Researchers have identified a group of microRNA biomarkers in saliva that successfully diagnosed concussion in professional male rugby players — a potential game-changer, given that it would be a noninvasive test for mild traumatic brain injury.
The diagnostic identified 96% of players with concussion in the Study of Concussion in Rugby Union through MicroRNAs (SCRUM) trial, a prospective, observational cohort study that enrolled 1028 male professional rugby athletes from the two top-tier leagues in England over two seasons.
“The results of our exciting and ground-breaking research shows that for the first time we have successfully identified that these specific salivary biomarkers can be used to indicate if a player has been concussed,” said study investigator Antonio Belli, MD, FRCS, FRCS, senior author, and professor of trauma neurosurgery, University of Birmingham, England, at a press briefing.
Belli noted that the study showed that the sncRNAs were released within minutes of brain injury.
“Although the biomarkers evolve over time, it is possible to make a diagnosis even at a really early timepoint,” he said, characterizing this as “one of the most exciting aspects of the study.”
The study was published online March 23 in the British Journal of Sports Medicine.
A True Signature of Concussion?
The researchers began the SCRUM trial soon after they identified a concentration of specific molecules in the saliva that change rapidly after brain injury. The sncRNAs, also known as microRNAs, play a role in mild and severe traumatic brain injury.
In SCRUM, researchers collected saliva samples from all athletes during the preseason. This baseline sampling helped eliminate any influence of pre-existing factors and led to “the selection of a narrow panel of sncRNA biomarkers that are likely to represent a true signature of concussion,” the authors write.
Samples were taken in-game during the first season, but not season two, as they were deemed not necessary. Samples were obtained immediately postgame and 36 to 48 hours postgame from players with head injuries, uninjured players, and players with musculoskeletal injuries. Analysis of saliva can be performed with standard qPCR equipment available in most accredited pathology laboratories.
All players with suspected head injury also went through the three-timepoint head injury assessment (HIA) protocol developed by World Rugby and used by English rugby teams. The timepoints are: in game, postgame within 3 hours of a head impact, and 36 to 48 hours after a head injury.
At the end of each season, independent clinicians reviewed HIA protocol documentation and video evidence. They were blinded to lab results and adjudicated each incident as HIA-positive or -negative. Forty-seven HIAs in 28 players were excluded as a result.
Over two seasons, 393 HIAs were conducted in 184 players. The clinical assessment determined that 106 players were HIA-positive, while 50 were HIA-negative.
The researchers found that 32 microRNAs were significantly differentially expressed across these two groups, with let-7f-5p showing the highest area under the curve (AUC) at 36 to 48 hours.
A combined panel of 14 microRNAs (let-7a-5p, miR-143-3p, miR-103a-3p, miR-34b-3p, RNU6-7, RNU6-45, Snora57, snoU13.120, tRNA18Arg-CCT, U6-168, U6-428, U6-1249, Uco22cjg1, and YRNA_255) was most accurate in differentiating those who had a concussion from all the other players, including those who were HIA-negative and controls.
This was true immediately postgame (AUC 0.91; 95% CI, 0.81 to 1) and 36 to 48 hours postgame (AUC 0.94; 95% CI, 0.86 to 1). This panel had a 96% correlation with the HIA adjudication.
In the second season, when the diagnostic was prospectively tested, the accuracy was slightly lower, at 94% — with an AUC of 0.96 (95% CI, 0.92 to 1) postgame and AUC of 0.93 (95% CI, 0.86 to 1) 36 to 48 hours later.
Meanwhile, players without concussion had biomarkers that “remained much closer to their baseline values throughout all timepoints after injury,” the authors note.
“A Substantial Advance”
The possibility of a rapid test would give be useful in sports, at the bedside, and for first responder and military needs, said Belli.
The diagnostic, being developed by Marker Diagnostics, a subsidiary of Marker AG (Zug Switzerland), “provides an invaluable tool to help clinicians diagnose concussion more consistently and more accurately,” he said.
“This is a very substantial and very novel finding,” said study co-author Simon Kemp, MD, the medical director of the Rugby Football Union, the national governing body for rugby in England.
“We’ve validated the biomarkers in our elite professional setting,” he said, adding that now players and medical staff will work together “to refine the value-adds and operationalize it to move from a laboratory-based test to a test that could deliver results in real time.”
