key: cord-0727889-l4b2nark
authors: Koskinen, J. M.; Antikainen, P.; Hotakainen, K.; Haveri, A.; Ikonen, N.; Savolainen-Kopra, C.; Koskinen, J. O.
title: Clinical Validation of Automated and Rapid mariPOC SARS-CoV-2 Antigen Test
date: 2021-02-11
journal: nan
DOI: 10.1101/2021.02.08.21250086
sha: 89498436a2c1896a2b6686f7c6338fbe83782071
doc_id: 727889
cord_uid: l4b2nark

Novel SARS coronavirus causing COVID-19 was recognized in late 2019. Diagnostics was quickly ramped up worldwide based on the detection of viral RNA. Based on the scientific knowledge for pre-existing coronaviruses, it was expected that the RNA of this novel coronavirus will be detected at significant rates from symptomatic and asymptomatic individuals due to existence of non-infectious RNA. To increase the efficacy of diagnostics, surveillance, screening and pandemic control, rapid methods, such as antigen tests, are needed for decentralized testing and to assess infectiousness. The objectives were to verify analytical sensitivity and specificity, and assess the clinical sensitivity, specificity and usability of a novel automated mariPOC SARS-CoV-2 test based on sophisticated optical laser technology detecting viral structure proteins. Analytical performance was verified using bacterial and viral preparations. Clinical performance of the test was evaluated against qRT-PCR in a retrospective study with nasopharyngeal swab specimens (N=211) collected from symptomatic patients suspected of acute SARS-CoV-2 infections. Sensitivity and specificity of the mariPOC test were 92.3% (12/13) and 100.0% (198/198), respectively. The test's limit of detection was 22 PFU/test and it had no cross-reactions with the tested respiratory microbes. Our study shows that the mariPOC can detect infectious individuals already in 20 minutes while clinical sensitivity close to qRT-PCR is achieved in two hours or less. The test targets conserved epitopes of SARS-CoV-2 nucleoprotein, making it robust against strain variations. The new test is a promising and versatile tool for syndromic testing of symptomatic cases and for high capacity infection control screening.

virus, isolated from patients mentioned to be pneumonic, was quickly sequenced to share 79.6% 35 full length genome similarity with the Severe Acute Respiratory Syndrome virus (SARS-CoV-1) 36 and 91.2% similarity between its nucleocapsid (N) proteins (1). The novel SARS-CoV-2, 37 causing COVID-19, was identified to be circulating in horseshoe bats for decades similarly to CC-BY-ND 4.0 International license It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity.

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Most often, the new qRT-PCR tests were adopted for clinical diagnostics with minimal 43 verification and validation against other diagnostic test methods, such as viral culture and 44 serology. 45 For the seasonal coronaviruses, the interpretation of gene positivity in clinical specimens has 46 been challenging since the viral RNA is detected at similar rates and qRT-PCR cycle threshold 47 (Ct) values from symptomatic and asymptomatic individuals. The viral RNA is also co-detected 48 with genomes of other respiratory viruses (4-7). This is also the case for the SARS-CoV-2 (8, 9). concluded for influenza that, "PCR...is not an appropriate method for indicating infectivity" and 54 "the antigen-detection test estimated the infectious period with comparable if not better accuracy 55 with culture" (17). In the case of COVID-19 diagnostics, the fact that viral RNA persistence can 56 be detected without viable virus for months, has been a known clinical challenge, as diagnostics 57 relied in the beginning of the pandemic solely on NAAT detection (18), the efficacy of which is 58 in ruling out positivity.

The expression of N-protein, which is the key pathogenicity factor of coronaviruses (19), is CC-BY-ND 4.0 International license It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. (which was not certified by peer review) there is viral RNA in the sample) they are prone to contaminations. A study of SARS-CoV-2 71 primer-probe sets from four major European suppliers found a significant level of contamination The different performance requirements of diagnostic, surveillance and screening testing have 86 been recently discussed by Mina and Andersen (2020) . There is a need for both super sensitive 87 . CC-BY-ND 4.0 International license It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. The mariPOC is an automated platform for the rapid multianalyte testing of acute infectious 100 diseases. It is based on a separation-free two-photon excitation assay technique (32, 33). CC-BY-ND 4.0 International license It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity.

The copyright holder for this preprint this version posted February 11, 2021. ; https://doi.org/10.1101/2021.02.08.21250086 doi: medRxiv preprint Page 6 mariPOC analyzer can be up to 300 single analyte tests or 100 multianalyte tests in 24 hours. The 111 results are reported in two phases, highly infectious cases in twenty minutes and low positive and 112 negative cases in two hours or less, depending on the test configuration.

Analytical sensitivity. Nasopharyngeal swab specimens from asymptomatic individuals were 115 suspended into mariPOC RTI sample buffer into volume corresponding to 1.3 mL per swab. This (Table 1) . Above the Ct value 25, the positivity rate of the mariPOC declined as typical for 177 an antigen test.

Clinical validation. Prevalence of SARS-CoV-2 in the study cohort was 6%, which is well in 179 alignment with the prevalence during the study time in the geographical area (5%). Considering Our verification cohort showed 90% positivity rate for the mariPOC below qRT-PCR Ct 25 217 (Table 1 and Figure 2) . This was an excellent result taking into account that the samples were 218 . CC-BY-ND 4.0 International license It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity.

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Our study together with other scientific data suggests that the mariPOC can detect most CC-BY-ND 4.0 International license It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity.

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We thank Vita laboratories Ltd and Seoul Clinical Laboratory for performing RT-PCR analyses. Evolutionary origins of the SARS-CoV-2 sarbecovirus lineage responsible for the COVID-19 pandemic. 300

Nat Microbiol 5:1408-1417. https://doi.org/10.1038/s41564-020-0771-4. 301 . CC-BY-ND 4.0 International license It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. (which was not certified by peer review)

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. CC-BY-ND 4.0 International license It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. (which was not certified by peer review)

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Rethinking Covid-19 Test Sensitivity -A Strategy for 434

Temporal dynamics in viral shedding and transmissibility of COVID-19

urine in one step by two-photon excitation assay technique

Field 444 evaluation of a rapid antigen test (Panbio™ COVID-19 Ag Rapid Test Device) for COVID-19 diagnosis 445 in primary healthcare centres

Virological assessment of hospitalized patients with COVID-2019

Assessing Viral Shedding and Infectivity of Asymptomatic or Mildly Symptomatic Patients with COVID-452 19 in a Later Phase

Cell-455 the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. (which was not certified by peer review) The copyright holder for this preprint this version posted

Van den Bruel A. 2020. Rapid, point-of-care antigen and molecular-based 460 tests for diagnosis of SARS-CoV-2 infection

. CC-BY-ND 4.0 International license It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. (which was not certified by peer review)The copyright holder for this preprint this version posted February 11, 2021. ; https://doi.org/10.1101/2021.02.08.21250086 doi: medRxiv preprint . CC-BY-ND 4.0 International license It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. (which was not certified by peer review)The copyright holder for this preprint this version posted February 11, 2021. ; https://doi.org/10.1101/2021.02.08.21250086 doi: medRxiv preprint