COVID-19 Serologic Assays: Study Using ELISA And Rapid Diagnostic Tests Show That Around 8.5 percent Of COVID-19 Patients Do Not Seroconvert
COVID-19 Serologic Assays
: A collaborative study led by St George’s University-London on the use of inexpensive and accurate ELISA and rapid diagnostic tests (RDT) to identify the demographic and clinical factors that affect the antibody response in COVID-19 patients, showed that up to 8.5% of COVID-19 patients do not seroconvert.
The research findings that are yet to have been peer-reviewed are published online on a preprint server. https://www.medrxiv.org/content/10.1101/2020.06.07.20124636v2
Serologic assays are just as crucial as RT-PCR Diagnostics or Antibody Tests as they can help with: vaccine development, the focused deployment of diagnostic testing, and the use of novel treatment. They may also help to understand the underlying disease process.
The utilization of the inexpensive enzyme-linked immunosorbent assay (ELISA) with RDT designed for lower to middle income countries makes this study unique in its applicability to developing regions. The researchers tested 645 clinical samples from 177 patients. About 34% and 35% were white and non-white, respectively. The median age was 64 years, with 57% being male. Almost three-fourth had one or more coexisting illnesses.
19 % or 34 patients were asymptomatic on admission and were diagnosed incidentally. For those with symptoms, there was a median delay of 6 days from the earliest symptom to testing. 94% were hospitalized, and a quarter died at a median interval of 19 days. 61% were discharged by the end of the study after a median hospital stay of 19 days. 14 (8%) were still in hospital when the study ended. 63 (36%) patients needed intensive care.
Significantly, from the IgG ELISA results, it is seen that about 8.5% did not seroconvert throughout the follow-up period. About 7% developed antibodies after enrolling in the study, while 84% had antibodies at the time of the first test.
The subsequent follow-up of the patients who did not develop antibodies up to the end of the study period, for another three weeks, shows that between 2% to 8.5% may fail to seroconvert for weeks despite having the infection.
Interestingly, after seroconversion, the mean IgG values remained constant for up to 2 months from the first symptom. The levels did not change by sex or the presence of respiratory symptoms. However, non-whites had a higher mean IgG titer at 1.06 vs 0.85 in whites.
Also seroconverters were older, at a median of 65 years than non-seroconverters, at 41 years, and more likely to have a coexisting disease. Notably, patients with hypertension were more likely to seroconvert, as were those with a higher body mass (BMI).
It was observed that rising C-reactive protein (CRP) levels predict a worse outcome due to the cytokine release syndrome. Non-seroconverters had lower CRP values compared to seroconverters. These differences were more pronounced if only peak CRP values were compared. Other markers of inflammation like peak D-dimer, fibrinogen, and ferritin followed the same trend.
These research findings relate to patients who present after being symptomatic for many days and presumably have reduced viral loads.
Serological tests can help pick up an infection in later phases of the disease when the RT-PCR is no longer sensitive enough. Again, serological tests can help understand which patients are at risk for infection and their immunity, as well as being immensely useful for epidemiological tests.
Importantly the stable antibody levels is another finding of significance, as well as the identification of factors related to seroconversion, namely, hypertension, symptomatic disease, increasing age, and increased BMI. Non-whites, those admitted to hospital, and higher inflammatory marker titers predict higher antibody levels, which in turn predict greater severity of the disease.
This implies that antibody titers and cytokines increase in parallel, signaling a higher risk of severe disease and death. Or on the other hand, it may be because the innate immune response is more robust in these individuals, with a resulting increased risk of severe disease and death. This could, of course, be due to a higher viral load or the activation of genes that regulate innate immunity. But higher viral loads are expected to stimulate acquired immunity, and this is more likely given the benefit shown with passive antibody therapy in small trials.
The 8.5% of patients who are non-seroconverters may truly never develop antibodies or may develop other forms of immune response such as T cell immunity or antibodies confined to other antigens. Again, very mild infections may involve only the respiratory mucosa, with dominant secretory immune responses, as a result, and, therefore, limited systemic IgG antibody production.
The research shows that many patients with COVID-19 remain seronegative for weeks after infection, and a proportion up to 60 days later is still negative for antibodies. This is the first such study outside China and shows how different races react to the infection. The advantages include the use of this first-generation ELISA assay to confirm infection purely on serological grounds, in a diverse group, and with infection at different phases from asymptomatic to fatal infection.
Importantly, the use of serological testing enhances diagnostic accuracy, especially when RT-PCR is negative, but symptoms suggest COVID-19. This may be a valuable procedure when late presentation becomes more prevalent, perhaps due to containment measures that lead patients to self-isolate rather than come to hospital.