However, neither scientists nor Marker Diagnostics have a rapid test yet.
The first stage was to confirm the concussion biomarkers exist in saliva, said Belli. “The next stage will be to then to make it faster and portable,” he said.
Belli said more studies will be conducted to further validate the test in rugby players, as well as to determine its usefulness in women, young athletes, and amateur players.
The SCRUM researchers are aiming to get the two English rugby leagues to widen testing so they can validate whether it can be used as a real-time diagnostic. Then, “if the results from next 300 cases match the results we’ve had, and there’s no reason to believe they won’t, it’s a very substantial advance,” said Kemp.
It’s not likely that the results in men could be directly extrapolated to women, said Patrick O’Halloran, MD, at the briefing. O’Halloran is a research scientist with Marker Diagnostics and a study investigator.
“There are a number of sources out there that suggest female response to concussion is different,” said O’Halloran. For women, “We think of ourselves as a step behind,” he said, but added that the company is trying to get more saliva samples from women to advance studies.
It’s also unclear whether the sncRNAs would be as effective in a setting where a clinical diagnosis is less certain, she said.
Marker Diagnostics has a CE Marking approval for its lab-based MDx.100 saliva biomarker concussion test, which means it has been deemed to meet European Union safety, health, and environmental protection requirements and can be sold in the EU.
The company is preparing to seek US Food and Drug Administration approval, said O’Halloran.
Need for More Research
Commenting on the findings, Christina Master, MD, FAAP, CAQSM, FACSM, a sports medicine pediatrician at Children’s Hospital of Philadelphia, Pennsylvania, said the microRNAs, also known as small non-coding RNAs “do appear to be associated with concussion in this specific population.”
Their diagnostic utility “appears to be high in this group of athletes, namely elite rugby players,” Master told Medscape Medical News. “It will be interesting to see how these biomarkers perform in a larger, more diverse cohort,” she said.
Master said given how little is known about the sncRNAs, “it is hard to predict whether they would be similar or different, expressed at greater or lesser levels, in younger individuals or women.”
She added that a rapid test “would have tremendous utility on the sidelines and in the athletic training rooms for sports medicine physicians and certified athletic trainers.” However, she said, she believes the data in this study only support its usefulness 36 to 48 hours postgame.
Studies should be extended across the age spectrum and to young athletes, nonathletes, females, and multiple races and ethnicities to understand how the biomarkers might perform in diagnosing concussion in these various groups, she added.
“It will be interesting to see how these biomarkers perform in a larger, more diverse cohort,” said Master.
Also commenting on the findings for Medscape Medical News, Julian Bailes, MD, chairman of neurosurgery at the NorthShore Neurological Institute in Chicago, Illinois, described the study as “really intriguing.”
“However, more work needs to be done, not only to clarify the science but also on the logistics of the application of the science to the field of play,” said Bailes, who has been a sideline physician for the National Football League and National Collegiate Atheletic Association for 30 years, and is also the medical director of the Center for Study of Retired Athletes at the University of North Carolina, Chapel Hill.
In US sports, trainers and clinicians make immediate on-field or on-court diagnoses based on multiple factors, with an eye toward taking the player out or keeping them in a game, said Bailes. “There’s no time to wait for a test” that has to be sent to a lab, he said.
However, a saliva-based lab test would still be valuable if it could give an objective postgame result and if, eventually, it “quantified as to how bad a concussion or how significant a concussion was,” Bailes added.
SCRUM was funded by the Midland Neurosciences Teaching and Research Fund, National Institutes for Health Research’s SRMC, Medical Research Council, Rugby Football Union, and Marker AG. Belli is a founding member and shareholder of Marker Diagnostics, a spinout company of the University of Birmingham. The Rugby Football Union has a financial interest in the intellectual property connected to the biomarkers studied. Master has disclosed no relevant financial relationships. Bailes has been a neurological consultant to the NFL Players’ Association and is the medical director of Pop Warner Football, a youth league.
Br J Sports Med. Published online March 23, 2021. Full text
Alicia Ault is a Lutherville, Maryland-based freelance journalist whose work has appeared in publications including Smithsonian.com, the New York Times, and the Washington Post. You can find her on Twitter @aliciaault.