Shiga toxin-producing Escherichia coli organisms (STEC) are pathogens that have ability of producing sporadic and epidemic diarrhea, hemorrhagic colitis, bloody diarrhoea (BD), non-bloody diarrhoea (NBD), and potentially life threatening hemolytic-uremic syndrome (HUS) in the United States. Non-O157 STEC serotypes can also cause serious illness, but their impact as pathogens remains unknown (Leotta et al., 2008; Nagy et al., 2008; Hedican et al., 2009). Shiga-like toxin (SLT)-producing E. coli has been associated with a spectrum of human illnesses, including hemorrhagic colitis and hemolytic uremic syndrome. Shiga-like toxins produced by E. coli include Shiga-like toxin I (SLT-I) and Shiga-like toxin II (SLT-II), which also known as verotoxin (Perera et al., 1988; Downes et al., 1989). Weeratna and Doyle (1991) say that verotoxin 1 (VT1) is a virulence factor of E. coli O157:H7, which causes severe food-borne disease.
2- Pathogenesis of STEC O157
People infected with Shiga toxin (Stx) producing E. coli (STEC) O157 strains present different immunological and physicochemical characteristics due to differences in stx genes carried by the strains; these are stx2vha alone, stx1 with stx2, stx1 with stx2vha, stx2 alone, and stx2 with stx2vha; however, no strain carry the stx1 gene alone. STEC strains carrying stx2 are more frequently associated with clinical manifestations of hemolytic-uremic syndrome (HUS) or bloody diarrhea than those carrying stx2vha (Kawano et al., 2008; Tanabe et al., 2010). Hedican et al. (2009) argued that differences in severity among STEC infections could not be explained by stx2, suggesting that additional factors are important in STEC virulence. Hara-Kudo et al. (2000) observed that if the organism put under starvation conditions for a long period, the immunologic methods based on O157 O antigenicity are unable to detect and isolate verotoxin (VT)-producing E. coli in foods and other environments.
3- Characterization of Shiga toxin producing E. coli O157 and the related Shiga-like toxins (SLT)-producing E. coli
Murinda et al (2004) suggested that isolates of the same serotypes of E. coli obtained from different sources and possessed the same marker profiles, could be cross-species transmissible. Therefore, they studied the characterization of E. coli isolates from dairy calves, cows/feedlots, mastitis, pigs, dogs, parrot, iguana, human disease, and food products for prevalence of Shiga toxin-producing E. coli (STEC) virulence markers. Four hundred E. coli isolates were tested to detect presence of genes encoding Shiga toxin 1 and 2 (stx1 and stx2), H7 flagella (flicC), enterohemolysin (hly) and intimin (eaeA) using multiplex polymerase chain reaction (PCR). They found that multiplex PCR was an effective tool for characterizing STEC pathogenic profiles and distinguished STEC O157:H7 from other STEC. They also reported that isolates from human and cattle disease shared similar toxigenic profiles, whereas isolates from other disease sources had few characteristics in common with the former isolates. They concluded that these data suggest interspecies transmissibility of certain serotypes, in particular, STEC O157:H7, between cattle and humans.
