A University of Washington study found the panel cross-reacts with 78 gut bacteria species, resulting in over 250,000 false-positive norovirus results since 2019.
A molecular diagnostic test widely used to identify causes of diarrhea frequently misidentifies normal gut bacteria as norovirus, according to a study by researchers at the University of Washington School of Medicine.
The study, published in the Journal of Clinical Microbiology, found that the BioFire FilmArray Gastrointestinal Panel, manufactured by biotech company bioMérieux, cross-reacts with 78 bacterial species commonly found in the human intestine.
Norovirus is the leading cause of gastroenteritis and foodborne illness globally, responsible for approximately 109,000 hospitalizations each year in the US and 685 million cases worldwide, according to the Centers for Disease Control and Prevention. While outbreaks occur most frequently during winter months in settings such as hospitals, schools, and long-term care facilities, clinicians began noticing abnormal reporting patterns roughly five years ago.
“Norovirus outbreaks typically happen in winter, so when labs started reporting lots of norovirus cases in the middle of July, people thought it was weird,” says Alexander Greninger, MD, PhD, professor of laboratory medicine and pathology at the University of Washington School of Medicine and senior author of the study, in a release.
According to the study, these testing discrepancies have resulted in more than 250,000 false-positive norovirus results since reports first emerged.
Impact on Clinical Workflow and Patient Care
Multiplex panels have gained widespread clinical adoption because they can rapidly detect 22 different bacterial and viral pathogens associated with diarrhea from a single patient sample. However, incorrect results create diagnostic and operational challenges for clinical laboratories and inpatient units.
“Diagnostic panels are very helpful, but if you’re misdiagnosed with norovirus, you may not get the appropriate treatment and the hospital has to implement expensive infection-control procedures,” says Jonathan Reed, MD, PhD, senior research scientist in the Greninger lab and co-first author of the paper, in a release.
To evaluate the cause of the errors, the researchers analyzed used BioFire test cartridges, known as pouches, that had tested stool samples from patients presenting with diarrhea across four Seattle-area healthcare facilities.
At these sites, the false-positive rate ranged between 31% and 74%.
“On average across the hospitals, there was about a 50% chance a positive test was false,” says Greninger in a release. “It was basically a coin flip whether your patient had norovirus or not.”
In response to initial clinician feedback, bioMérieux updated its instructions regarding how to interpret norovirus test results, but did not modify the assay evaluated in the study, according to the researchers.
Identifying the Mispriming Mechanism
The panel relies on reverse transcription polymerase chain reaction (RT-PCR), which uses short synthetic DNA primers designed to match specific target sequences and produce copies known as amplicons.
To pinpoint the source of the false signals, the research team opened used test pouches and sequenced the amplicons produced during testing. They found that one of the norovirus primers bound to and amplified DNA sequences from 78 distinct species, most of which represent normal stool microbiota.
“We were a bit surprised that so many bacterial species were being amplified by the assay,” says Reed, in a release.
The researchers determined that the primer binds to segments of non-target bacterial DNA that partially mirror target norovirus sequences. This mispriming event initiates off-target PCR amplification, yielding a detectable signal in the absence of the virus.
“Our findings demonstrate the need to redesign this assay to ensure reliable testing and improve patient care,” says Clarice Mauer, research scientist in the Greninger lab and co-first author of the study, in a release.
bioMérieux Develops Updated GI Panels
In June, bioMérieux submitted its new BioFire FilmArray Gastrointestinal 1.1 Panel and GI1.1 Panel Mid to the FDA for 510(k) review. The company says the new versions, which include enhanced norovirus detection and increased assay specificity, were developed following a comprehensive investigation into the root cause of the observed risk of false-positive norovirus results.
“Our commitment to continuous improvement is central to our purpose to improve public health and we look forward to bringing these new tests to market in the future,” the company said in a statement to CLP.
ID 119680835 © Jirapatch Iamkate | Dreamstime.com
(Editor’s note: This article has been updated to include additional information from bioMérieux about its updated gastrointestinal panels currently under FDA review.)
