Secondary Biomarkers in COVID-19

The coronavirus spike protein has served us well in both diagnostic and therapeutic efforts to combat COVID-19. PCR infectivity tests amplify regions on this protein, and vaccines mimic this antigen in conferring immunity. PCR tests that detect the spike protein perform well enough, but they lack the power to answer the most critical questions regarding COVID-19 cases: Who will get sick, and how badly. Similarly, antibody testing informs on a patient's previous exposure to the virus, so it is a reactive versus a proactive assay.

Pre-existing conditions affect the course of COVID-19 infection, but making predictions based on age and health status is fraught with uncertainty. A listing of fatal complications from COVID uncovers myriad symptoms not directly related to virus or antibody titers, including a host of inflammatory conditions (often leading to acute respiratory distress), organ injury or failure, secondary infections, and coagulation disorders.

With direct testing for infection or immunity already well-covered, the search is on for predictive biomarkers that can alert physicians to take extra precautions that prevent serious disease progression and thereby save lives.

The Best Glossary Resource, a comprehensive repository of biomarker information maintained by the FDA-NIH Biomarker Working Group, defines a biomarker as "A defined characteristic that is measured as an indicator of normal biological processes, pathogenic processes, or biological responses" that "may include molecular, histologic, radiographic, or physiologic characteristics."

What's cool about molecular biomarkers, the subject of this article, is their potential simplicity. For example, dysregulation of two common metabolites, arginine and kynurenine, distinguishes COVID-infected from uninfected patients with 98% accuracy, without the uncertainties of PCR tests.

Stormy weather

Many people who succumb to the virus experience a cytokine storm, essentially an immune system gone haywire, while those who remain asymptomatic show normal immune function. These two subtypes are increasingly viewed as distinct phenotypes: one likely to develop acute respiratory distress and multi-organ failure, the other at low risk. Identifying these groups correctly allows clinicians to administer preventive treatments, such as steroids, to avoid the worst COVID complications.

For these insights, investigators turn to secondary—non-spike—biomarkers, of which several categories have been identified from blood (lymphocytes, neutrophils, neutrophil-lymphocyte ratio), among the inflammatory apparatus (C-reactive protein, erythrocyte sedimentation rate, procalcitonin), from the immune system (interleukins and related molecules), and among circulating biochemicals (D-dimer, troponin, creatine kinase, aspartate aminotransferase), particularly those markers related to coagulation and acute respiratory distress syndrome.

Proteomic biomarkers

Immune system hyperactivation is a recurring theme in the COVID-19 story. Many hospitalized patients recover but an alarming number do not and, increasingly, researchers look to their immune status at admission for clues on which patients will go home and which will become seriously ill. Cytokine levels are a direct measure of hyperactivation but relying on them is a reactive strategy, as elevated levels mainly tell physicians what they already know, that patients are sick.

A Yale group has reported on an AI-based approach to identify a prominent proteomic signature of neutrophil activation composed of granulocyte colony-stimulating factor and interleukin-8, and the neutrophil-derived effectors resistin, lipocalin-2, and hepatocyte growth factor to identify patients likely to progress to critical stage. Together, these biomarkers "were the strongest predictors of critical illness."

Since evidence of neutrophil activation is observable early in the course of the illness, it precedes the onset of the most severe complications, it could be used to identify which patients would likely be admitted to intensive care. "Early elevations in developing and mature neutrophil counts also predicted higher mortality rates" and, according to the authors, could serve as "molecular markers that distinguish patients at risk of future clinical decompensation."

Exosomes

Exosomes are extracellular vesicles, ranging in size from about 30 nm to 150 nm, which cells release under various health- and non-health conditions. The contents or "payloads" carried within exosomes, which includes genes, proteins, and small molecules, are rich in information but not always accessible in quantity or purity sufficient for making life-and-death decisions.

A multinational research group recently published a comprehensive overview of exosomes released during COVID infection, which suggests that exosome genomics and/or proteomics can provide some answers to why some individuals remain asymptomatic while others fall dangerously ill.

Exosomes released during COVID-19 infection apparently modulate the immune response and other aspects of the disease. Researchers found correlations between disease severity and progression, and proteomic contents related to immunity, inflammation, and activation of coagulation and complement pathways. COVID-19–associated tissue damage and organ damage are believed to occur through these mechanisms.

The group also identified potential proteomic biomarkers (e.g., fibrinogen, fibronectin, complement C1r subcomponent, and serum amyloid P-component) that are diagnostic for infection or disease severity. They also identified, for the first time, coronavirus RNA in the exosomal cargo, which suggests that the virus employs endocytosis to spread. They wrote: "Our findings indicate circulating exosomes’ significant contribution to several processes—such as inflammation, coagulation, and immunomodulation—during SARS-CoV-2 infection."

Metabolomic biomarkers

In March 2021, French researchers published a landmark paper on metabolomic biomarkers associated with ambulatory infection, mild lung involvement, hospitalization (with oxygen administration), and severe illness requiring intensive care.

Investigators identified close to 80 metabolites, including amino acids, lipids, polyamines, sugars, and derivatives, that were altered in critical vs. mild COVID patients. Among their most significant observations were increases in anthranilic acid and 5-hydroxy-DL-tryptophan, a derivative of L-tryptophan.

Also that month, Chinese researchers reported on a strong correlation between key metabolites and pro-inflammatory cytokines, particularly IL-6, M-CSF, IL-1α, and IL-1β. They identified 36 metabolites, through both targeted and untargeted metabolomics, that showed "consistent alterations along with the increasing disease severity" and "observed distinct metabolic profiles among healthy controls, mild, and severe cases…reflecting markedly dysregulated metabolic status of COVID-19.”

COVID-19 is a complex disease whose course is dictated by how the body reacts to infection. Molecular biomarkers, of which dozens or even hundreds might be relevant to predicting patient outcomes, are a prominent avenue of research that will pay dividends as the pandemic winds down, and perhaps beyond, to the next global health crisis.