A University of Birmingham-led study of top-flight U.K. rugby players has identified a biomarker panel of small, noncoding RNAs (sncRNAs) in saliva that represent a diagnostic signature for sport-related concussion. Reported in British Journal of Sports Medicine, findings from SCRUM (Study of Concussion in Rugby Union through MicroRNAs) could lead to the development of the first laboratory-based, noninvasive clinical salivary test for concussion, both for use in sport, but also for use in much wider healthcare and military settings.

The study, part of the REpetitive COncussion in Sport (ReCoS) research program, was carried out by researchers at the University of Birmingham, through the National Institute for Health Research’s Surgical Reconstruction and Microbiology Research Centre (NIHR SRMRC) based at University Hospitals Birmingham NHS Foundation Trust (UHB), in collaboration with the Rugby Football Union (RFU), Premiership Rugby, and Marker Diagnostics.

Senior study author Antonio Belli, MD, stated, “The ability to rapidly diagnose concussion using biomarkers in addition to existing tools solves a major unmet need in the sporting world as well as in military and healthcare settings, particularly in injuries without significant visible symptoms … Conducting a study in a professional contact sports setting has meant we have been able to collect invaluable data enabling us to make significant advances in our biological knowledge and understanding of concussion and its diagnosis … Crucially, the differences in the salivary concentration of these biomarkers are measurable within minutes of injury, which means we can make rapid diagnoses.” Belli is professor of trauma neurosurgery at the University of Birmingham, consultant neurosurgeon at UHB, and director of NIHR SRMRC.

The researchers reported their findings in a paper titled, “Unique diagnostic signatures of concussion in the saliva of male athletes: The Study of Concussion in Rugby Union through MicroRNAs (SCRUM).”

Sport-related concussion is defined as “a traumatic brain injury induced by biomechanical forces that typically results in the rapid onset of short-lived impairment of neurological function that resolves spontaneously,” the authors wrote. In the absence of objective diagnostic tests for concussion, identifying when someone has a concussion currently relies on a clinician’s interpretation of the signs and symptoms, together with the results of formal clinical assessments. However, concussion can be hard to diagnose, particularly at grass-roots sporting level, which is where it mostly occurs, but where gold standard medical assessment by trained clinicians during and after a game isn’t readily available.

The short-term consequences of a missed diagnosis range from a long recovery time, often with protracted and pervasive symptoms, to a heightened risk of further injuries, and this can—although rarely, the authors noted—progress to catastrophic brain swelling. And the possibility that a high percentage of concussions may be misdiagnosed or even missed has raised concerns about the long-term brain health of athletes exposed to repeated concussions.

Recent advances in high-throughput technologies such as next-generation sequencing (NGS), have opened the way to studying different classes of molecules, including RNA species, as potential biomarkers, the investigators noted. “Among these, microRNAs (miRNAs, miRs), which belong to the sncRNAs (20–200 nucleotides in length), are the most studied ….” sncRNAs can have roles in regulating the expression of different cellular proteins that are linked to various diseases, such as cancer and Alzheimer’s disease. And there is also “… evidence of a miRNA signature that varies according to TBI [traumatic brain injury] severity in blood, cerebrospinal fluid (CSF) and saliva,” the authors continued.

In professional male rugby union in England, clinicians’ evaluations of athletes who have sustained a head injury with the potential to result in concussion are supported by a standardized head injury assessment (HIA) protocol, and this offers “… an excellent setting in which to examine biomarkers of concussion and clinical outcomes,” the authors explained. The aim of their newly reported study was to try and identify sncRNAs that might be differentially expressed in the saliva of players who have had a clinical diagnosis of concussion, compared with other groups. The study in addition aimed to investigate over what time period any differential sncRNA expression response might be detected. The team wanted to find out whether they could identify a combination of sncRNAs in saliva could effectively predict whether a professional clinical assessment would make a diagnosis of concussion. “In this study, we sought to investigate the role of salivary sncRNAs as a new class of molecules to serve as potential biomarkers of sport-related concussion,” they wrote. “We assessed these against a standard tool used to diagnose concussion in professional rugby.”

