Quantitative SARS-CoV-2 antigen test as a tool able to predict the stage of the infection.

Dear Editor,

Fowler et al. proposed RNA RT-LAMP as a rapid and accurate tool to promptly identify highly contagious individuals during the pandemic era. In the current vaccination era, it would be useful to have a reliable tool to provide information on the infectivity/contagiousness of individuals.

FDA approved antigen (Ag) test as a fast and convenient alternative to PCR but, as known, this approach can be effective at symptoms onset, when viral antigen is abundant, otherwise false negative results can occur; moreover, positive antigenic results need to be confirmed by molecular test.

These assays are mostly qualitative and, even when a numerical value is provided, no straightforward correlation with the virological and clinical parameters has ever been demonstrated.

We evaluated an Ag test based on chemiluminescence (CLEIA), Lumipulse®G SARS-CoV-2 Ag (Fujirebio INC), in an extensive population with different characteristics.

This comparative study included 1000 nasopharyngeal samples (NPS), analyzed during the period fall/winter 2020-2021 at the Virology laboratory of Bambino Gesù Pediatric Hospital.

NPS were collected in Universal Transport Medium (UTM, Copan) and immediately analyzed for molecular SARS-CoV-2 detection by AllplexTM SARS-CoV-2 Assay (Seegene), according to which, 850 samples resulted positive (Cycle threshold, Ct<40) and 150 negative. Referring to an external standard curve (y = −3.179x + 42.28; R2 = 0.939), defined on the basis of serial dilutions of a commercial standard (EDX SARS-CoV-2 Standard, Exact Diagnostics LLC), for each Ct, the corresponding RNA viral load was calculated and expressed in log10 copies/ml.

Antigen detection was performed by Lumipulse®G SARS-CoV-2 Ag, using the automated Lumipulse G1200 System (Fujirebio).

Samples were considered negative when SARS-CoV-2 Ag concentration was <1 pg/ml, in gray zone when ≥1.00 and <10 pg/ml and positive when ≥ 10 pg/ml, according to manufacturer's instruction.

Considering molecular test as the reference standard, Ag showed a specificity of 95.33% (143/150 samples resulted negative, 7/150 resulted positive, 6 of which, included in the gray zone) and a sensitivity of 64% (541/850 samples had a SARS-CoV-2 Ag concentration <1 pg/ml, 131/850 between 1.00 and < 10 pg/ml, and 178/850 ≥ 10 pg/ml).

SARS-CoV-2 RNA viral load distribution versus antigen concentration, showed a clear difference in mean CTs and viral loads between the three groups (Fig. 1 ): negative (<1 pg/ml), gray-zone (≥ 1 and < 10 pg/ml) and positive (≥10 pg/ml) antigen, corresponded to 1.84, 3.15 and 6.31 log10 copies/ml, respectively (P value for trend < 0.001).

Fig. 1

SARS-CoV-2 RNA viral load (Ct or copies/ml) versus Ag concentration (pg/ml)

Samples were grouped according to antigen concentration in: Ag <1.00 pg/ml, Ag ≥ 1.00 and <10 pg/ml and Ag ≥ 10 pg/ml.

(A) Lines represent median SARS-CoV-2 RNA viral load expressed in Ct in the three groups. It was 35.86 (IQR: Ct 34.12–38.19), 32.28 (IQR: Ct 30.37–34.30) and 22.23 (IQR: Ct 18.48–26.05), respectively. P value for trend <0.001

(B) Box plots represent medians and quartiles of SARS-CoV-2 RNA viral load expressed in copies/ml in the three groups. It was 1.84 log10 copies/ml (IQR: 1.29–2.57 log10 copies/ml), 3.15 log10 copies/ml (IQR: 2.51–3.76 log10 copies/ml) and 6.31 log10 copies/ml (IQR: 5.11–7.49 log10 copies/ml), respectively. The ends of the box are the upper and lower quartiles, the median is marked by a rhombus inside the box. The whiskers extend to 5–95%. P value for trend < 0.001.

