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Researchers in Boston Find COVID-19 Spike Protein Lingers in Long COVID-19 Patients

Viral reservoir could be behind persistence, says study, which also suggests a blood biomarker could be found for clinical laboratory testing

Microbiologists and virologists working closely with physicians treating long COVID-19 patients will gain new insights in a study that found coronavirus spike protein in COVID-19 patients’ blood up to 12 months after diagnosis. The researchers believe their findings could be used to develop a clinical laboratory biomarker for long COVID-19.

Researchers at Brigham and Women’s Hospital and Massachusetts General Hospital said medical experts are not sure why some people have unwelcome symptoms weeks and months after a positive COVID-19 diagnosis, while others clear the infection without lingering effects.

The scientists believe if this work is validated, clinical laboratories might gain an assay to use in the diagnosis of long COVID-19.

“The diagnosis and management of post-acute sequelae of COVID-19 (PASC) poses an ongoing medical challenge. … Strikingly, we detect SARS-CoV-2 spike antigen in a majority of PASC patients up to 12 months post-diagnosis, suggesting the presence of an active persistent SARS-CoV-2 viral reservoir,” the researchers wrote in their published study, which can be found on the preprint server medRxiv, titled, “Persistent Circulating SARS-CoV-2 Spike Is Associated with Post-Acute COVID-19 Sequelae.”

David Walt, PhD

“The half-life of spike protein in the body is pretty short, so its presence indicates that there must be some kind of active viral reservoir,” said David Walt, PhD (above), Professor of Pathology, Brigham and Women’s Hospital, and lead author of the study that found coronavirus spike protein in long COVID patients. The study findings indicate a potential clinical laboratory biomarker for long COVID-19. (Photo copyright: Brigham and Women’s Hospital.)

Viral Reservoir Possibly Behind Long COVID-19

The study suggests that SARS-CoV-2 finds a home in the body, particularly the gastrointestinal tract, “through viral reservoirs, where it continues to release spike protein and trigger inflammation,” Medical News Today reported.

Lead author of the study David Walt, PhD, Professor of Pathology, Brigham and Women’s Hospital and the Hansjörg Wyss Professor Biologically Inspired Engineering at Harvard Medical School, told The Guardian he “was motivated to carry out the study after earlier research by his colleagues detected genetic material from the COVID virus (viral RNA) in stool samples from children with multisystem inflammatory syndrome (a rare but serious condition that often strikes around four weeks after catching COVID) as well as spike protein and a marker of gut leakiness in their blood.”

Long COVID—also known as long-haul COVID, post-COVID-19, or its technical name, post-acute sequelae of COVID-19 or PASC—can involve health problems continuing weeks, months, or even years after a positive diagnosis, according to the federal Centers for Disease Control and Prevention (CDC).

Symptoms of long COVID, according to the researchers, include:

  • fatigue,
  • loss of smell,
  • memory loss,
  • gastrointestinal distress, and
  • shortness of breath. 

“If someone could somehow get to that viral load and eliminate it, it might lead to resolution of symptoms,” Walt told the Boston Globe, which noted that the researchers may explore a clinical trial involving antiviral drugs for treatment of long COVID-19.

Clues from Earlier Studies on Long COVID-19

Medical conditions that persisted following a COVID-19 infection have been studied for some time. In fact, in an earlier study, Walt and others found children who developed a multisystem inflammation syndrome weeks after being infected by SARS-CoV-2, according to their 2021 paper published in The Journal of Clinical Investigation, titled, “Multisystem Inflammatory Syndrome in Children Is Driven by Zonulin-Dependent Loss of Gut Mucosal Barrier.”

Although these earlier studies provided clues, the cause of PASC remains unclear, the researchers noted. They planned to take a more precise look at PASC biology by using appropriate sampling and patient recruitment.

“Disentangling the complex biology of PASC will rely on the identification of biomarkers that enable classification of patient phenotypes. Here, we analyze plasma samples collected from PASC and COVID-19 patients to determine the levels of SARS-CoV-2 antigens and cytokines and identify a blood biomarker that appears in the majority of PASC patients,” the researchers wrote.

Finding a Marker of a Persistent Infection

The researchers used plasma samples from 63 people with a previous SARS-CoV-2 diagnosis (37 also had PASC), Medical News Today reported. Over a 12-month period, the researchers’ findings included:

  • Detection in 65% of PASC samples of full-length spike, S1 spike, and nucleocapsid throughout the year of testing.
  • Spike detected in 60% of PASC patient samples, and not found in the COVID-19 samples.

In an interview with Scientific American, bioengineer Zoe Swank PhD, post-doctoral researcher, Brigham and Women’s Hospital, and co-author of the study, said, “Our main hypothesis is that the spike protein is not causing the symptoms, but it’s just a marker that is released because you still have infection of some cells with SARS-CoV-2.” 

