx antigen detection.(This study was presented in part at the 48th Annual Interscience Conference on Antimicrobial Agents and Chemotherapy, Washington, DC, 25 to 28 October 2008.)     

Direct comparison of the BD ProbeTec ET system with in-house LightCycler PCR assays for detection of Chlamydia trachomatis and Neisseria gonorrhoeae from clinical specimens

The commercial BD ProbeTec ET (BDPT) system for the detection of Neisseria gonorrhoeae and Chlamydia trachomatis from clinical specimens was compared with our in-house LightCycler real-time PCR (LC-PCR) assays. Specimens initially positive by the BDPT system were retested by our LC-PCR assays. Our results for C. trachomatis testing indicate a 91.2% agreement when the results of 114 clinical specimens, initially positive by BDPT over a wide range of method-other-than-acceleration (MOTA) scores, were retested by our LC-PCR assay. The agreement between the two systems improved to 96% when only MOTA scores of >30,000 were retested by the LC-PCR assay. The overall agreement between the two systems for Neisseria gonorrhoeae detection from 155 clinical specimens was only 77.4%, with agreement particularly low (24.1%) for MOTA scores ranging from 2,000 to 19,999. Repeat testing of specimens with the BDPT only closely correlated with that seen by others demonstrating that reproducibility of the BDPT system for specimens initially within the MOTA score range from 2,000 to 9,999 is problematic, especially for Neisseria gonorrhoeae testing. With our study, we proposed an algorithm for C. trachomatis and N. gonorrhoeae testing which involves screening with the BDPT system followed by selective use of our in-house LC-PCR assays.     

Real-time fluorescence PCR assays for detection and characterization of Shiga toxin,intimin, and enterohemolysin genes from Shiga toxin-producing Escherichia coli

PCR assays have proved useful for detecting and characterizing Shiga toxin-producing Escherichia coli (STEC). Recent advances in PCR technology have facilitated the development of real-time fluorescence PCR assays with greatly reduced amplification times and improved methods for the detection of amplified target sequences. We developed and evaluated two such assays for the LightCycler instrument: one that simultaneously detects the genes for Shiga toxins 1 and 2 (stx(1) and stx(2)) and another that simultaneously detects the genes for intimin (eae) and enterohemolysin (E-hly). Amplification and sequence-specific detection of the two target genes were completed within 60 min. Findings from the testing of 431 STEC isolates of human and animal origin, 73 isolates of E. coli negative for stx genes, and 118 isolates of other bacterial species with the LightCycler PCR (LC-PCR) assays were compared with those obtained by conventional block cycler PCR analysis. The sensitivities and specificities of the LC-PCR assays were each 100% for the stx(1), eae, and E-hly genes and 96 and 100%, respectively, for the stx(2) gene. No stx(2) genes were detected from 10 stx(2f)-positive isolates because of significant nucleotide differences in their primer annealing regions. Melting curve analyses of the amplified Shiga toxin genes revealed sequence variation within each of the tested genes that correlated with described and novel gene variants. The performance characteristics of the LC-PCR assays, such as their speed, detection method, and the potential subtyping information available from melting curve analyses, make them attractive alternatives to block cycler PCR assays for detecting and characterizing STEC strains.     

Diversity of virulence patterns among shiga toxin-producing Escherichia coli from human clinical cases-need for more detailed diagnostics

Intestinal infections due to shiga toxin-producing Escherichia coli bacteria (STEC) reveal a broad range of clinical symptoms and a large scale of virulence properties of the respective pathogens. The question whether all STEC variants or only a particular group of them need to be considered for clinical and epidemiological purposes was answered throughout this study. Using the PCR technique for the identification of 25 different virulence-associated genes, 266 E. coli strains belonging to 81 different E. coli serotypes from various clinical origins were investigated. A great genetic diversity of the virulence properties and a broad range of virulence marker combinations have been identified. However, distinct virulence marker combinations (e.g. Stx2/LEE/pO157 as well as Stx2dac/pO113) were found to be associated with the same notified clinical symptoms (e.g. HUS). Such an association speaks either for the "shiga toxin-only concept" or for several redundant, but clinically or epidemiologically important virulence properties.     