Mora et al. (2004) run an epidemiological subtyping of a collection of Shiga toxin-producing E. coli (STEC) O157:H7 strains isolated in Spain between 1980 and 1999. They used DNA macrorestriction fragment analysis by pulsed-field electrophoresis (PFGE) and phage typing. They reported that PT8 and PT2 were the most frequently found among strains from both humans (51%) and bovines (46%). Moreover, they detected a significant association between PT2 and PT14 and the presence of acute pathologies. They concluded that cattle are a main source of strains pathogenic for humans. PT8 and PT2 strains formed two groups which differed from each other in their motilities, stx genotypes, PFGE patterns, and the severity of the illnesses that they caused. Khan et al. (2002) investigated the prevalence of Shiga toxin-producing E. coli (STEC) in beef that were collected from the city's abattoir, as well as healthy domestic cattle and hospitalized diarrhea patients, Calcutta, India. They used multiplex polymerase chain reaction using primers specific for stx1 and stx2 detected STEC. They found that among various virulence genes in the STEC isolates, stx1 allele is the most detected gene. They also evaluated the cytotoxic effect of the Shiga toxins produced by the strains by performing bead enzyme-linked immunosorbent assay and Vero cell assay. They argued and concluded that STEC is not a causative factor of diarrhea in India; however, its presence in domestic cattle and beef samples suggests that this enteropathogen may become a major public health problem in the future. Leotta et al. (2008) compared the phenotypic and genotypic characteristics of STEC O157 strains, which were detected between 1993 and 1996, from humans in Australia, New Zealand and Argentina. They found that STEC O157 strains isolated in Australia, New Zealand and Argentina differed from each other in terms of stx-genotype and phage type. Moreover, no common PFGE patterns were reported for the isolated strains in the three countries. International collaborative studies of the type reported here are needed to detect and monitor potentially hypervirulent STEC clones. Koitabashi et al. (2006) argued that E. coli O157: H7strains, which are stx(2)-positive and produce little or no Stx2, may be widely distributed in the Asian environment.
4- Detection of Shiga toxin-producing E. coli (STEC) and the related Shiga-like toxins (SLT)-producing E. coli
To best detect infected patients and potential outbreaks, clinical laboratories must have methods to accurately and quickly detect STEC in stool specimens (Grys et al., 2009). However, the main problem in large-scale epidemiological investigations of the incidence of Shiga toxin and Shiga-like toxins producing E. coli in diarrheal stools is the lack of a sensitive specific and fast test to detect toxin (Perera et al., 1988). Shiga toxin and the closely related Shiga-like toxins produced by E. coli represent a group of very similar cytotoxins that may play an important role in diarrheal disease and hemolytic uremic syndrome. Moreover, they possess similar biological activities and also share the same binding receptor, globotriosyl ceramide (Gb3) (Ashkenazi & Cleary, 1989). Shelton et al. (2008) believed that infections caused by Shiga toxin-producing Escherichia coli (STEC) are undiagnosed, particularly non-O157 STEC. They evaluated the use of a multiple protocol approach to improve isolation, diagnosis, and characterization of STEC strains. They concluded that a multiple protocol approach is necessary to reliably diagnose and isolate STEC strains, and that PCR profiling of strains could allow for more rapid identification of outbreaks.
4:1- Culture Methods
Culture confirmation of Shiga toxin-producing Escherichia coli (STEC) is very important for epidemiologic analysis (Hu et al., 2009). Sugiyama et al. (2001); El Sayed Zaki and El-Adrosy (2007) and Hu et al. (2009) reported that isolation and differentiation of the Shiga toxin (Stx)-producing strains of E. coli (STEC) serovar O157:H7 can be confirmed easily by their late fermentation of sorbitol and lack of β-glucuronidase activity; however, detection of non-O157 STEC strains by culture method can't be done due to their biochemical diversity and because of heavy growth of competing bacteria and its phenotypical similarity to commensal nonpathogenic E. coli. Sugiyama et al. (2001) say that many STEC strains, except Stx, produce enterohaemolysin (Ehly) regardless of their serovars; therefore, washed sheep blood agar containing Ca2+ ions (WBA) can be used to detect