The researchers obtained saliva samples from more than 1,000 male professional players in the top two tiers of England’s elite rugby union across two seasons (2017–19) of competition. Samples were collected before the season began from 1,028 players, and during standardized “gold standard” head injury assessments at three time points—during the game, immediately afterwards, and 36–48 hours later, in 156 of these players. Saliva samples were also collected from a comparison group of 102 uninjured players, and another 66 who had sustained muscle or joint injuries, and so had not been assessed for head injury.

Using samples collected during the 2017–18 season, the team identified a panel of a combination of 14 salivary sncRNAs—that was highly accurate (96%) at identifying concussed players from all other groups. This sncRNA panel differentiated concussed players from those with suspected traumatic brain injury, but whose head injury assessments had ruled out concussion, and from the comparison group, both immediately after the game and 36–48 hours later.

When the sncRNA panel was then assessed prospectively during the 2018–19 season, the results confirmed that the biomarkers could successfully predict whether players would be positive or negative for concussion via the HIA protocol, in 94% of cases. Co-author Matt Cross, head of science and medical operations at Premiership Rugby, commented, “The findings from the study are clearly promising and highlight the potential for salivary biomarkers to further support clinical decision making and the accurate identification and diagnosis of concussion in a range of different sporting and nonsporting settings.”

The authors acknowledged that their observational study doesn’t suggest that sncRNA biomarkers outperform the gold standard clinical assessment. “It is clear that in this study design, the biomarkers cannot outperform the clinical assessment,” they stated. “As a consequence, we believe the biomarkers are likely to prove most useful in nonprofessional sport settings, where the overwhelming majority of sport-related concussions occur.”

Scientists think that saliva can receive cellular signals directly from cranial nerves in the mouth and throat, and so can rapidly register traumatic brain injury, making a saliva test particularly suitable for pitch-side diagnosis. And as the biology of concussion is still not fully understood, sncRNAs might help to shed light on the response to injury as this evolves over time. “Concussion can be hard to diagnose and is often missed, especially where a structured evaluation by an expert clinician is not possible—for example, at grass-root level,” they wrote. “Small noncoding RNAs can provide a diagnostic tool that might reduce the risk of missing this type of injury at all levels of participation … In community sport, small noncoding RNAs may provide a noninvasive diagnostic test that is comparable in accuracy to the level of assessment available in a professional sport setting.” And in an elite sport setting, the test could be added to current head injury evaluation protocols.

“The detection of signatures of concussion at early time points in saliva (a noninvasively sampled biofluid) presents both at the pitch side, and in primary care and emergency medicine departments, an opportunity to develop a new and objective diagnostic tool for this common clinical presentation,” they concluded.

Marker Diagnostics, a subsidiary of Swiss biotechnology company Marker AG, is in the process of is working to commercialize the patented salivary concussion test as an over-the-counter test for elite male athletes. It has also obtained a CE Mark for test, MDx.100. “A patented salivary concussion test is in the process of being commercialized as an over-the-counter test for elite male athletes,” the authors stated in comments further to their published paper. “Meanwhile our research team aims to collect further samples from players in two elite men’s rugby competitions to provide additional data to expand the test and develop its use. This will guide the prognosis and safe return to play after concussion and further establish how the test will work alongside the head injury assessment process.”

Marker Diagnostics and the University of Birmingham are also currently carrying out several additional studies to further validate and expand the test for use in different groups that were not included in the SCRUM study, including women, young athletes, and community sports players.

Tinus Maree, CEO of Marker AG, added: “This ground-breaking validation of the biomarker panel shows that we can use the simple swab collection of saliva to accurately and specifically diagnose concussion. It is a biological measure of mild traumatic brain injury and will contribute to a new global standard of care for the injury and a meaningful reduction of the cost and health burden associated with concussion.”