Isogenic lysogens of diverse shiga toxin 2-encoding bacteriophages produce markedly different amounts of shiga toxin

We produced isogenic Escherichia coli K-12 lysogens of seven different Shiga toxin 2 (Stx2)-encoding bacteriophages derived from clinical Shiga toxin-producing E. coli (STEC) isolates of serotypes O157:H7, O145, O111, and O83 to assess the variability among these phages and determine if there were phage-related differences in toxin production. Phage genomic restriction fragment length polymorphisms (RFLP) and superinfection resistance studies revealed significant differences among these phages and allowed the seven phages to be placed into five distinct groups. Experiments revealed striking differences in spontaneous phage and toxin production that were correlated with the groupings derived from the RFLP and resistance studies. These results suggest that the genotype of the Stx2 prophage can influence the level of phage release and toxin expression by host strains and thus may be relevant to STEC pathogenesis.     

Prevalence and clinical manifestations of shiga toxin-producingEscherichia coli infections in Austrian children

The prevalence and clinical manifestations of infections associated with Shiga toxin-producingEscherichia coli (STEC) among Austrian children were assessed. Stool samples from 280 pediatric patients were analyzed by enzyme immunoassay (EIA) for the presence of free fecal Shiga toxin (Stx) 1 and 2, and by culture on sorbitol MacConkey agar. Specimens testing positive by the EIA were subjected to a cytotoxicity assay, polymerase chain reaction analysis, and a colony hybridization test. Direct culture on MacConkey agar demonstrated the presence of threeEscherichia coli O157:H7-positive stools. These were also positive by EIA and by the DNA-based methods. An additional six samples were positive by EIA, and in four of these, non-O157 STEC of serotypes O111H^–, O146: H^–, and O113:H53 could be isolated. Analysis of stools for a variety of enteric pathogens demonstrated that STEC was the third most common bacterial pathogen. The clinical manifestations of STEC infections were difficult to distinguish from those of infections caused by other enteric pathogens, as most patients presented with watery diarrhea. The median age of children with STEC infections was 27.6 months (range, 7 months to 5.75 years); children withSalmonella orCampylobacter infections were younger on average, while those withRotavirus infections were older. This study demonstrated that althoughEscherichia coli O157:H7 could be identified with the same sensitivity by both EIA or agar-based methods, the identification of non-O157 STEC strains was enhanced by the use of EIA followed by colony hybridization. Analysis of overnight cultures from 53 STEC isolates revealed that all strains producing Stx1, Stx2, or Stx2c reacted in the EIA. However, culture supernatants from Stx2e-producingEscherichia coli O101 were negative in the EIA. Despite this disadvantage, the EIA is easy to perform and time efficient and can be recommended as a screening test for non-O157 STEC in children with diarrhea.     

Development of a PCR-restriction fragment length polymorphism assay for the epidemiological analysis of Shiga toxin-producing Escherichia coli

Six characteristic regions (I to VI) were identified in Shiga toxin 2 (Stx2) phages (T. Sato, T. Shimizu, M. Watarai, M. Kobayashi, S. Kano, T. Hamabata, Y. Takeda, and S. Yamasaki, Gene 309:35-48, 2003). Region V, which is ca. 10 kb in size and is located in the upstream region of the Stx operons, includes the most distinctive region among six Stx phages whose genome sequences have been determined. In this study, we developed a PCR-restriction fragment length polymorphism (RFLP) assay for the epidemiological analysis of Shiga toxin-producing Escherichia coli (STEC) on the basis of the diversity of region V. When region V was amplified by long and accurate-PCR (LA-PCR) with five control E. coli strains carrying six different Stx phages such as E. coli strains C600 (Stx1 phage), C600 (933W phage), C600 (Stx2 phage-I), C600 (Stx2 phage-II), and O157:H7 Sakai strain RIMD0509952 (VT1-Sakai phage and VT2-Sakai phage), an expected size of the band was obtained. Restriction digest of each PCR product with BglI or EcoRV also gave the expected sizes of banding patterns and discriminated the RFLPs of five control strains. When a total of 204 STEC O157 strains were analyzed by LA-PCR, one to three bands whose sizes ranged from 8.2 to 14 kb were obtained. Two STEC O157 strains, however, did not produce any bands. Subsequent restriction digest of the PCR products with BglI or EcoRV differentiated the RFLPs of 202 STEC O157 strains into 24 groups. The RFLP patterns of pulsed-field gel electrophoresis (PFGE) of representative strains of STEC O157 divided into 24 groups were well correlated with those of PCR-RFLP when STEC O157 strains were isolated in the same time period and in the close geographic area. To evaluate the PCR-RFLP assay developed here, ten strains, each isolated from four different outbreaks in different areas in Japan (Tochigi, Hyogo, Aichi, and Fukuoka prefecture), were examined to determine whether the strains in each group showed the same RFLP patterns in the PCR-RFLP assay. In accordance with the results of PFGE except for strains isolated in an area (Fukuoka), which did not produce any amplicon, ten strains in each group demonstrated the same RFLP pattern. Taken together, these data suggest that the PCR-RFLP based on region V is as useful as PFGE but perhaps more simple and rapid than PFGE for the molecular epidemiological analysis of STEC strains during sporadic and common source outbreaks.     

