This long case definition aims to describe the methods presently available for screening specimens and confirming the identity of STEC and to provide guidance for laboratories, which might wish to introduce tests for STEC. Isolation and presumptive identification of STEC O in the clinical diagnostic laboratory is relatively straightforward because of its inability, in most cases, to ferment sorbitol, with definitive identification achieved by serotyping and demonstrating one or both of the toxin genes stx 1 and stx 2. For sorbitol fermenting serotypes, isolation and presumptive identification presents more of a challenge and either a toxin test or NAA for virulence factors is essential.
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All presumptive STEC isolates should be referred to a reference laboratory for definitive identification and subtyping. Faecal specimens may be cultivated directly to the following media and incubated at o C: the media selected will depend on the detection strategy chosen.
Faeces, rectal swabs, urine are suitable specimens; sweeps on MacConkey agar are preferred to selected single colonies as the STEC may well be in the minority. Chromogenic agars do not offer significantly improved sensitivity for isolation of E. A subset of these organisms, enterohaemorrhagic E. For health and safety reasons, toxin-producing control strains should be used only for those tests for which the demonstration of toxin production makes their use unavoidable.
Commercial latex agglutination kits for E. For confirmation of serotype by titration, a toxin-negative strain of E. This strain can also be used as a control for the demonstration of typical haemolysis on EHEC plates. Note: There is a low infective dose for humans and laboratory acquired infections have been reported. STEC - the demonstration of Shiga toxin or Shiga toxin genes in an isolate that has been confirmed biochemically as E. There is no external quality control programme at present for the detection of STEC in specimens. Nucleic acid amplification NAA methods can rapidly determine the presence of genes encoding determinants for serotype and virulence factors such as Stx, attachment effacing factors or haemolysin in microbiologically complex samples without prior amplification by culture.
They have been used to screen faeces and foodstuffs directly or can be used to screen cultures. Sequences of stx 1 do not vary significantly unlike stx 2 which has at least 3 variants stx 2, stx 2c, stx 2e 7. In-house methods are based on real time TaqMan technology or standard PCR followed by identification of product using gel electrophoresis. Most NA assays used in Australia are based on the PCR method of Paton and Paton 13 , which targets a conserved region found in both stx 1 and s tx 2 followed by hybridisation of specific probes for each of the toxin genes.
Variants of the method use a second round amplification with specific primer sets for each toxin differentiation of product size after gel electrophoresis is not possible as products are too similar in size - and bp. Real time assays can deliver a result within 24 hours of the specimen being received. NA extracts positive for toxin genes can then be tested for the presence of accessory virulence factors found in STEC, including eae and EHEC hly A - markers for the locus of enterocyte effacement LEE pathogenicity island and the STEC megaplasmid, respectively, and the rfb region which specifies serotype.
Presently, sequences for the O, O and O rfb regions are commonly used in Australia. Multiplex PCR assays containing primers for these targets and alternate toxin gene sequences are often used to provide supplementary information about specimens, which have been found to be positive by the initial screening PCR.
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The main impediment to the routine use of NA methods is the laborious nature of the NA extraction procedure directly from faeces and food. Less inhibitory extracts are obtained if the samples are inoculated into broth and extractions performed after overnight incubation either onsite or after transportation to a reference laboratory. This process suits the work-flow of a diagnostic laboratory where broth cultures may be set up over the course of the day or evening as the specimen arrives in the laboratory. Only a small amount of specimen about the size of a match head should be inoculated into the broth so that there is adequate dilution of inhibitory faecal material.
With practice, inhibition can be reduced to close to zero. If the sweep is positive, separate colonies are selected for confirmation by PCR or dot blot assay using specific toxin gene sequences as probes. PCR-positive isolates should always be referred to a reference laboratory for definitive identification.
Faeces from cases of HUS or bloody diarrhoea. Direct NAA detection of the toxin genes in a suitable specimen is extremely sensitive if the initial NA extraction procedure is effective in removing inhibitory substances. The limit of sensitivity of the TaqMan system approached organisms in a background of 10 9 enteric organisms in seeding experiments using LB broths from negative human faecal specimens. However, due to the high sensitivity of PCR, a positive result does not imply the organism will be isolated. If a sample of food implicated in an outbreak or specimen from an HUS case is positive by PCR up to colonies may need to be screened to find a positive colony on subculture.
