Q&A: Researchers identify concussion biomarkers in saliva

A large prospective observational study examining non-invasive biomarkers identified unique indicators of concussion in the saliva of adult male athletes, according to results published in British Journal of Sports Medicine.

The diagnostic tests available for concussion are “extremely poor” objectively, Valentina Di Pietro, PhD, a molecular neuroscientist at the Institute of Inflammation and Aging and a Birmingham-Illinois BRIDGE fellow at the University of Birmingham in England, and colleagues wrote. This leaves individuals at risk for further concussive events prior to the resolution of the initial concussion. Conventional neuroimaging, including CT and MRI, provides normal results, according to the researchers, and the diagnosis of concussion currently relies on the clinician’s interpretation of observed signs and symptoms reported by the patient, as well as cognitive/neuropsychometric and/or physical evaluations such as balance or oculo-vestibular assessments.

Recent advances in high-throughput technology, such as next-generation sequencing, have enabled researchers to examine new classes of molecules, including RNA species, as potential biomarkers. Di Pietro and colleagues examined the role of salivary small non-coding RNAs in diagnosing sports-related concussion from an all-men group of professional rugby players in England across two seasons. Healio Neurology spoke with Di Pietro to learn more about the results, which confirmed concussion in 106 players.

Healio Neurology: What prompted this research?

Di Pietro: Sport-related concussion is a mild traumatic brain injury that typically results in the rapid onset of short-lived impairment of neurological function that usually resolves spontaneously. However, concussion is difficult to diagnose, and cases may be misdiagnosed or unidentified. Meanwhile, there are concerns regarding the long-term brain health of athletes exposed to repeated concussions.

Healio Neurology: Can you describe the study methodology?

Di Pietro: Saliva was obtained from male professional players in the top two tiers of England's elite rugby union competition across two seasons (2017-2019). Samples were collected pre-season from 1,028 players and during standardized head injury assessments (HIAs) at three time points (in-game, post-game, and 36-48 hours post-game) from 156 of them. Samples were also collected from controls, including 102 uninjured players and 66 players who sustained a musculoskeletal injury. Diagnostic small non-coding RNAs were identified with next-generation sequencing and validated using quantitative PCR in 882 samples. A predictive logistic regression model was built on 2017-2018 data (the training dataset) and prospectively validated the following season (the test dataset).

Healio Neurology: What were the primary findings?

Di Pietro: The team identified a panel of salivary biomarkers that can accurately distinguish concussed players from all other groups, including players with suspicion of concussion who had a concussion ruled out after a structured HIA, uninjured controls from the same game, and players with musculoskeletal injuries.

The panel was successfully prospectively tested during the 2018-2019 season to predict whether players would be positive or negative for concussion via the HIA protocol. The panel was highly accurate at differentiating concussed players from all controls (96% accuracy). More specifically, it could predict which players would pass or fail the HIA in 94% of cases.

Healio Neurology: How could a more precise test for diagnosing concussions positively impact treatment?

Di Pietro: This large study of non-invasive concussion biomarkers has identified unique signatures of concussion in saliva of male athletes, validated by a diagnosis of concussion by the existing HIA. This means it is now possible to accurately identify concussions simply by analyzing players' saliva, extending the ability to safeguard the brains of injured players outside professional ranks.

Healio Neurology: What are the next steps for this research — and how generalizable are the current findings?

Di Pietro: We hope to collect data to provide prognostic information, particularly regarding when it may be safe to return a player to contact training or competition. In addition, it would be wrong to extrapolate the results of our study to groups that were not included, such as women and children, due to biological differences. Specific data will need to be collected from these cohorts. Further research is ongoing and is planned by Marker Diagnostics and the University of Birmingham to expand the populations in which the test can be used, including women, junior athletes and individuals in community-level sports.