key: cord-0992923-kh6dzlam
authors: Menchinelli, G.; Bordi, L.; Marzialiotti, F.; Palucci, I.; Capobianchi, M. R.; Sberna, G.; Lalle, E.; Romano, L.; De Angelis, G.; Marchetti, S.; Sanguinetti, M.; Cattani, P.; Posteraro, B.
title: Lumipulse G SARS-CoV-2 Ag Assay Evaluation for SARS-CoV-2 Antigen Detection Using 594 Nasopharyngeal Swab Samples from Different Testing Groups
date: 2021-01-29
journal: nan
DOI: 10.1101/2021.01.26.21250533
sha: 6ae076e6243826956252b1e4b36700357f89ed2c
doc_id: 992923
cord_uid: kh6dzlam

Compared to RT-PCR, lower performance of antigen detection assays, including the Lumipulse G SARS-CoV-2 Ag assay, may depend on specific testing scenarios. We tested 594 nasopharyngeal swab samples from individuals with COVID-19 (RT-PCR cycle threshold [Ct] values [≤]40) or non-COVID-19 (Ct values [≤]40) diagnoses. RT-PCR positive samples were assigned to diagnostic, screening, or monitoring groups of testing. With a limit of detection of 1.2 x 104 SARS-CoV-2 RNA copies/ml, Lumipulse showed positive percent agreement (PPA) of 79.9% (155/194) and negative percent agreement of 99.3% (397/400), whereas PPAs were 100% for samples with Ct values of <18 or 18-<25 and 92.5% for samples with Ct values of 25-<30. By three groups, Lumipulse showed PPA of 87.0% (60/69), 81.1% (43/53), or 72.2% (52/72), respectively, whereas PPA was 100% for samples with Ct values of <18 or 18-<25, and was 94.4%, 80.0%, or 100% for samples with Ct values of 25-<30, respectively. RT-PCR positive samples were also tested for SARS-CoV-2 subgenomic RNA and, by three groups, testing showed that PPA was 63.8% (44/69), 62.3% (33/53), or 33.3% (24/72), respectively. PPAs dropped to 55.6%, 20.0%, or 41.7% for samples with Ct values of 25-<30, respectively. All 101 samples with a subgenomic RNA positive result had a Lumipulse assays antigen positive result, whereas only 54 (58.1%) of remaining 93 samples had a Lumipulse assays antigen positive result. In conclusion, Lumipulse assay was highly sensitive in samples with low RT-PCR Ct values, implying repeated testing to reduce consequences of false-negative results.

Antigen testing has recently been added to the landscape of clinical laboratory methods to detect 43 and combat the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), which is the 44 notorious cause of coronavirus disease 2019 (COVID-19) (https://www.cdc.gov/coronavirus/2019-45 ncov/lab/resources/antigen-tests-guidelines.html#anchor_1597523027400). Like the molecular-46 relying on real-time reverse-transcription polymerase chain reaction (RT-PCR) and, to date, the standard method for the etiological COVID-19 diagnosis-antigen testing detects the presence of 48 SARS-CoV-2 in the acute infection phase only (1) . Unlike the molecular or antigen, antibody 49 testing is relevant in the convalescent or recovered infection phases only (1) . 50 Theoretically, antigen-based assays are advantageous in terms of fast turnaround times and 51 reduced costs but are less sensitive than RT-PCR-based assays (2). Additionally, the former have 52 the disadvantage to provide false-positive results, which leads false-positive patients to be managed 53 as patients with true SARS-CoV-2 infection (3, 4). However, the false-positive result likelihood 54 seems to depend on specific testing scenarios (e.g., those to identify infected persons who are 55 asymptomatic and without known or suspected exposure to SARS-CoV-2) 10 min at 37°C to allow specific binding to the antigen of aforementioned immunocomplexes, and 131 then to form additional immunocomplexes. Finally (after washing), a substrate solution is added 132 and incubated for 5 min at 37°C, and the resulting chemiluminescence signals are automatically 133 read by the analyzer and used to calculate the SARS-CoV-2 antigen's amount in the sample through 134 the interpolation with a SARS-CoV-2 Ag calibrator curve. 135 We determined the limit of detection (LOD) of the Lumipulse assay according to a previously 136 described protocol (9). Briefly, aforementioned contrived samples were spiked with a dilution series 137 of Vero E6 cell-cultured SARS-CoV-2 (INMI-1 strain) at a concentration range of 1.0 × 10 5 50% 138 tissue culture infective dose (TCID50)/ml (4.0 × 10 8 RNA copies/ml) to 1.0 TCID50/ml (4.0 × 10 3

RNA copies/ml), and then tested in replicates (Fig. S1 ). plotted the probability (y-axis) against the SARS-CoV-2 concentration's logarithm (x-axis), and we 145 calculated the 95% LOD value, which was the lowest concentration at which the replicates yielded 146 positive detection 95% of the time (Fig. S1 ).

