By Ben Butkus
German scientists have developed a real-time PCR assay to detect the Shiga toxin-producing Escherichia coli strain that caused last summer's fatal outbreak in Europe, and have demonstrated that it is more sensitive and nearly as specific as commonly used microbiological assays.
The researchers believe their results support the potential use of real-time PCR assays to detect STEC strains directly from stool samples much more rapidly than current methods to enable earlier treatment, particularly in an outbreak situation.
In addition, they have begun evaluating a commercial real-time assay from Germany's Altona Diagnostics to improve the specificity and time to result of their in-house-developed assay, according to one of the research team members.
The scientists, from the Medical Laboratory Bremen, published data demonstrating the robustness of their assay in a paper appearing in the current issue of Clinical Laboratory; and also detailed their findings in a correspondence published in the latest issue of The Lancet Infectious Disease.
According to Andreas Gerritzen, first author on the study, the Medical Laboratory Bremen served as a key diagnostic facility during last year's outbreak in Germany of Shiga toxin-producing E. coli, or STEC. The outbreak caused more than 800 patients to develop hemolytic uremic syndrome, and proved fatal in 49 cases.
"The Bremen region was a center for the STEC outbreak in Northern Germany, with our lab being the most important diagnostic facility in the region," Gerritzen wrote in an e-mail to PCR Insider. "We isolated a large number of relevant clinical strains but were not involved in the first description and publication of the outbreak strain."
That honor went to researchers from the University of Münster, who, along with collaborators from Life Technologies, isolated the STEC strain and characterized its genome using an Ion Torrent PGM sequencer. That group diagnosed patients by using microbiological culture as a gold-standard assay and by performing broth enrichment of fecal samples followed by Shiga-toxin immunoassay or PCR to detect the Shiga toxin 1 and 2 genes (stx1 and stx2).
However, the Bremen researchers noted in their publications that these procedures, which are recommended by the Robert Koch Institute and are still the most commonly used methods to diagnose STEC, only display adequate sensitivity and specificity when coupled with the broth enrichment step, which is time-consuming to perform and requires at least an overnight incubation period.
In particular, previously developed PCR-based molecular tests for various STEC strains have generally necessitated overnight enrichment of samples because DNA extracts taken directly from human stool specimens contain large amounts of PCR inhibitors that have been shown to confound test results.
Nevertheless, given the rapidity with which STEC infections can cause serious illness or death, Gerritzen and colleagues set out to develop a PCR-based testing protocol that could detect the bacteria directly from stool samples without a prior broth enrichment step.
The Bremen group's protocol entailed suspending a stool sample in sterile water, creating a supernatant, and isolating and purifying nucleic acid with the ExtraStar Purification Kit 1.0 from Germany's Astra Diagnostics, recently renamed Altona Diagnostics, operating on a ThermoFisher KingFisher Flex magnetic particle processor. Using previously described primers for stx1 and stx2, they performed real-time PCR assays on the purified samples using a Roche LightCycler mastermix and instrument platform.
They processed 754 fecal samples from 481 patients presenting with symptoms in the Bremen region between May 23 and June 10, 2011. Of these, 112 samples were STEC positive with the gold standard of microbiological culture. They tested these samples using Stx immunoassays and another established technique, Vero cell cytotoxicity assays, which detected 86 and 94 of the 112 positive samples for sensitivities of 76.8 percent and 83.9 percent, respectively.
Meantime, their stx1 and stx 2 molecular assay detected 108 of 112 positive samples for a sensitivity of 96.4 percent. This result led the researchers to conclude that their method could be worth considering in the case of a future STEC outbreak.
"The direct fecal stx real-time PCR [assay] proved superior to enrichment-based [Vero cell toxicity assay] and [enzyme immunoassays] and can be recommended as a quick and sensitive tool for the early diagnosis of STEC infection, in addition to microbiological culture," Gerritzen said. "First findings could be reported on the very day of specimen collection and clinical action be taken immediately while awaiting a confirmatory report not before the next day."
The Bremen researchers did, however, report some issues with specificity — though not enough to recommend against use of the assay. According to Gerritzen, the researchers observed additional melting peaks in about 10 percent of the PCR products. These were caused by primer-dimer formation or other non-specific products, he said. "In these samples, PCR products were analyzed by length with a [base pair] ladder in gel electrophoresis," he said.
In order to eliminate this step, the Bremen researchers have tested the RealStar STEC PCR Kit 1.0 from Hamburg-based Altona, which enables parallel detection of stx1, stx2, and an internal control.
According to Gerritzen, Altona's assay thus far has demonstrated comparable sensitivity to the researchers' in-house real-time PCR assay. The in-house PCR works with an intercalating dye to detect the PCR products. However, because the Real-Star test works with hydrolysis probes, as opposed to an intercalating dye for the in-house PCR assay, it requires no additional gel electrophoresis steps.
Jens Dannenbeg, a sales manager with Altona, told PCR Insider that his company quickly developed the RealStar STEC assay in June 2011 in response to the outbreak. The assay uses the same real-time PCR chemistry as several existing CE IVD marked Altona products bearing the RealStar brand name.
Dannenberg noted that the Bremen researchers' findings could help drive use of Altona's assay in the case of a future outbreak, or even as a test to rule out STEC if patients begin presenting with STEC-related symptoms. However, he also said that microbiologists have generally been slow to embrace molecular testing in favor of more tried and true methods.
"It's a situation where we would like to get more access to microbiologists," Dannenberg said. "Often in Europe virology and microbiology are somewhat separated. Virologists traditionally for many years have used real-time PCR since it offers so many advantages over viral titer. In microbiology, on the other hand, many are still using more traditional non-molecular tests, since you can easily cultivate microbiological targets. It takes time, but it's easy to do. You see your colonies or you don't."
Dannenberg added that during last year's outbreak, the fact that many labs still employed these classical microbiological techniques may have worked against efforts to track and control the spread of the STEC strain.
"To my impression this indicated that molecular diagnostics are less commonly used among microbiologists, and this was an opportunity for us to enter microbiology," he said. "For us to develop an assay like this, it doesn't really take us a long time once we have some kind of sequence information available, and reference samples to check for specificity and so on.
"How soon this STEC assay could be used again, or how quickly an assay could be developed for a future outbreak — this is always difficult to answer," he added. "I think we are quite fast in developing the assay, and then we can produce it under qualified conditions, but it would still be for [research use only]."
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