non-O157 STEC strains, but some of non-O157 STEC strains failed to produce Ehly on this medium. Sugiyama et al. (2001) added mitomycin C to WBA (WBMA) in expectation of the same effect on Ehly production and found a marked increase in the number of Ehly-positive strains. They used 185 serovars and 17 α-haemolytic E. coli isolated from urinary tract infections. They used three types of washed blood agar media. These are: (1) WBA, which was prepared from 33 g of tryptose blood agar base (Difco) supplemented with 5 g of tryptose (Difco), 10 mM of CaCl2 and 5% defibrinated sheep blood washed three times in phosphate-buffered saline, pH 7·2, (2) WBVCCA that was prepared by addition of 30 mg of vancomycin hydrochloride (Sigma), 20 μg of cefixime (Fujisawa) and 3 mg of cefsulodin (Sigma) to WBA and (3) WBMA that was made by the addition of 0·5 μg ml-1 of mitomycin C to WBA. In addition to these three media, ordinary sheep blood agar (BA) was also used as a control. They also included 10 faecal samples from healthy people to compare the selectivity of WBMA and WBVCCA media. The results showed that WBAM markedly enhanced Ehly activity of non-O157 STEC 96.7% compared to 77.7% on WBA and 71.1% on WBAVCCA. Only three of the non-O157 STEC strains produced haemolysis on BA. There was no difference observed between growth of faecal flora from the 10 healthy people on both WBMA and WBVCCA media. Six STEC strains that did not produce haemolysis on WBMA were tested for Stx production by the VT-RPLA and all produced either Stx I or Stx II or both. All the 17 α-haemolysin-producing strains of E. coli showed strong haemolysis on all five media. There were different appearances of the haemolytic zones produced by STEC strains compared with that of the 17 α-haemolytic strains of E. coli. STEC strains formed small, umbilicated colonies surrounded by characteristic narrow, turbid zones of haemolysis after incubation for 18-24 h at 37 °C. In contrast, the α-haemolytic strains formed low-convex colonies surrounded by clear zones visible after 3-8 h incubation. All, except the three of the STEC strains, failed to produce haemolysis on BA, whereas α-haemolysin-producing strains lysed erythrocytes on BA. However, those three STEC strains produced narrow clear zones of haemolysis visible after 20 h incubation on WBA as well as on BA. Sugiyama et al. (2001) suggested that the original WBA may be insufficient to detect Ehly production in non-O157 STEC strains. They concluded that enterohaemolysin (Ehly) production can be used as a tool for the detection and presumptive identification considerably, not only of STEC O157 but also of non-O157 STEC strains. Sugiyama et al. (2001) also concluded that differentiation between Ehly and α-haemolysin can be easily made by the appearance of the haemolytic zones on both enterohaemolysin and ordinary blood agar plates.
Hu et al. (2009) introduced an acid enrichment procedure to facilitate detection of STEC from patients who were symptomatic. They used both conventional and the acid enrichment methods for the isolation of STEC from 47 clinical fecal broths, which tested positive for Shiga toxin. The results showed that the culture method and acid enrichment method recovered STEC from 70% (33/47) and 91% (43/47) of the fecal broths, respectively. They reported that the overgrowth of competitor colonies on Rainbow agar plus tellurite and novobiocin (RTN) and Sorbitol MacConkey agar plus cefixime and tellurite (TCSMAC) were greatly reduced by the using of acid enrichment protocol. They concluded that incorporation of an acid enrichment procedure in clinical testing improved the isolation of STEC in fecal specimens.
4:2- Enzyme-linked immunosorbent assays (ELIZAs)
Ashkenazi and Cleary (1989) say that Shiga toxin and Shiga-like toxins are detected on the basis of their ability to damage several cell lines, by using expensive and tedious assays that require facilities for and experience with tissue cultures, which are more suitable for research laboratories. Ashkenazi and Cleary (1989) improved a rapid method to detect Shiga toxin and Shiga-like toxin I based on specific binding to their Gb3 natural receptor, which was coated onto microdilution plates. Bound toxin was then detected by enzyme-linked immunosorbent assay (ELISA) with monoclonal antibodies. They reported that the sensitivity of this method was 2 ng/ml and it was very specific in that no cross-reactivity was noted with purified toxin. Downes et al. (1989) developed two sandwich enzyme-linked immunosorbent assays (ELISAs) based on