Very high specificity if target sequences are well chosen. Negative predictive value will depend on the inhibition rate and the primers, which should be designed to detect all variants present in the geographical area.
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Positive predictive value of an assay for the toxin genes is influenced by the primer design, specimen quality and test sensitivity. Qualitative NA tests are considered positive if a PCR product of the appropriate size is present following gel electrophoresis and negative control lanes are clear. All positive PCR products should be verified by second round PCR, dot blot analysis, restriction enzyme digest or sequencing to confirm the result.
A Ct value of more than 35 cycles is generally considered to be negative. Very weak positives with Ct values of up to 37 cycles may be discernible if the increase in fluorescence is sufficiently steep. Ideally an internal inhibition control should be included in each specimen tube. In practice specimens are analysed for inhibition by the inclusion of a separate specimen tube spiked with positive control DNA at two concentrations, one of which should be near the lower limit of detection.
Inhibitory specimens are identified by the absence of a PCR product of the appropriate size in this sample after gel electrophoresis or absence of a fluorescent signal in the case of real-time PCR. Applied Biosystems now has an internal control that can be spiked into samples. However, TaqMan protocols can only detect four dyes, which are presently used at maximum capacity ie two test probes, an internal fluorescent dye and the quencher.
Positive and negative control organisms should be included in each run.
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The gold standard assay for the presence of toxin in faecal specimens and isolates remains Vero cell culture. Neutralisation tests in Vero cell cultures have shown that there is little, if any, crossreactivity between antibodies raised against each of the toxin types. Cell-free filtrates of enrichment broth cultures are applied to freshly split monolayers of Vero cells and observed for cytopathogenicity following hours incubation. Neutralisation tests, other antibody-based tests such as EIA or nucleic acid testing should be performed to differentiate cytopathogenicity due to Shiga-toxin production from non-specific toxic effects that may be seen in monolayers especially following inoculation of enrichment broths from primary faecal specimens.
This specialised test is unlikely to be available in diagnostic laboratories, and results are not available for hours, making rapid diagnosis impossible. They are specific, reasonably sensitive and easy to use, although expensive. Results are available in 1. As with all EIA assays, care must be taken during the washing process to avoid false positives.
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The detectable toxin concentration can be increased by incubating the cell pellet obtained after incubation in broth culture with 0. The prevalence was calculated from the number of samples confirmed for STEC and total population of each study. The incidence was calculated from the number of STEC samples to the population of the city of study. The statistics were taken from population and housing census of the statistical center of Iran.
Moreover, prevalence was pooled using the random effects model with the reverse variance technique of DerSimonian and Laird for between-study variance estimation. At the preliminary search, articles were detected; 16 studies were relevant Figure 1. After reviewing the abstract, a total of six potentially relevant articles containing 1, positive E. Amongst them, 32 were positive for STEC.
The design was cross sectional. One study evaluated mixed groups of patients with either diarrhea or urinary tract infection Median duration of studies was 10 months range 5 to 12 months Table 1. Detail of extracted data is shown in Table 2. Funnel plot Figure 3 was asymmetric toward positive axis in favor of publication bias and population heterogeneity and methodology design, thus the studies with positive results were underestimated.
Data from six cross—sectional studies with good to poor quality of acceptance and low to moderate of bias were used. The prevalence of urinary STEC was approximately 3. Only one study performed a serotype for O The dominant virulence factors were stx1 and stx2. Because of high infectivity of this pathogen even with low dose of this organism, the physician should be well informed to consider this possibility and check VTEC in cases with aggressive manifestation of urinary tract infection, diarrhea negative HUS, or TTP.
The central laboratories should be enlightened to be assembled to investigate both O and non-O STEC in suspicious victims. Limitations: The heterogeneity of studies was one of the major drawbacks. None of the studies followed the patients for occurrence of STEC complications, such as hemorrhagic colitis or hemolytic uremic syndrome, changes in blood cells, or kidney function.
None of the studies reported the outcome of affected and non affected cases. Small sample size was another limitation of the study. Resistance pattern of breakthrough urinary tract infections in children on antibiotic prophylaxis. J Infect Public Health. Hooman N. J Ped Nephrol.
Short-term and long-term outcome of hemolytic uremic syndrome in Iranian children. J Nephrol. Mohkam M. Nephro Urol Mon.