Before testing with the Lumipulse assay, samples were centrifuged at 3000 × g for 15 min to allow 148 separation of the supernatant from the remaining viscous UTM material, and 100 µl were analyzed 149 for the antigen quantification as above described. Samples with an antigen level exceeding the 150 detection limit (i.e., 5000 pg/ml) were diluted, and dilutions were used to quantify the original perpetuity.

preprint (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in

The copyright holder for this this version posted January 29, 2021. ; https://doi.org/10.1101/2021.01.26.21250533 doi: medRxiv preprint RT-PCR assay for SARS-CoV-2 subgenomic RNA detection. To determine the presence of 156 SARS-CoV-2 subgenomic RNA (i.e., E gene subgenomic RNA), samples were subjected to a 157 previously developed in-house RT-PCR assay (13). This is an adaptation from the method described 158 by Wölfel et al. (14) that looks specifically at the E gene subgenomic RNA to indicate active virus 159 infection/transcription (15) . Briefly, SARS-CoV-2 RNA (also including genomic RNA) was 160 extracted from samples using the Seegene Nimbus automated system and then used for the RT-PCR perpetuity.

preprint (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in preprint (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. preprint (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in The copyright holder for this this version posted January 29, 2021. ; https://doi.org/10.1101/2021.01.26.21250533 doi: medRxiv preprint for-priority-diagnostics-to-support-response-to-the-covid-19-pandemic-v.0.1). Furthermore, we 332 limited oversampling of samples with laboratory-confirmed SARS-CoV-2 infection, which 333 accounted for the high risk of bias affecting patient selection in many published studies (17).

Stratifying our study participants by days from the symptom onset (5) was impracticable for us.

However, we compensated for this limitation by including testing groups that were comparable for 336 size (~60 RT-PCR positive samples per group), and we assumed that RT-PCR negative samples 337 were almost equally distributed across testing groups.

To summarize, our results show that Lumipulse assay's performance was satisfactory, confirming perpetuity. preprint (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in The copyright holder for this this version posted January 29, 2021. ; (15) 15 ( RT-PCR, real-time reverse-transcription polymerase chain reaction; SARS-CoV-2, severe acute respiratory syndrome coronavirus 2; Ct, cycle threshold; NA, not applicable. a Groups were established according to the Centers for Disease Control and Prevention (CDC) definitions for testing settings (https://www.cdc.gov/coronavirus/2019-ncov/lab/resources/antigen-tests-guidelines.html#anchor_1597523027400), and were further stratified by viral load (i.e., Ct values) as indicated. b SARS-CoV-2 antigen was detected in nasopharyngeal swab samples of groups' individuals by the Lumipulse G SARS-CoV-2 Ag assay, which provides a 0.01-5000 pg/ml measurement range. Using the manufacturer's cutoff of 1.34 pg/ml, results were expressed as negative, gray-zone positive, or positive when antigen concentrations in the samples were <1.34, 1.34-10, or >10-5000 pg/ml, respectively. Samples with antigen concentrations above 5000 pg/ml were rounded to 5000 pg/ml for convenience reasons. c SARS-CoV-2 subgenomic RNA was detected in nasopharyngeal swab samples of groups' individuals by in-house RT-PCR assay for the presence of replicative (E gene) RNA (Liotti, 2020c). d For comparisons between percent agreement rates. Testing from COVID-19 diagnosis, mean days ± SD 1.6 ± 3.3 NA d 6.1 ± 7.0 7.7 ± 8.6 <0.001 a All samples were from diagnostic (n = 59), screening (n = 63), or monitoring (n = 72) testing groups (see Table 2 ). Testing for SARS-CoV-2 subgenomic RNA was performed using an in-house RT-PCR assay to assess the presence of replicative (E gene) RNA, as previously described (Liotti, 2020c). b The time period between SARS-CoV-2 RT-PCR (to which Ct values refer) used to diagnose COVID-19 and testing for SARS-CoV-2 subgenomic RNA (and antigen) ranged from 0 days in the diagnostic or screening groups to 32 days in the monitoring group. Only in the last group, consequently, two temporally different samples were tested. c For comparisons between the RT-PCR positive/Antigen positive groups herein listed. d NA, not applicable.

COVID-19 testing

COVID-19 with prior negative results

Epub ahead of print

Virological assessment of hospitalized 407 patients with COVID-2019

SARS-CoV-2 genomic and subgenomic 410

RNAs in diagnostic samples are not an indicator of active replication

Routine use of point-of-care tests: usefulness and application in 413 clinical microbiology

The authors are grateful to the Reale Group and the Fondazione Valentino Garavani & Giancarlo 347