toxin-specific murine monoclonal capture antibodies and rabbit polyclonal second antibodies which are specific for SLT-I and SLT-II. The SLT-I ELISA and the SLT-II ELISA were sensitive and can detect as 200 pg of purified SLT-I, and 75 pg of purified SLT-II, respectively. However, they reported that the ELISAs were not sufficiently sensitive to detect low levels of toxin (less than 50 CD50 per ml) found in fecal extracts. From their findings, they concluded that both ELISAs appeared to detect significant levels of SLT-I (> 100 CD50 per ml) and SLT-II (> 50 CD50 per ml) in E. coli culture extracts and should be useful diagnostic tools in many microbiology laboratories. Law et al. (1992) believe that current techniques available to detect Shiga-like toxin (SLT)-producing E. coli lack sensitivity or require specialized equipment and facilities, and in some cases detect only strains belonging to serotype O157. Therefore, they used an ELISA technique, capable of detecting both SLTI and SLTII with crude P1 glycoprotein from hydatid cysts, in combination with enhancement of toxin production by culture with mitomycin C. They mixed E. coli O157:H7 in culture with other organisms and they found that SLTI could be detected when the proportion of toxigenic organisms represented 1% of the mixture, and SLTII when the proportion was 0.025%. They used the same technique to examine faecal samples with added E. coli O157:H7 and they reported that SLTII-producing strains were detected when they comprised less than 0.1% of the coliform population. They concluded that this technique is a sensitive and specific assay for detecting low numbers of SLT-producing bacteria in mixed growth such as occurs in cases of haemolytic uraemic syndrome and haemorrhagic colitis. In two years later, Law et al. (1994) compared the efficacy of two methods, (i) the culture of faeces on sorbitol MacConkey agar (SMA), in the detection of infections caused by SLT-producing E. coli, and (ii) the culture of faeces in broth that contained mitomycin C followed by enzyme-linked immunosorbent assay (ELISA) for SLTs. They isolated the SLT-producing E. coli O157 strains on SMA from 42 of 475 faecal samples; however, they detected SLTs by ELISA in culture supernates or lysates of 54 of 475 samples. Interestingly, they reported that SLT-producing E. coli of serogroups other than O157 were isolated in four cases. Again, they reported and concluded that the ELISA is a sensitive and rapid technique for the diagnosis of SLT-producing E. coli infection, especially where low numbers of the organism are present in faeces and when the infection is caused by a serogroup other than O157. Weeratna and Doyle (1991) modified a sensitive method for detection of VT1 in milk and in ground beef and to determine the conditions for VT1 production in these foods. They used a sandwich enzyme-linked immunosorbent assay (ELIZA). They developed VT1-specific monoclonal antibody 9C9F5 as the capture antibody, and a rabbit polyclonal antibody was developed against VT2 as the detection antibody for the detection and quantification of VT1 in milk and in ground beef. They found that ELIZA was sensitive to a minimum of 0.5 ng of VT1/ml of milk and 1.0 ng of VT1/g of ground beef and they also noted that VT1 production was greater in ground beef than in milk. They reported that ground beef is a better medium for VT1 production than milk.
4:3- Polymerase Chain Reaction (PCR) assays
Kai et al. (1999) used a flow injection-type sensor based on surface plasmon resonance to detect polymerase chain reaction (PCR) products. They also used asymmetric PCR to amplify the target DNA sequence, and two products with different length were produced. The significance of their DNA detection system was that the target DNA was double stranded but the probe binding site, located in the 3'-terminus, was single stranded. They concluded that the two products will avoid the formation of intra- and intermolecular complexes permitte not only to detect PCR product but also to develop a rapid detection system for the detection of the verotoxin 2 gene of E. coli O157:H7. In the following year, Kai et al. (2000) isolated and amplified strains possessing Shiga toxin-2 (stx-2) genes from stool samples. Stool samples from healthy carriers and patients were tested and showed a high correlation between positive results for a PCR and the presence of verotoxin-producing E. coli O157:H7, confirmed by isolation of serotype O157:H7 on sorbitol MacConkey medium (10/10 stool samples). They applied a BIAcore 2000 surface plasmon