Real-Time Multiplex PCR for Detecting Shiga Toxin 2-Producing Escherichia coli O104:H4 in Human Stools

Zhang, Wenlan; Bielaszewska, Martina; Bauwens, Andreas; Fruth, Angelika; Mellmann, Alexander; Karch, Helge

doi:10.1128/jcm.06817-11

Cited by 21 publications

(20 citation statements)

References 16 publications

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“…A limitation of the two pooled NAAT strategies was that the detection threshold was greater with pooling than with individual specimen testing. However, the LoD of the postenrichment pooled NAAT is comparable to the LoD of other described inhouse and commercial PCRs for STEC detection (21)(22)(23). To overcome any potential loss of detection, it may be possible to make method improvements, such as by concentrating enriched pools with centrifugation prior to lysis.…”

Section: Discussionsupporting

confidence: 60%

Pooled Nucleic Acid Amplification Test for Screening of Stool Specimens for Shiga Toxin-Producing Escherichia coli

et al. 2016

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c Shiga toxin-producing Escherichia coli (STEC)-associated enteric illness is attributed to O157 and non-O157 serotypes; however, traditional culture-based methods underdetect non-O157 STEC. Labor and cost of consumables are major barriers to implementation of the CDC recommendation to test all stools for both O157 and non-O157 serotypes. We evaluated the feasibility of a pooled nucleic acid amplification test (NAAT) as an approach for screening stool specimens for STEC. For retrospective evaluation, 300 stool specimens were used to create pools of 10 samples each. The sensitivity was 83% for the preenrichment pooling strategy and 100% for the postenrichment pooling strategy compared with those for individual NAAT results. The difference in cycle threshold (C T ) between individual and pooled NAAT results for specimens was significantly lower and more consistent for postenrichment pooling (stx 1 mean ‫؍‬ 3.90, stx 2 mean ‫؍‬ 4.28) than those for preenrichment pooling (excluding undetected specimens; stx 1 mean ‫؍‬ 9.34, stx 2 mean ‫؍‬ 8.96) (P < 0.0013). Cost of consumables and labor time savings of 48 to 81% and 6 to 66%, respectively, were estimated for the testing of 90 specimens by the postenrichment pooled NAAT strategy on the basis of an expected 1 to 2% positivity rate. A 30-day prospective head-to-head clinical trial involving 512 specimens confirmed the sensitivity and labor savings associated with the postenrichment pooled NAAT strategy. The postenrichment pooled NAAT strategy described here is suitable for efficient large-scale surveillance of all STEC serotypes. Comprehensive detection of STEC will result in accurate estimation of STEC burden and, consequently, appropriate public health interventions. Shiga toxin-producing Escherichia coli (STEC) is a major cause of sporadic and outbreak-associated enteric illness worldwide. While more than 100 STEC serotypes can cause illness in humans (1), traditional testing methods focus primarily on the most common serotype, O157:H7, owing to the ease of its detection using culture-based media. These methods underdetect non-O157 serogroups, and as such, their clinical burden is not well understood (2-4). Moreover, recent evidence suggests that infections attributed to non-O157 STEC may be more prevalent than those attributed to O157 (1, 5), with common serogroups being O26, O45, O103, O111, O121, and O145 in the United States (6, 7) and in other countries (1). Although non-O157 STEC serotypes are generally associated with milder disease than O157 STEC, infections caused by non-O157 STEC can also lead to hemolytic uremic syndrome (1) and have been associated with major outbreaks, most notably a 2011 outbreak in Germany that involved 3,816 cases and 54 deaths (8). Enhanced surveillance evidence is needed to determine the true burden of non-O157 STEC for public health investigations of potential exposure sources. Such efforts are ongoing in Canada (9) but require improved and comprehensive screening methods.The Centers for Disease Control and Prevention (CDC) recom...

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Section: Discussionsupporting

confidence: 60%

Pooled Nucleic Acid Amplification Test for Screening of Stool Specimens for Shiga Toxin-Producing Escherichia coli

et al. 2016

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“…The C T values of 9 replicates indicated that the assay was highly reproducible. Multiplex PCRs targeting the major virulence genes (stx 2 , aggR, and aggA) of these strains were used in association with those associated with the somatic (rfb O104 and wzx O104 ) and H4 flagellar (fliC H4 ) antigens (4,12,21,26). In this study, the PCR assays targeting the O104 antigen sequences tested positive for all E. coli O104 strains having H types other than H4 and showed crossreactivity with E. coli strains carrying the K9 capsular antigen and thus were not specific to the O104:H4 strains.…”

Section: Resultsmentioning

confidence: 99%

“…The used PCR assays (4,12,21,26) combine multiple pairs of primers targeting, for example, genes encoding Shiga toxin 2 (stx 2 ), O104 (rfb O104 ) and H4 (fliC H4 ) antigens, tellurite resistance (terD), and AggR (aggR), which is the master regulator of EAEC plasmid, as well as chromosomally inherited virulence genes (18). However, none of these gene targets was unique to the O104:H4 outbreak strain.…”

