Skip to main content
Premium Trial:

Request an Annual Quote

Stanford Team Validates Cheap, Easy-to-Use Multiplex PCR Assay Method for STEC Screening


Clinical researchers from the Stanford University School of Medicine have published a study demonstrating how their laboratory-developed multiplex real-time PCR-based assay to detect Shiga toxin-producing E. coli of all serotypes could be a useful screening tool to help stem community-acquired diarrhea outbreaks and better manage individual patients that develop potentially life-threatening conditions from infection with the bacteria.

During the course of their study, the researchers also found evidence from a Northern California epidemiological study that infection with either O157 or non-O157 Shiga toxin-producing E. coli, or STEC, can cause severe complications such as hemolytic uremic syndrome.

These findings also underscore recommendations from the US Centers for Disease Control and Prevention that all diarrheal stool samples from patients with acute community-acquired diarrhea be tested for both non-O157 STEC and the O157 serotype — recommendations that many laboratories have yet to follow, the researchers claim.

The new assay and its evaluation are described in a paper published this month in the Journal of Molecular Diagnostics. The study's authors include researchers from Stanford and Santa Clara County Public Health Laboratory.

As the researchers explained in the paper, STEC are a major cause of bacterial gastroenteritis and often cause severe complications such as hemolytic uremic syndrome, or HUS, and hemorrhagic colitis. One particular serotype of STEC, E. coli O157, had long been thought to be the primary culprit of these complications.

However, in recent years it has come to light that non-O157 serotypes can also be responsible for sporadic cases and community outbreaks of diarrhea, the most well-known recent example being an outbreak of E. coli O104:H4-associated diarrheal illness in Germany in May and June 2011 that led to HUS in more than 800 patients and resulted in 54 deaths.

Even prior to this notorious outbreak, the CDC in 2009 issued recommendations for diagnosing STEC, including the specific recommendation that all stools submitted for routine testing from patients with acute community-acquired diarrhea be simultaneously cultured for E. coli O157:H7 and tested with an assay that detects Shiga toxins to detect non-O157 STEC.

In recent years, molecular methods, and especially real-time PCR-based methods, have proved to be the most sensitive types of assays to detect the Shiga toxin genes, stx1 and stx2, as well as a majority of STEC serotypes, and several commercial versions of these assays now exist.

However, according to the study's authors, many of these tests use fluorescently labeled probes that can be prohibitively expensive to use in a large-scale screening capacity. The Stanford scientists instead developed an assay that uses SYBR Green chemistry, which they claim is cheaper and less complicated than competing fluorescent probe-based methods, and which other laboratory groups have in recent years adopted as a simpler alternative to fluorescent probe-based PCR.

Further, their assay uses a simple sample prep procedure that essentially obviates the use of pricey kits to extract and purify DNA from stool samples.

"I think this assay has relatively simple chemistry, and the way it's set up in our lab is also simple, where we take a drop or two of stool and put it in a broth that sits in an incubator overnight," Niaz Banaei, an assistant professor of pathology and infectious disease at Stanford University School of Medicine and corresponding author on the paper, told PCR Insider. "The extraction part is also extremely simple – it's basically boiling a portion of that broth after diluting it in water."

Banaei added that the assay "costs very little because of the chemistry and the reaction volume is relatively small, so we can screen all of our stool samples for Shiga toxin-producing E. coli without spending a tremendous amount of resources."

The assay protocol provides an algorithm for confirmation of presumed positives by an independent set of primers that target different regions of the stx1 and stx2 genes, the researchers wrote. They also noted that the test has a turnaround time of between 24 and 36 hours from specimen collection, a time window during which serotyping by a public health laboratory can take place.

The researchers validated their assay using 65 known STEC-negative and 40 known STEC-positive samples, as well as 15 clinical specimens that were culture-positive for Shigella or Salmonella but not STEC. Overall, their assay demonstrated a sensitivity of 100 percent and specificity of 98.5 percent.

The group then began routinely using the assay to test all stool samples sent to its laboratory and collected data over a two-year period. During that time, the assay identified a greater number of stx-positive clinical specimens than did a conventional PCR method that had previously been used by the public health laboratory. Overall, the researchers calculated a positive predictive value of 95.6 percent for their assay.

"We went live with this assay in 2010, about a year before … the German outbreak … precisely for the reason that we wanted to be able to detect not just the most common serotype — the O157 serotype — but essentially all serotypes," Banaei said. "And so a year later when that German outbreak occurred, I realized that if that outbreak had occurred here, we would have been ready for it, because we would not have missed a non-O157."

The researchers also found that more than half of the STEC strains they detected during that time were non-O157 serotypes, which would have been missed with the conventional PCR assay. Further, they discovered that although O157 serotypes were overwhelmingly most responsible for complications such as HUS, non-O157 strains also played a part.

"There is conflicting evidence in the literature regarding the frequency of hemolytic uremic syndrome with O157 vs. non-O157," Martina Lefeterova, a pathology resident at Stanford University School of Medicine and first author on the paper, told PCR Insider.

"Especially after the German outbreak we assumed that it's equally likely to occur with both, but our data actually indicate that, at least in the sample population that we had, that O157 is more frequently associated with HUS," she said. "After we reviewed our data we have now actually also recorded in our algorithm the testing for O157, to be able to — after identifying the Shiga toxin — find out whether it's O157 to monitor the patients more closely."

Nevertheless, the researchers pointed out the fact that HUS and related syndromes can occur in non-O157 cases, underscoring the need to screen stool samples for all STEC serotypes and lending additional credence to the CDC recommendations.

Banaei, Lefterova, and colleagues also conducted a telephone survey as part of their study that revealed that nearly half of all clinical laboratories in Northern California do not screen all stool specimens for the presence of Shiga toxins — data that jibe with other published surveys of physicians.

One major reason labs aren't routinely following the recommendations is cost, Lefterova said, "and I think that's one of the advantages of an assay like ours. A lot of labs will already have a setup for some kind of molecular assay, and as long as it's a real-time PCR-based instrument, the reagent costs for this assay are relatively low." Further, she said, a lack of experience and training may be a contributing factor.

The researchers concede that a limitation of their assay is the time it takes — around a day — to provide a result. The overnight enrichment step may actually improve the assay's sensitivity, Banaei said, but noted that "it would be nice to know [an answer] within an hour or two of the time the sample is submitted. Because if someone is at risk for developing HUS or is on their way to developing it, and they knew that this is a Shiga toxin-producing E. coli, the recommendation is not to give antibiotics, which worsens the symptoms of HUS."

He added that testing directly from stool to speed up the assay is "totally doable — we just haven’t validated it yet because we wanted to keep it simple."

The group is making its assay protocol available for use by public health laboratories everywhere with the hope that more people will adopt it in order to adhere to CDC guidelines.

However, Banaei said that his team is "working with a company that has an on-demand system to turn this into an on-demand test along with some other causes of GI disease." He declined to identify the partnering company, citing the very early stage of the relationship, but noted the firm is located in the Bay Area.

"This would be more of an on-demand type of system where … you could add the stool to a cartridge and then have the results back in an hour," he said.