resonance device using peptide nucleic acid as a sensor probe to detect PCR products. They used this method for the rapid detection of DNA from significant pathogenic organisms. Sharma and Dean-Nystrom (2003) say that a multiplex real-time PCR (R-PCR) assay was designed and evaluated on the ABI 7700 sequence detection system (TaqMan) to detect enterohemorrhagic E. coli (EHEC) O157:H7 in pure cultures, feces, and tissues. They analyzed 67 bacterial strains using three sets of fluorogenic probes and primers for real-time detection and amplification of a 106-bp region of the eae gene encoding EHEC O157:H7-specific intimin, and 150-bp and 200-bp segments of genes stx1 and stx2 encoding Shiga toxins 1 and 2, respectively. The results showed that the R-PCR assay perfectly differentiated EHEC O157:H7 serotype from non-O157 serotypes and provided accurate profiling of genes encoding intimin and Shiga toxins. Moreover, bacterial strains lacking these genes were not detected with this assay. They concluded that the R-PCR assay for eae(O157:H7), stx1, and stx2 proved to be a quick method for detection of EHEC O157:H7 in complex biological matrices and could also potentially be used for quantification of EHEC O157:H7 in foods or fecal samples. Depending on the sequences of the rfb(E. coli O157) and stx2 genes, Hsu et al. (2005) designed two combinations of primers and fluorescent probes. They analyzed 217 bacterial strains using real-time PCR assays and the results showed that the duplex real-time PCR assay successfully distinguished the E. coli O157 serotype from non-E. coli O157 serotypes. Furthermore, this method gave an accurate means of profiling the genes encoding O antigen and Shiga-like toxin 2. They proved that the real-time PCR assays for rfb(E. coli) (O157) and stx2 can be rapid tests for the detection of E. coli O157 in food and can be used for the quantification of E. coli O157 in foods or fecal samples.
4:4- Other Advanced Detection Essays
Noller et al. (2003) introduced Multiple-Locus Variable-Number Tandem-Repeats Analysis (MLVA) method, which used as typing tool for Shiga-toxin-producing E. coli (STEC) O157 isolates. This method is performed by using a single fluorescent dye and the different patterns were assigned using a gel image. Lindstedt et al. (2004) significantly developed this method using multiple dye colours and improved PCR multiplexing to speed up, and ease the interpretation of the results. Lindstedt et al. (2004) used 72 strains of Shiga-toxin-producing E. coli O157 for the development of the improved MLVA assay. The essay is based on capillary separation of multiplexed PCR products of VNTR loci in the E. coli O157 genome labeled with multiple fluorescent dyes. The different alleles at each locus were then assigned to allele numbers, which were used for strain comparison. They found and reported that the different MLVA patterns are based on allele sizes entered as character values, thus removing the uncertainties introduced when analyzing band patterns from the gel image. Moreover, they proposed an easy numbering scheme for the identification of separate isolates that will facilitate exchange of typing data. In 2006 Hyytiä-Trees et al. did more improvements and modifications in MLVA scheme targeting 29 polymorphic VNTR regions of STEC O157. They included nine VNTR loci in the final protocol, which were amplified in three PCR reactions, after which the PCR products were sized using capillary electrophoresis.
Gavin et al. (2004) argued that STEC non-O157 serotypes cannot be detected in stool by sorbitol-MacConkey agar culture (SMAC). They 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. Rabbit polyclonal anti-Shiga toxin 1 and 2 capture antibodies and a horseradish peroxidase-labeled monoclonal mouse anti-Shiga toxin 1 and 2 conjugate were involved in ProSpecT assay. The results showed that twenty-nine stool specimens were STEC positive by ProSpecT assay. The ProSpecT assay was 100% specific and sensitive for detection of E. coli O157 in stool (7 of 7) compared to SMAC. Moreover, the ProSpecT assay detected doubles as many STEC as SMAC. They also noticed that fifty-two percent of confirmed STEC-positive stools were non-bloody; therefore, screening strategies that test only visibly bloody stools for STEC would miss a majority of cases. They concluded that the ProSpecT assay is highly 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. They also suggest that Shiga toxin assays should be considered for routine use in settings where high prevalence of STEC disease is present.