In that article, Swank shared the scientists’ intent to do more research involving hundreds of samples over the course of the COVID-19 pandemic from many hospitals and people.

COVID-19 Not the Only Virus That Hangs On

Having a long-haul COVID-19 marker is a “game-changer,” according to an infectious disease expert who was not involved in the study.

“There has not so far been a clear, objective marker that is measurable in the blood of people experiencing long COVID-19,” Michael Peluso, MD, Assistant Professor, Medicine, University of California San Francisco, told Scientific American. “I hope their findings will hold up. It really would make a difference for a lot of people if a marker like this could be validated,” he added.

However, COVID-19 is not the only virus that could persist. Ebola also may linger in areas that skirt the immune system, such as the eye interior and central nervous system, according to a World Health Organization fact sheet.

Thus, medical laboratory leaders may want to follow the Brigham and Women’s Hospital research to see if the scientists validate their finding, discover a biomarker for long-haul COVID-19, and pursue a clinical trial for antiviral drugs. Such discoveries could have implications for how diagnostic professionals work with physicians to care for long COVID patients.   

Donna Marie Pocius

Related Information:

Persistent Circulating SARS-CoV-2 Spike Is Associated with Post-Acute COVID-19 Sequelae

Long COVID: “Viral Reservoir” of Spike Protein May Explain Long-Term Symptoms

Are Pockets of COVID in the Gut Causing Long-Term Symptoms?

CDC: COVID-19: Long-Term Effects

Boston Researchers May Have Found Biomarker for Long COVID

Multisystem Inflammatory Syndrome in Children Is Driven by Zonulin-Dependent Loss of Gut Mucosal Barrier

People with Long COVID May Still Have Spike Proteins in Their Blood

WHO: Ebola Virus Disease

Multiple Studies Raise Questions About Reliability of Clinical Laboratory COVID-19 Diagnostic Tests

In the absence of a “gold standard,” researchers are finding a high frequency of false negatives among SARS-CoV-2 RT-PCR tests

Serology tests designed to detect antibodies to the SARS-CoV-2 coronavirus that causes the COVID-19 illness have been dogged by well-publicized questions about accuracy. However, researchers also are raising concerns about the accuracy of molecular diagnostics which claim to detect the actual presence of the coronavirus itself.

“Diagnostic tests, typically involving a nasopharyngeal swab, can be inaccurate in two ways,” said Steven Woloshin, MD, MS, in a news release announcing a new report that “examines challenges and implications of false-negative COVID-19 tests.” Woloshin is an internist, a professor at Dartmouth Institute, and co-director of the Geisel School of Medicine at Dartmouth.

“A false-positive result mistakenly labels a person infected, with consequences including unnecessary quarantine and contact tracing,” he stated in the news release. “False-negative results are far more consequential, because infected persons who might be asymptomatic may not be isolated and can infect others.”

Woloshin led a team of Dartmouth researchers who analyzed two studies from Wuhan, China, and a literature review by researchers in Europe and South America that indicated diagnostic tests for COVID-19 are frequently generating false negatives. The team published their results in the June 5 New England Journal of Medicine (NEJM).

For example, one research team in Wuhan collected samples from 213 hospitalized COVID-19 patients and found that an approved RT-PCR test produced false negatives in 11% of sputum samples, 27% of nasal samples, and 40% of throat samples. Their research was published on the medRxiv preprint server and has not been peer-reviewed.

The literature review Woloshin’s team studied was also published on medRxiv, titled, “False-Negative Results of Initial Rt-PCR Assays for COVID-19: A Systematic Review.” It indicated that the rate of false negatives could be as high as 29%. The authors of the review looked at five studies that had enrolled a total of 957 patients. “The collected evidence has several limitations, including risk of bias issues, high heterogeneity, and concerns about its applicability,” they wrote. “Nonetheless, our findings reinforce the need for repeated testing in patients with suspicion of SARS-Cov-2 infection.”

Another literature review, published in the Annals of Internal Medicine, titled, “Variation in False-Negative Rate of Reverse Transcriptase Polymerase Chain Reaction–Based SARS-CoV-2 Tests by Time Since Exposure,” estimated the probability of false negatives in RT-PCR tests at varying intervals from the time of exposure and symptom onset. For example, the authors found that the median false-negative rate was 38% if a test was performed on the day of symptom onset, versus 20% three days after onset. Their analysis was based on seven studies, five of which were peer-reviewed, with a total of 1330 test samples.

Doctors also are seeing anecdotal evidence of false negatives. For example, clinicians at UC San Diego Health medical center treated a patient with obvious symptoms of COVID-19, but two tests performed on throat samples were negative. However, a third test, using a sample from a bronchial wash, identified the virus, reported Medscape.

The lesson for clinicians is that they can’t rely solely on test results but must also consider their own observations of the patient, Joshua Metlay, MD, PhD, of Massachusetts General Hospital told Medscape.