“Serological testing, and the ability to detect viral antigens, may increase diagnostic accuracy for COVID-19 and our findings support early studies suggesting that these diagnostic modalities should be combined, particularly when RT-PCR results are negative but symptoms resemble those of COVID-19,”said the study team.
The research however has some limitations, such as the inclusion of only hospitalized patients, with about 20% being asymptomatic. Patients with mild infection must also be included in future studies, which will also address the association between the viral load as measured by RT-PCR and serological response, as well as the duration of antibody production.
Finally, the clinical contribution of antibody responses in the recovery or protection from this infection requires more intensive study.
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Understanding Seroconverstion Properly
Briefly, seroconversion is the transition from the point of viral infection to when antibodies of the virus become present in the blood. Given that many diagnostic tests use the presence of antibodies to infer illness, understanding seroconversion becomes a very important part of immunology and virology.
Why Seroconversion is important?
Though antibodies can be important for diagnosing disease in several viral contexts, such as with HIV and also COVID-19, in many cases, accurate diagnosis of COVID-19 is done by nucleic acid tests. However, there is some dispute over this, with some research indicating that detection of seroconversion can detect virus-specific antibodies despite negative nucleic acid tests.
Whatever it maybe, determining seroconversion can be important for understanding the immune response, infection rates, and identification of potential serum donors.
Comprehending all three aforementioned benefits of studying seroconversion is critical for understanding disease proliferation and spread. For example, information on the rate of infection is needed to determine the infection fatality rate with any accuracy.
Understanding the seroconversion in a quantitative manner can allow for the detection of individuals who have strong antibody responses to viruses, and can, therefore, be donors. Similarly, studying seroconversion can aid in understanding which antibody responses are associated with protection from the virus in question.
However seroconversion is not limited to clear cases of infection. Asymptomatic patients can also undergo seroconversion. Similarly, detection of seroconversion does not mean antibodies are present for an indefinite amount of time, nor that all individuals with a disease will undergo seroconversion.
For COVID-19, current research indicates that the production of disease-specific antibodies, or the completion of seroconversion, is completed within 17-19 or 20-22 days of the onset of symptoms, depending on the antibody in question. The median for undergoing seroconversion for COVID-19 immunoglobin G and M (IgG and IgM) is 13 days after symptoms appear. https://www.nature.com/articles/s41591-020-0897-1
The Link Between Seroconversion & Infectiousness
Typically in most diseases, infectiousness is at its highest before seroconversion. This is true for HIV, where most seroconversion research has focused, but there is also evidence that this is occurring in SARS-CoV-2. Certain cases also indicate that virus shedding can continue after seroconversion. https://www.journalofinfection.com/article/S0163-4453(20)30190-0/pdf
Though the research on COVID-19 is extensive and mostly very new, the importance of seroconversion is highlighted in many papers. Where viral shedding continues past seroconversion, there are implications that the contagious period is extended by as much as a week after clinical recovery.
Almost similar results have been found for asymptomatic patients. This can be very damaging for infectious spread and for developing containment strategies.
Though seroconversion and the presence of antibodies can, in some diseases, confer immunity from re-infection, this may not always be the case. For example, there is so far not enough data to accurately determine whether seroconversion of COVID-19 leads to protection from re-infection.
Importantly, the basic reproduction number (R0) indicates how contagious a disease is. Studies on seroconversion and cross-reactivity have shown that when testing for seroconversion, there is little or no cross-reactivity from other human coronaviruses. This indicates that humans are ‘serologically naïve’ to COVID-19, meaning it has not been encountered before (unlike other viral diseases like influenza). This naivety to COVID-19 can be a contributing factor to the disease’s relatively high R0 number.
Most of the time seroconversion is detected based on the presence of antibodies. However, different diseases can trigger different types of antibodies. For example, for COVID-19, there have been documented cases of seroconversion of immunoglobin G and M (IgG and IgM, respectively). There can also be different combinations of seroconversion occurring. https://covid19.elsevierpure.com/en/publications/a-serological-assay-to-detect-sars-cov-2-seroconversion-in-humans
In one study of 26 COVID-19 patients, nine patients had synchronous seroconversion of IgG and IgM, seven patients had IgM seroconversion before IgG seroconversion, and ten patients had seroconversion of IgM later than that of IgG.
A way in which seroconversion can be detected is via a replication-competent virus, but this process can take several days and requires strict biosafety regulations. Other methods, which are more recently developed, include the use of traditional ELISAs in combination with pseudotyped viral particle-based entry assays. This type of method does not need to involve live viral particles, and therefore has fewer strict regulations involved and can be carried out easier.