Nagy et al. (2008) say that Stx1 and Stx2 are both composed of one enzymatically active "A" subunit and five identical copies of a "B" subunit. The pentamer B can bind to the terminal Gal-α1,4-Gal disaccharides on the surface of host cells. They believed that this carbohydrate-toxin interaction can be applied to detect STEC. They mention that glycopolymers, glycodendrimers and glycol conjugated nanoparticles have been widely used as anti-adhesion molecules for toxins and bioprobes to control the interactions of carbohydrate-protein. Glyconanoparticles show the highest potential for the application of carbohydrate-protein interactions. Glycopolydiacetylene (GPDA) nanoparticles were produced by the polymerization of 1,3-diacetylenic acid derivatives. The unique "blue-to-red" colorimetric transition of GPDA nanoparticles bound with macromolecules has been utilized to monitor the ligand-receptor binding events for antibody-receptor interactions, bacteria, toxins, and viruses (Nagy et al., 2008). Therefore, Nagy et al. (2008) applied the GPDA nanoparticles to detect shiga toxin or related toxins. They used the GPDA nanoparticles with Gal-α1,4-Gal disaccharides on the surface to detect the Shiga toxins from E. coli O157 on 96-well plates. They found that the minimum detectable concentration of E. coli was 1200 unit/µL. as this method showed highly selective, quantitative, sensitive and fast responsive colorimetric biosensor to monitor the STEC, Nagy et al. (2008) reported that the GPDA nanoparticles can be use in routine checking and monitoring of STEC in food and water supplies and to environmental samples.
Based on the fact that the toxins bind cell-surface oligosaccharides, Uzawa (2009) describe new detection methods applying carbohydrate epitopes as the toxin ligands. The Shiga toxin has an affinity for globobiosyl (Gb(2)) disaccharide. Surface plasmon resonance (SPR) was applied to detect Shiga toxin. A polyanionic Gb(2)-glycopolymer was designed for this purpose, and it was used for the assembly of Gb(2)-chips using alternating layer-by-layer technology. He concluded that the method allowed todetect the toxin at a low concentration. He argued that the present approaches provide a highly effective way to counter bioterrorism.
Jyoti et al. (2010) argued that detection of EHEC using fluorogenic-substrate based culture media and/or nucleic acid amplification based Real-Time polymerase chain reaction assays are either time consuming or need expensive instrumentation. Jyoti et al. (2010) believed that stx2 gene representing EHEC can be detected by using gold nanoparticles (GNPs) of 20 +/- 0.2 nm, which were synthesised by citrate reduction method and characterised by spectroscopy and transmission electron microscopy. The GNPs were functionalised with 19 and 22 bp of thiolated single stranded DNA complementary to target highly conserved 149 bp region of stx2 gene. Transmission Electron Microscopy revealed the hybridization, aggregation and reduction in the interparticle distances of the GNP probes in the presence of target DNA. They reported that the aggregation and the spectral shift in the plasmon band observed with 10(6) copies of target DNA indicates feasibility of a simple and quick colorimetric 'spot and read' test in contrast to amplification based detection methods. Due to some limitations in sensitivity for the detection of Shiga toxin or Shiga-like toxin by the present conventional methods, including ELISA and PCR, Tanabe et al. (2010) used a ProteoChip, which requires smaller volumes of reagents. They reported that this technique allows detection of lower concentrations of the toxins, compared with the conventional assay.
4:5- Comparison between some of used detection methods
El Sayed Zaki and El-Adrosy (2007) aimed to assess verotoxin gene detection (VT1/VT2) within STEC PCR compared with the Vero cells cytotoxicity among O157 and non-O157 STEC serotypes. They cultured all stool samples on Tryptic Soy Broth and Sorbitol MacConkey agar with cefixitime and tellurite supplements which were identified as E. coli by BBL crystal. They also performed further identifications including verotoxin production assessment by Vero cells cytotoxicity assay, PCR for specific VT1/VT2 genotyping, and isolates were plated on blood agar and tested for enterohemolysis. The results showed that Vero cells cytotoxicity assay revealed that 58 of E. coli isolates (71.6%) were STEC. In PCR, 33 (56.9%) of the 58 strains were positive for the VT2 gene, 24 (41.4%) were positive for the VT1 gene and one isolate was positive for both genes. The sensitivity and specificity of PCR were 100% compared to Vero cells cytotoxicity. They concluded that enterohemolysin production can be used as surrogate marker for STEC. They also believed that molecular method is the most rapid and promising approach for detection of STEC.