Sensitivity and Specificity of COVID-19 Clinical Laboratory Tests

The key measures of test accuracy are sensitivity, which refers to the ability to detect the presence of the virus, and specificity, the ability to determine that the targeted pathogen is not present. “So, a sensitive test is less likely to provide a false-negative result and a specific test is less likely to provide a false-positive result,” wrote Kirsten Meek, PhD, medical writer and editor, in an article for ARUP Laboratories.

“Analytic” sensitivity and specificity “represent the accuracy of a test under ideal conditions in which specimens have been collected from patients with either high viral loads or a complete absence of exposure,” she wrote. However, “sensitivity and specificity under real-world conditions, in which patients are more variable and specimen collection may not be ideal, can often be lower than reported numbers.”

In a statement defending its ID Now molecular point-of-care test, which came under scrutiny during a study of COVID-19 molecular tests by NYU Langone Health, Northwell Health, and Cleveland Clinic, according to MedTech Dive, Abbott Laboratories blamed improper sample collection and handling for highly-publicized false negatives produced by its rapid test. An FDA issued alert about the test on May 14 noted that Abbott had agreed to conduct post-market studies to identify the cause of the false negatives and suggest remedial actions.

Issues with Emergency Use Authorizations

In their NEJM analysis, Woloshin et al point to issues with the FDA’s process for issuing Emergency Use Authorizations (EUAs). For example, they noted variations in how manufacturers are conducting clinical evaluations to determine test performance. “The FDA prefers the use of ‘natural clinical specimens’ but has permitted the use of ‘contrived specimens’ produced by adding viral RNA or inactivated virus to leftover clinical material,” they wrote.

When evaluating clinical performance, manufacturers ordinarily conduct an index test of patients and compare the results with reference-standard test, according to the Dartmouth researchers. For people showing symptoms, the reference standard should be a clinical diagnosis performed by an independent adjudication panel. However, they wrote, “it is unclear whether the sensitivity of any FDA-authorized commercial test has been assessed in this way.” Additionally, a reference standard for determining sensitivity in asymptomatic people “is an unsolved problem that needs urgent attention to increase confidence in test results for contact-tracing or screening purposes.”

Stephen Rawlings, MD, PhD
“To truly determine false negatives, you need a gold standard test, which is essentially as close to perfect as we can get,” Stephen Rawlings, MD, PhD, (above), a resident physician of internal medicine and infectious diseases fellow at UC San Diego’s Center for AIDS Research (CFAR), who has been working on SARS-CoV-2 test validation since March. “But there just isn’t one yet for coronavirus,” he told Medscape. (Photo copyright: University of California, San Diego.)

In a perspective for Mayo Clinic Proceedings, Colin P. West, MD, PhD; Victor M. Montori, MD, MSc; and Priya Sampathkumar, MD, offered four recommendations for addressing concerns about testing accuracy:

  • Continued adherence to current measures, such as physical distancing and surface disinfection.
  • Development of highly sensitive and specific tests or combinations of tests to minimize the risk of false-negative results and ongoing transmission based on a false sense of security.
  • Improved RT-PCR tests and serological assays.
  • Development and communication of clear risk-stratified protocols for management of negative COVID-19 test results.

“These protocols must evolve as diagnostic test, transmission, and outcome statistics become more available,” they wrote.

Meanwhile, clinical laboratories remain somewhat on their own at selecting which COVID-19 molecular and serology tests they want to purchase and run in their labs. Complicating such decisions is the fact that many of the nation’s most reputable in vitro diagnostics manufacturers cannot produce enough of their COVID-19 tests to meet demand.

Consequently, when looking to purchase tests for SARS-CoV-2, smaller medical laboratory organizations find themselves evaluating COVID-19 kits developed by little-known or even brand-new companies.

—Stephen Beale

Related Information:

New Report Examines Challenges and Implications of False-Negative COVID-19 Tests

Questions about COVID-19 Test Accuracy Raised Across the Testing Spectrum

COVID-19 Test Results: Don’t Discount Clinical Intuition

FDA Provides New Tool to Aid Development and Evaluation of Diagnostic Tests That Detect SARS-CoV-2 Infection

EUA Authorized Serology Test Performance

Emergency Use Authorization (EUA) Information and List of All Current EUAs 

Coronavirus (COVID-19) Update: FDA Provides Promised Transparency for Antibody Tests

Understanding Medical Tests: Sensitivity, Specificity, and Positive Predictive Value

Webinar Part 1: Quality Issues Your Clinical Laboratory Should Know Before You Buy or Select COVID-19 Serology Tests

Webinar Part 2: Achieving High Confidence Levels in the Quality and Accuracy of Your Clinical Lab’s Chosen COVID-19 Serology Tests, featuring James Westgard, PhD

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