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Specific Detection of Enteroaggregative Hemorrhagic Escherichia coli O104:H4 Strains by Use of the CRISPR Locus as a Target for a Diagnostic Real-Time PCR

et al. 2012

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In 2011, a large outbreak of an unusual bacterial strain occurred in Europe. This strain was characterized as a hybrid of an enteroaggregative Escherichia coli (EAEC) and a Shiga toxin-producing E. coli (STEC) strain of the serotype O104:H4. Here, we present a single PCR targeting the clustered regularly interspaced short palindromic repeats locus of E. coli O104:H4 (CRISPR O104:H4 ) for specific detection of EAEC STEC O104:H4 strains from different geographical locations and time periods. The specificity of the CRISPR O104:H4 PCR was investigated using 1,321 E. coli strains, including reference strains for E. coli O serogroups O1 to O186 and flagellar (H) types H1 to H56. The assay was compared for specificity using PCR assays targeting different O104 antigen-encoding genes (wbwC O104 , wzx O104 , and wzy O104 ). The PCR assays reacted with all types of E. coli O104 strains (O104:H2, O104:H4, O104:H7, and O104:H21) and with E. coli O8 and O9 strains carrying the K9 capsular antigen and were therefore not specific for detection of the EAEC STEC O104:H4 type. A single PCR developed for the CRISPR O104:H4 target was sufficient for specific identification and detection of the 48 tested EAEC STEC O104:H4 strains. The 35 E. coli O104 strains expressing H types other than H4 as well as 8 E. coli strains carrying a K9 capsular antigen tested all negative for the CRISPR O104:H4 locus. Only 12 (0.94%) of the 1,273 non-O104:H4 E. coli strains (serotypes Ont:H2, O43:H2, O141:H2, and O174: H2) reacted positive in the CRISPR O104:H4 PCR (99.06% specificity).

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“…The number of strains and the diversity of serotypes and pathogroups that were investigated in this study provide a solid base for a future utilization of the CRISPR real-time PCR tests not only on pure bacterial isolates but also on food and stool samples. These assays would work potentially with DNA extracted from enriched food (19) or stool samples (26) suspected to be positive. This needs to be confirmed with further evaluation of the CRISPR PCR tests on spiked and naturally contaminated samples.…”

Section: Discussionmentioning

confidence: 99%

Use of Clustered Regularly Interspaced Short Palindromic Repeat Sequence Polymorphisms for Specific Detection of Enterohemorrhagic Escherichia coli Strains of Serotypes O26:H11, O45:H2, O103:H2, O111:H8, O121:H19, O145:H28, and O157:H7 by Real-Time PCR

2012

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bWe explored the genetic diversity of the clustered regularly interspaced short palindromic repeat (CRISPR) regions of enterohemorrhagic Escherichia coli (EHEC) to design simplex real-time PCR assays for each of the seven most important EHEC serotypes worldwide. A panel of 958 E. coli strains investigated for their CRISPR loci by high-throughput real-time PCR showed that CRISPR polymorphisms in E. coli strongly correlated with both O:H serotypes and the presence of EHEC virulence factors (stx and eae genes). The CRISPR sequences chosen for simplex real-time PCR amplification of EHEC strains belonging to the top 7 EHEC serogroups differentiated clearly between EHEC and non-EHEC strains. Specificity estimates for the CRISPR PCR assays varied from 97.5% to 100%. Sensitivity estimates for the assays ranged from 95.7% to 100%. The assays targeting EHEC O145: H28, O103:H2, and O45:H2 displayed 100% sensitivity. The combined usage of two simplex PCR assays targeting different sequences of the O26 CRISPR locus allowed detection of EHEC O26:H11 with 100% sensitivity. By combining two simplex PCR assays targeting different sequences of the EHEC O157 CRISPR locus, EHEC O157:H7 was detected with 99.56% sensitivity. EHEC O111:H8 and EHEC O121:H19 were detected with 95.9% and 95.7% sensitivity, respectively. This study demonstrates that the identification of EHEC serotype-specific CRISPR sequences is more specific than the mere identification of O-antigen gene sequences, as is used in current PCR protocols for detection of EHEC strains. Shiga toxin-producing Escherichia coli (STEC) strains are a diverse group of E. coli strains belonging to over 400 E. coli O:H serotypes, some of which cause outbreaks and sporadic cases of food-borne illnesses ranging from diarrhea to hemorrhagic colitis (HC) and the hemolytic-uremic syndrome (HUS) (11,15,16). According to their pathogenicity for humans, the latter strains were also designated enterohemorrhagic E. coli (EHEC) (17, 18). Numerous cases of HC and HUS have been attributed to EHEC O157:H7 strains (25), but it has now been recognized that other STEC serotypes belong to the EHEC group (8, 24). Cumulative evidence from numerous countries indicates that up to 30 to 60% of human EHEC infections are caused by non-O157 EHEC strains (7). There are seven "priority" EHEC serotypes most frequently implicated in outbreaks and sporadic cases of HC and HUS (8, 24). These comprise serotypes O26:H11, O45:H2, O103:H2, O111:H8, O121:H19, O145:H28, and O157:H7 and their nonmotile derivatives.Although regulations are disparate throughout the world, many food inspection programs aim at detecting STEC strains that pose a significant threat to human health in foods that are the most likely to disseminate EHEC and to be consumed raw or undercooked. Some beef products are thus of particular interest in that aspect. The U.S. regulations have precisely been revised to add 6 additional serogroups (O26, O103, O45, O111, O121, and O145) to the existing O157:H7 regulation (22,23). This regulation imposes testin...

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Real-Time Multiplex PCR for Detecting Shiga Toxin 2-Producing Escherichia coli O104:H4 in Human Stools

Cited by 21 publications

References 16 publications

Pooled Nucleic Acid Amplification Test for Screening of Stool Specimens for Shiga Toxin-Producing Escherichia coli

Pooled Nucleic Acid Amplification Test for Screening of Stool Specimens for Shiga Toxin-Producing Escherichia coli

Specific Detection of Enteroaggregative Hemorrhagic Escherichia coli O104:H4 Strains by Use of the CRISPR Locus as a Target for a Diagnostic Real-Time PCR

Use of Clustered Regularly Interspaced Short Palindromic Repeat Sequence Polymorphisms for Specific Detection of Enterohemorrhagic Escherichia coli Strains of Serotypes O26:H11, O45:H2, O103:H2, O111:H8, O121:H19, O145:H28, and O157:H7 by Real-Time PCR

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