Gilmour et al. (2009) say that the development of molecular reagents is a helpful tool for the identification of both toxin and serogroup-specific genetic determinants and can lead to a more comprehensive characterization of stool samples and isolation of STEC strains. They performed cytotoxicity assay, commercial immunoassay and conventional PCR targeting Shiga-toxin determinants to study and screen 876 stool samples for STEC from paediatric patients with gastroenteritis. They also included routine culture methods for isolating O157 STEC. The results showed that the screening assays detected 45 stools presumptively having STEC, and a total of 20 O157 and 22 non-O157 strains were isolated using non-differential culture techniques. They also isolated multiple STEC serotypes from two clinical stool samples. These data were compared to molecular serogroup profiles determined directly from the stool enrichment cultures using a LUX real-time PCR assay targeting the O157 fimbrial gene lpfA, a microsphere suspension array targeting allelic variants of espZ and a gnd-based molecular O-antigen serogrouping method. Gilmour et al. (2009) reported that the lpfA real-time PCR assay and espZ microsphere array may precisely predict the presence and provide preliminary typing for the STEC strains present in clinical samples. They concluded that this toolbox of molecular methods provided strong detection capabilities for STEC in clinical stool samples, including co-infection of multiple serogroups.
Grys et al. (2009) agreed that culture on sorbitol MacConkey agar is an inexpensive, effective, and widely used method based on lack of sorbitol fermentation by E. coli O157:H7; however, they argued that there are some limitations that limit the use of culture such as slow turnaround, false-negative results in antibiotic-treated patients, and false-negative results due to emerging serotypes of non-O157 STEC that ferment sorbitol. There are other methods that detect the Stx antigen from stool, either after broth enrichment or directly, for example, enzyme immunoassay (EIA) in which the optimal sensitivity and specificity are achieved only when a broth enrichment step is employed; this results in slow turnaround. Grys et al. (2009) evaluated a Stx gene real-time PCR assay using hybridization probes and the LightCycler device for the detection of Stx directly from stool specimens. They evaluated 204 prospectively collected stool specimens, which were also tested for Stx by enzyme immunoassay (EIA), and by culturing on chromogenic agar, and 85 archived stool specimens previously tested for Stx (by EIA) and/or E. coli O157:H7 (by culture) were tested by PCR. The results showed that 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). The inhibition studies demonstrated that none of the 53 specimen extracts tested contained substances that would cause a false-negative result in a low-positive sample. They proved that the real-time PCR assay can detect STEC directly from stool with 100% sensitivity (as less as 1,000 CFU in a small sample of stool) and specificity and same-day results compared to EIA and/or culture and more rapid turnaround than either EIA or culture. Moreover, The PCR assay has the ability to detect non-O157 STEC organisms, as well as culture-negative stools. Other advantages of the real-time PCR method are: (1) the two sets of oligonucleotide primers and probes allow excellent selection of the desired target and minimize false-positive results and (2) detection and amplification of nucleic acid is performed in closed system, which minimizes the potential for contamination and false positives. Grys et al. (2009) say one disadvantage of the PCR method is that there is no isolate to characterize, so culture isolates are vital to public health efforts to detect outbreaks and track the epidemiology of STEC organisms. They also mentioned other disadvantages associated with Stx antigen assays; these are: (1) a false positive may set off an inappropriate and potentially expensive investigation, and (2) a false-negative result can be a problem, as a patient may receive antibiotic treatment, which could increase the risk of HUS; however, the results of this assay showed 0% inhibition rate stool which means false-negative results will be rare. They concluded that the assay is the first comparison to culture and EIA of a LightCycler real-time PCR assay using a non-enriched stool specimen for STEC detection.
5- Conclusion
