What is long COVID?

There have been over half billion cases of SARS CoV-2 (COVID-19) globally since the pandemic began. The majority of infected persons have been either asymptomatic or have experienced a mild acute infection from which they recover within a few weeks. However, some, even those who had mild cases, suffer symptoms that linger. This phenomenon of so-called long COVID (also called as post-acute sequelae of SARS CoV-2 infection (PASC), post-COVID-19 syndrome, or long-haul COVID) has been one of the most disturbing trends to emerge from the pandemic, even as the overall virulence of the prevalent dominant strains of SARS-CoV-2, the virus responsible for COVID-19 disease, has abated significantly. Our lack of knowledge of how and why long COVID occurs is perhaps its scariest aspect, and medical scientists worldwide have been scrambling to better understand the syndrome. However, there is no current consensus on the diagnosis, definition, symptoms, or duration of long COVID.

Even the estimates of what percent of patients develop long COVID vary widely (0.2% to 30%), in part because of methodological differences between studies. However, another confounding issue is that the symptoms most often attributed to long COVID – fatigue, fever, cough, difficulty breathing, or shortness of breath are shared with many other medical conditions, e.g., other post-viral syndromes and chronic fatigue.

A host of other symptoms have also been associated with long COVID: neurological symptoms, difficulty thinking or concentrating, depression, anxiety, headache, sleep disturbance, joint or muscle pain, cardiac symptoms, digestive problems including diarrhea and stomach pain, and vascular issues that include pulmonary embolism. This difficulty in even diagnosing long COVID, let alone treating it, arises from the fact that no test exists that can differentiate long COVID from other causes of the observed symptoms. An important aspect for ruling out the possibility of other causes is ascertaining whether a prospective long COVID patient was actually exposed to SARS-CoV-2 in the past.

Establishing previous SARS-CoV-2 infection

A key issue in determining if a person had COVID-19 in the past is the amount of time that may have transpired since the infection. RT-PCR tests for detecting SARS-CoV-2, while sensitive, are ideally performed at the time of peak viral load to avoid unacceptably high rates of false negatives. Serological assays for detecting blood-borne antibodies made by B cells against SARS-CoV-2 proteins can be useful, particularly for epidemiological surveys, in assessing prior contact with the virus. However, not only do antibodies to SARS-CoV-2 decrease over time, sometimes to undetectable levels, but widespread vaccination has led to a high prevalence of anti-COVID-19 antibodies in the general population.

A third option for retrospective diagnosis of SARS-CoV-2 infection involves antigen-specific memory T cells generated by the immune system following infection. Medical scientists at the University of Cambridge recently published results of a study conducted at a clinic for patients with long COVID symptoms. Most of them were thought to have been infected in the early spring of 2020, before COVID-19 testing was widely available.

Cell-based cytokine immunoassay for confirming the previous infection in long COVID patients

The researchers at the University of Cambridge developed a cell-based assay for measuring levels of cytokines interleukin-2 (IL-2) and interferon-γ (IFNγ) released by patients’ memory T cells after exposure to SARS-CoV-2 peptides in vitro (Fig. 1). In this case, blood was drawn and peripheral blood mononuclear cells (PBMCs) that include both B cells and T cells were isolated. The PBMCs were exposed to pools of peptides from various SARS-CoV-2 proteins, then levels of IL-2 and IFN-γ released by the memory T cells were measured. Samples from patients with SARS-CoV-2 infection proven by RT-qPCR were used as positive controls.

In the course of the study, the researchers determined that background in the IFN-γ response was unacceptably high. Therefore, the assay was focused on IL-2 to lower the rate of false positives. Using the IL-2 assay, they determined that 42.5% of their long COVID patients who had never been tested for COVID-19 initially and who were seronegative for SARS-CoV-2 antibodies during screening had indeed been infected with COVID19. Results from the positive control group revealed assay sensitivity of 75% and specificity of 88% for previous SARSCoV-2 infection more than six months after a positive PCR test.This method of confirming previous infection in patients suspected of having long COVID can provide reliable evidence of previous infection for up to 12 months, including in patients without detectable COVID-19 antibodies.

Fig. 1: Peripheral blood mononuclear cell (PBMC)-based assay for the detection of previous SARS-CoV-2 infection in seronegative patients with long COVID

Development of this assay provides an important diagnostic tool for clinicians tasked with treating patients who report symptoms consistent with long COVID. The outcome also demonstrates the power and potential of well-designed cell-based assays. In view of the published sensitivity and specificity of the assay, the number of presumptive long COVID patients found to be negative for previous COVID-19 infection is just as important as those determined to be positive. Because of the variety of potential causes for such symptoms, choosing an appropriate course of treatment is of the utmost importance.

Journal reference: Evidence of previous SARS-CoV-2 infection in seronegative patients with long COVID. Krishna BA, Lim EY, Mactavous L et al. EBioMedicine. 2022 Jun 27; 81:104129.

Related blogs

Drug Lot Release Potency Assay / Testing

Strategies For Generating Stable Cell Lines For Producing Therapeutic Antibodies