Subtyping method for Escherichia coli shiga toxin (verocytotoxin) 2 variants and correlations to clinical manifestations

Shiga toxin 2 (Stx2) from Shiga toxin-producing Escherichia coli (STEC) was subtyped by a method involving partial sequencing of the stxAB2 operon. Of 255 strains from the Danish STEC cohort, all 20 cases of hemolytic-uremic syndrome were associated with subtype Stx2 (11 cases), subtype Stx2c (1 case), or the two combined (8 cases).     

Isolation of a lysogenic bacteriophage carrying the stx(1(OX3)) gene, which is closely associated with Shiga toxin-producing Escherichia coli strains from sheep and humans.

A specific PCR for the detection of a variant of the gene encoding Shiga toxin 1 (stx(1)) called stx(1(OX3)) (GenBank accession no. Z36901) was developed. The PCR was used to investigate 148 Stx(1)-producing Escherichia coli strains from human patients (n = 72), cattle (n = 27), sheep (n = 48), and a goat (n = 1) for the presence of the stx(1(OX3)) gene. The stx(1(OX3)) gene was present in 38 Shiga toxin-producing E. coli (STEC) strains from sheep belonging to serogroups O5, O125, O128, O146, and OX3 but was absent from Stx(1)-positive ovine STEC O91 strains. The stx(1(OX3)) gene was also detected in 22 STEC strains from humans with nonbloody diarrhea and from asymptomatic excreters. Serotypes O146:H21 and O128:H2 were most frequently associated with stx(1(OX3))-carrying STEC from sheep and humans. In contrast, Stx(1)-producing STEC strains from cattle and goats and 50 STEC strains from humans were all negative for the stx(1(OX3)) gene. The stx(1(OX3))-negative strains belonged to 13 serotypes which were different from those of the stx(1(OX3))-positive STEC strains. Moreover, the stx(1(OX3)) gene was not associated with STEC belonging to enterohemorrhagic E. coli (EHEC) serogroups O26, O103, O111, O118, O145, and O157. A bacteriophage carrying the stx(1(OX3)) gene (phage 6220) was isolated from a human STEC O146:H21 strain. The phage was able to lysogenize laboratory E. coli K-12 strain C600. Phage 6220 shared a similar morphology and a high degree of DNA homology with Stx(2)-encoding phage 933W, which originates from EHEC O157. In contrast, few similarities were found between phage 6220 and Stx(1)-encoding bacteriophage H-19B from EHEC O26.     

Comparison of LightCycler-based PCR,COBAS amplicor CMV monitor,and pp65 antigenemia assays for quantitative measurement of cytomegalovirus viral load in peripheral blood specimens from patients after solid organ transplantation

In order to evaluate the LightCycler-based PCR (LC-PCR) as a diagnostic assay technique, a classical pp65 antigenemia assay and the commercially available COBAS Amplicor CMV Monitor (CACM) assay were compared to the LC-PCR assay for the detection and quantitation of cytomegalovirus (CMV) load in 404 parallel specimens of peripheral blood from 66 patients after solid organ transplantation. A good correlation existed among these three assays (r congruent with 0.6, P < 0.0001). The LC-PCR assay was the most sensitive (54% of specimens positive) compared to the CACM (48.6%) and the pp65 antigenemia (26%) assays. The LC-PCR assay detected all samples found positive by using both the CMV pp65 antigenemia assay and the CACM assay. The LC-PCR also had the widest dynamic range (from 250 to 10(7) DNA copies/ml of plasma). No cross-reactions were found among CMV and Epstein-Barr virus, varicella-zoster virus, or herpes simplex virus in the LC-PCR by using amplification with specifically designed primer pairs. Precision, expressed as the coefficient of variation, was <3% with standard DNA from cell cultures and between 6.55 and 14.1% with clinical specimens in repeat LC-PCR runs. One run of the LC-PCR took half of the time required for the semiautomated CACM procedure. Because of its sensitivity, specificity, cost-effectiveness, and simplicity, the LC-PCR assay could replace the pp65 antigenemia and the CACM assays as the preferred technique for the surveillance, diagnosis, and monitoring of response of CMV diseases in high-risk populations.     

