A better predictor of the severity of COVID-19
Diagnostic tests played a crucial role during the global wave of COVID-19. The gold standard for infection with severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) continues to remain molecular tests such as reverse transcriptase polymerase chain reaction (RT-PCR) performed on nasopharyngeal swabs (NP). More recent tests, for example – chemiluminescent immunoassays (CLIA) and simple molecular matrices (Simoa), have also shown a good correlation with RT-PCR on NP swabs, at least for cycle thresholds.
Despite the high sensitivity of RT-PCR, the correlation between RT-PCR results of NP swabs and disease severity has been disputed. In addition, the variability of cycle cutoff (Ct) values depending on the sampling procedure, as well as the likelihood of false negatives, are some of the disadvantages associated with RT-PCR results from NP swabs collected. before symptoms appear. As a result, it is possible to improve the diagnosis of SARS-CoV-2 infection. The ideal diagnostic test should be able to detect viral presence with similar or greater sensitivity than RT-PCR performed on NP swabs, with a superior estimate of prognostic value, in addition to requiring readily accessible biological samples. .
Routinely, blood samples are used to determine the concentration of antibodies. Yet this technique is rarely used in cases of acute infection or because of the possibility of pre-existing circulating antibodies. Another alternative is to examine the antigen in non-respiratory fluids like the bloodstream, which is supported by evidence that SARS-CoV-2 migrates from the lungs to the bloodstream.
In this perspective, a new study published on the medRxiv* preprint server analyzed the clinical efficacy of two serum antigen tests to identify SARS CoV-2 infection and assess the kinetics of serum nucleocapsid (N) antigen in severe and non-severe patients for the prediction of the severity of the disease.
Here, the serum N antigen was assessed using CLIA and Simoa, and severity cutoffs were established. This study involved 90 patients and 243 blood samples taken at various times after the onset of symptoms. The severity of the disease was assessed using the World Health Organization (WHO) Clinical Progression Scale. The flowchart of the study (in accordance with the Declaration of Helsinki) has been illustrated below:
Study flow diagram
The commercial SARS-CoV-2 N-Protein Advantage kit, a sandwich enzyme immunoassay (ELISA) based on paramagnetic microbeads, was used to examine the samples. CLIA detected the SARS-CoV-2 N antigen in the serum of patients. RT PCR for the detection of SARS-CoV-2 in NP swab samples was performed by targeting the N2 and E genes. Data were analyzed using descriptive statistics. All longitudinal samples of the research population were used to estimate the temporal kinetics curves using smoothing splines with four nodes.
It was found that in individuals with severe symptoms, the maximum antigenic response was detected on day 7 using both tests. Following this, a decrease was recorded in the antigenic reaction up to day 20. The antigenic response in non-severe individuals corresponded to a plateau phase which gradually decreased over time. Using the Simoa test, the difference in kinetics between severe and non-severe individuals was more discernible.
Using these severity thresholds in kinetic models determined the optimal time from symptom onset in severe patients (i.e. 4-10 days). For both antigen assays, the clinical sensitivity was 100% and the clinical specificity was 92.3%.
In addition, when NP RT-PCR was used, the mean Ct value of asymptomatic patients was significantly higher than that of severe patients. At the same time, no significant difference was found between mild, moderate and severe patients. Serum antigen assays revealed that severe patients had higher antigen levels. The distinction between severe and non-severe patients was most evident between days 4 and 10.
On the Simoa and iFlash tests, the cut-offs for identifying patients at higher risk of severe disease were estimated at 5,043 pg / mL and a cut-off index (COI) of 313.8. The likelihood ratios for these cutoffs were 30.0 and 10.9, respectively, indicating their ability to differentiate severe from non-severe patients from day 2 to day 14.
Remarkably, patients with SARS-CoV-2 Spike IgG levels above the positive cutoff had statistically higher Ct values and decreased serum antigen levels. While the majority of patients (96.6%) with a negative SARS-CoV-2 Spike IgG were antigen positive in serum with both tests.
The analysis leads to the following conclusions.
- Detection of sensitive N antigen in serum offers an important new marker for the diagnosis of COVID-19 that is accessible in all clinical laboratories and requires only a blood test.
- It enables further prospective improvements to create rapid antigenic blood tests or integrated ELISAs that detect both antigens and antibodies.
- Significantly, assessing antigenemia in the first two weeks after symptom onset can help identify those at risk for developing severe COVID-19.
- These tests are more convenient as testing procedures for patients and may potentially improve clinical triage to optimize intensive care use.
medRxiv publishes preliminary scientific reports which are not peer reviewed and, therefore, should not be considered conclusive, guide clinical practice / health-related behavior, or treated as established information.