IQR abbreviation for interquartile range.

Of interest, only 5/541 samples with a negative antigen value showed >4 log10 copies/ml RNA viral load (0.9%). Lumipulse Ag assay showed a remarkable high sensitivity (97.4%) when considering samples with medium-high viral load (>4 log10 copies/ml).

A strong positive correlation (R2 = 0.841) was evident between RNA viral load (log10 copies/ml) and antigen concentration (log10 pg/ml).

For 278 patients, it was possible to reconstruct the history of infection and to correlate antigen detection with days after first SARS-CoV-2 detection (Fig. 2 ): all 58 samples with antigen levels ≥ 50 pg/ml were collected from patients tested within ten days from first positivity. Of interest, only 3/58 samples referred to antigen detected from 8 to 10 days from first positivity.

Fig. 2

Antigen concentration and timing of infection

Representation of the day of sample collection in relation to timing of infection and Ag concentration (log10 pg/ml) for 58 samples with Ag ≥ 50 pg/ml (orange dots), 20 samples with Ag ≥1 pg/ml and <50 pg/ml (gray dots) and 207 samples with Ag <1 pg/ml (blue dots). For each group, trend between Ag concentration and days after first SARS-CoV-2 Ag/RNA detection is indicated by dot line. P value for the two major groups < 0.001.

Conversely, 205/207 samples with Ag <1 pg/ml, referred to samples collected later than 10 days from the first SARS-CoV-2 detection, the remaining 2/207 samples were collected at the 9th day from diagnosis.

Finally, the 20 samples with antigen levels ≥1 pg/ml and <50 pg/ml, were distributed over a period ranging from the acute to the convalescence phase.

Both in symptomatic and asymptomatic subjects, SARS-CoV-2 RNA can be detectable up to 3,4 weeks or longer in nasopharynx. , During convalescence, in presence of low amount of RNA (Ct > 35), only in 2–5% of cases virus isolation is possible and the risk to transmit infection is negligible. However, it is fundamental to find a tool able to give indication on timing of infection.

Our data indicate that Lumipulse antigen quantification allows a definition of the period of the infection: antigen levels > 50 pg/ml characterize the early/acute phase, while antigen levels <1 pg/ml the late/convalescent phase.

The 99% of samples presenting an antigen concentration <1 pg/ml (538/543) had a viral load <4 log10 copies/ml, reported as associated to a post-acute phase and were collected in a late/convalescent period of the infection. Analyzing the clinical course of the infection in the patients with a viral load >4 log10 copies/ml (5/543), negative antigen NPS were collected at least two weeks after the first SARS-CoV-2 detection, thus, in the post-acute phase.

Moreover, samples with Ag concentration ≥10 pg/ml showed a strong linear correlation with the corresponding RNA viral load (R2 = 0.841). Since high viral load is related to the early stages of infection, we could assume the same for antigen detection. In support to this hypothesis, all samples with antigen levels >50 pg/ml were taken within 10 days from the first positivity (infection onset).

Overall, Lumipulse® Ag results well correlate to the timing of infection, showing a net demarcation (P value < 0.001) between samples with Ag concentration >50 pg/ml, associated with early stages, and those with Ag concentration <1 pg/ml, related to late/convalescent phases.

Our results go beyond the classical utilization of the qualitative antigen test, as reported by Young et al. in their letter, and offer a new and clinically relevant role for the quantitative antigen, as a parameter able to define the timing of the infection. This might be particularly useful in those patients with unknown status of infection, and/or for those without a molecular test at symptoms onset, and/or for those asymptomatic with a positive molecular test and/or for vaccinated subjects with low viral shedding.

In conclusion, while real time RT-PCR remains the cornerstone for diagnosis of SARS-CoV-2 infection, Lumipulse quantitative Ag can be useful to define the stage of the disease. In particular, a positive molecular test with a negative Ag test can reasonably indicate a convalescent phase, identifying those subjects with low chances of being contagious.