Use of ramification amplification assay for detection of Escherichia coli O157:H7 and other E. coli Shiga toxin-producing strains

Escherichia coli O157:H7 and other Shiga toxin-producing E. coli (STEC) strains are important human pathogens that are mainly transmitted through the food chain. These pathogens have a low infectious dose and may cause life-threatening illnesses. However, detection of this microorganism in contaminated food or a patient's stool specimens presents a diagnostic challenge because of the low copy number in the sample. Often, a more sensitive nucleic acid amplification method, such as PCR, is required for rapid detection of this microorganism. Ramification amplification (RAM) is a recently introduced isothermal DNA amplification technique that utilizes a circular probe for target detection and achieves exponential amplification through the mechanism of primer extension, strand displacement, and ramification. In this study, we synthesized a circular probe specific for the Shiga toxin 2 gene (stx(2)). Our results showed that as few as 10 copies of stx(2) could be detected, indicating that the RAM assay was as sensitive as conventional PCR. We further tested 33 isolates of E coli O157:H7, STEC, Shigella dysenteriae, and nonpathogenic E. coli by RAM assay. Results showed that all 27 STEC isolates containing the stx(2) gene were identified by RAM assay, while S. dysenteriae and nonpathogenic E. coli isolates were undetected. The RAM results were 100% in concordance with those of PCR. Because of its simplicity and isothermal amplification, the RAM assay could be a useful method for detecting STEC in food and human specimens.     

Antibody response to Shiga toxins Stx2 and Stx1 in children with enteropathic hemolytic-uremic syndrome

A Western blot (immunoblot) assay (WBA) for the detection of immunoglobulin G antibodies to Shiga toxins Stx2 and Stx1 in sera from 110 patients with enteropathic hemolytic-uremic syndrome (53 culture confirmed to have Shiga toxin-producing Escherichia coli [STEC] infection) and 110 age-matched controls was established by using a chemiluminescence detection system. Thirty-nine (74%) of the 53 culture-confirmed cases were infections with STEC serotype O157, and 14 (26%) were associated with infection by other STEC serotypes. The frequency of an anti-Stx2 response following infection by a Stx2-producing strain (34 of 48 cases; 71%) was higher than that of an anti-Stx1 response following Stx1-producing STEC infection (4 of 10). Furthermore, the frequency of an anti-Stx2 response in 110 control sera (10%) was significantly higher than the frequency of an anti-Stx1 response (1.8%) (P = 0.0325). For STEC O157 culture-confirmed cases WBA for toxin detection had a diagnostic sensitivity of 71% and a specificity of 90%. Because of its high specificity the assay might be a helpful tool for diagnosing suspected STEC infection when tests of stool samples or serological tests against various lipopolysaccharide antigens are negative. Furthermore, the prevalence of anti-Stx antibodies in healthy controls probably reflects the population immunity to systemic Stx-associated disease. It can thus serve as a basis for comparing immunity levels in different populations and for considering future Stx toxoid immunization strategies.     

Rapid detection of Shiga toxin-producing bacteria in feces by multiplex PCR with molecular beacons on the smart cycler

We have developed a rapid (1-h) real-time fluorescence-based PCR assay with the Smart Cycler thermal cycler (Cepheid, Sunnyvale, Calif.) for the detection of Shiga toxin-producing Escherichia coli (STEC), as well as other Shiga toxin-producing bacteria. Based on multiple-sequence alignments, we have designed two pairs of PCR primers that efficiently amplify all variants of the Shiga toxin genes stx(1) and stx(2), respectively. These primer pairs were combined for use in a multiplex assay. Two molecular beacons bearing different fluorophores were used as internal probes specific for each amplicon. Assays performed with purified genomic DNA from a variety of STEC strains (n = 23) from diverse geographic locations showed analytical sensitivities of about 10 genome copies per PCR. Non-STEC strains (n = 20) were also tested, and no amplification was observed. The PCR results correlated perfectly with the phenotypic characterization of toxin production in both STEC and non-STEC strains, thereby confirming the specificity of the assay. The assay was validated by testing 38 fecal samples obtained from 27 patients. Of these samples, 26 were PCR positive for stx(1) and/or stx(2). Compared with the culture results, both the sensitivity and the negative predictive value were 100%. The specificity was 92%, and the positive predictive value was 96%. Moreover, this assay detected STEC from a sample in which the STEC concentration was at the limit of detection of the conventional culture methods and from a sample in which STEC was not detected by the conventional culture methods. This real-time PCR assay is simple, rapid, sensitive, and specific and allows detection of all Shiga toxin-producing bacteria directly from fecal samples, irrespective of their serotypes.