If the findings of their study are confirmed by other research centers, the biomarker could be the first specific and quantifiable indicator for confirming long COVID. Currently, clinicians confer a diagnosis of long COVID based upon a collection of symptoms that patients develop after SARS-CoV-2 infection.

"If a patient arrives in clinic and they relate the persistence of typical signs and symptoms of long COVID, 12 weeks or more after COVID -19 infection, I give them a presumptive diagnosis, but I don't have any blood tests or biomarkers to confirm this diagnosis," said William Stringer, M.D., a Lundquist Institute investigator and senior author on the study.

The study results, reported in the journal Infection, detail the detection of SARS-CoV-2 protein fragments within extracellular vesicles (EVs) -- tiny, naturally occurring packages that help cells share proteins, metabolites, and other materials. The researchers collected and analyzed blood samples from 14 patients over 12 weeks of aerobic exercise training (56 samples in all) in a clinical trial led by Stringer in long COVID.

The researchers found 65 distinct protein fragments from SARS-CoV-2 inside the EVs. These fragments come from the virus's Pp1ab protein, an RNA Replicase enzyme which is key to how the virus copies itself and makes other viral particles. This protein is found uniquely in SARS-CoV-2, and not in uninfected human cells, noted Asghar Abbasi, Ph.D., a Lundquist Institute investigator and first author of the study.

Significantly, the researchers found that these viral peptides were demonstrated in each subject, but not each blood draw, in the EVs of Long COVID patients and were not detected in a separate control group of pre-pandemic EV samples.

These findings add to growing evidence that suggests that SARS-CoV-2 may persist in certain body tissues long after the initial infection. Some groups hypothesize these lingering viral reservoirs could play a role in Long COVID. How the virus reaches tissues without its usual entry points -- such as the brain -- remains an open question, and may be related to EV particles.

"We thought that maybe if the virus is circulating or moving in the body, we should try to see if EVs are carrying those viral fragments," Abbasi explained.

This idea became part of an ongoing clinical trial led by Drs. Abbasi and Stringer, which was already studying EVs to see if they are linked to changes in immune function related to exercise and post-exertional malaise, a common symptom in these patients.

"While promising, the molecular signal of the viral peptides within the study samples was observed to be subtle and not consistently detected at every blood collection time point," said Patrick Pirrotte, Ph.D., associate professor at TGen, director of the Integrated Mass Spectrometry Shared Resource at TGen and City of Hope, and co-senior author of the study. "There's still a lot to unpack that we don't know at this point."

For instance, he added, the researchers don't know if the exercise itself drives the expression of viral programs intracellularly, and then those viral programs result in proteins that are going to be shed, or if there is a permanent reservoir in those cells, and it's just a matter of detecting it at a certain time point. Although the identified peptides originated from one of the virus' largest proteins, the researchers did not detect other comparably large proteins indicative of active viral replication. It's possible that the peptides contained in the EVs are just molecular "trash" leftover after the formation of new viral proteins.

"We haven't run [our tests] on people without long COVID symptoms who are currently, or who were, infected with COVID," said Stringer. "This raises the question: is this just continuing to take out the trash from the COVID infected cell or is this really ongoing replication someplace? I think that's the mechanistic issue that needs to be resolved in future studies."

The Pulmonary Education and Research Foundation (PERF) and the UCLA David Geffen School of Medicine (DGSoM)-Ventura County Community Foundation (VCCF) funded this research.