Emerging pandemic coronavirus (CoV) was recognized in Wuhan, China, in late 2019. The virus, isolated from patients mentioned to be pneumonic, was quickly sequenced to share 79.6% full length genome similarity with the Severe Acute Respiratory Syndrome virus (SARS-CoV-1) and 91.2% similarity between its nucleocapsid (N) proteins1. The novel SARS-CoV-2, causing COVID-19, was identified to be circulating in horseshoe bats for decades similarly to SARS-CoV-12. Diagnostic nucleic acid amplification tests (NAAT), mostly quantitative real-time polymerase chain reaction (qRT-PCR), were quickly developed worldwide, based on protocol provided for World Health Organization3. Diagnostic qRT-PCR capacities were ramped up quickly in central laboratories because such tests are fast to develop for new targets. Most often, the new qRT-PCR tests were adopted for clinical diagnostics with minimal verification and validation against other diagnostic test methods.
For the seasonal coronaviruses, the interpretation of gene positivity in clinical specimens has been challenging since the viral RNA is detected at similar rates and qRT-PCR cycle threshold (Ct) values from symptomatic and asymptomatic individuals. The viral RNA is also co-detected with genomes of other respiratory viruses4–7. This is also the case for the SARS-CoV-28, 9. Moreover, recent scientific evidence indicates that qRT-PCR positivity has poor correlation for assessment of SARS-CoV-2 infectiousness10–16. Whereas, Pekosz et al. (2020) showed that the detection of N-protein by an antigen test correlates with SARS-CoV-2 viral culture more accurately than qRT-PCR13. Already half a decade ago Inagaki et al. (2016) unequivocally concluded for influenza that, “PCR...is not an appropriate method for indicating infectivity” and “the antigen-detection test estimated the infectious period with comparable if not better accuracy with culture”17. In the case of COVID-19 diagnostics, the fact that viral RNA persistence can be detected without viable virus for months, has been a known clinical challenge, as diagnostics relied in the beginning of the pandemic solely on NAAT detection18, the efficacy of which is in ruling out positivity.
The expression of N-protein, which is the key pathogenicity factor of coronaviruses19, is essential for the coronavirus replication and transcription of the viral RNA20, 21. Without the accumulation of the N-protein, the coronaviral mRNA is degraded by the nonsense-mediated decay (NMD) pathway of eukaryotic cells19. Alexandersen at al. (2020) concluded that the detection of RNA is not an indicator of actively replicating SARS-CoV-2. Their data suggests that virion and subgenomic RNAs are stable in cellular double-membrane vesicles and, therefore, can be detected long after the acute infection22. Furthermore, Zhang et al. (2021) found that parts of the reverse-transcribed SARS-CoV-2 RNA can integrate into the human genome without the ability to yield infectious viruses and suggest that this could explain at least partly the long term RNA shedding23.
Shortly after viral exposure, viral concentration is low and qRT-PCR Ct values are high. When the virus starts replication, it happens fast. In a cell model, extensive coronavirus RNA transcription has occurred in 6 to 8 hours after the infection24. In addition, NAATs being prone for reporting clinically insignificant findings (analytically the detection may be correct, there is viral RNA in the sample) they are prone to contaminations. A study of SARS-CoV-2 primer-probe sets from four major European suppliers found a significant level of contamination from the reagents. False positives as low as qRT-PCR Ct 17 were obtained25. Low levels of SARS-CoV-2 RNA contamination has also been found from surfaces and air in rooms where mildly ill individuals were isolated without notable viable virus26, 27. It has also been shown that environmental contamination may yield in positive test results in PCR among individuals sampled in the same area where intranasal influenza vaccine dosing was done28. These data suggests that individuals having presence near symptomatic patients can be contaminated by RNA without being infected with viable virus. Thus, methods detecting the viral RNA by amplification are prone for clinically insignificant positive results, especially when significant part of the population has been infected recently. The fact that a positive NAAT result is not a reliable biomarker of active infection or COVID-19, is a true challenge for clinicians and decision making for quarantine. It is not only that a missed necessary quarantine has health and epidemic costs but also that a falsely imposed quarantine has social and financial consequences29.
The different performance requirements of diagnostic, surveillance and screening testing have been recently discussed by Mina and Andersen (2020). There is a need for both super sensitive PCR based tests and rapid and appropriately sensitive antigen tests to fight the COVID-19 pandemic30. The use of the two methodologies should supplement one another in clinical practice and pandemic fight.
In the present study, we analytically and clinically validated the performance of a novel 2nd generation mariPOC SARS-CoV-2 test (ArcDia International Ltd, Finland), which is a promising test to decentralize and speed up coronavirus testing31, as intended for rapid and automated detection of viral acute phase proteins when there is a clinical suspicion of acute COVID-19. Monoclonal antibodies of the test are designed to target a conserved epitope in the N-protein, which is the most abundant protein in coronaviruses. We have previously shown that the presence of coronavirus OC43 N-protein in the nasopharynx correlates with the respiratory tract infection symptoms32. It has been shown that clinical presentations of seasonal coronavirus OC43 infections can be similar to those of coronaviruses that are considered as severe viruses (SARS and MERS)33.
The mariPOC is an automated platform for the rapid multianalyte testing of acute infectious diseases. It is based on a separation-free two-photon excitation assay technique34, 35. Subsequently to nasopharyngeal sampling, the mariPOC test’s operational steps are: combining the swab with the sample buffer from a bottle-top dispenser, vortexing the sample tube to release the sample from the swab (Fig. 1), followed by automated analysis and objective fluorescent result read out. The analysis is performed by an automated analyzer with sophisticated autoverification functions, and the result can be transferred automatically to the laboratory information system or as anonymized epidemiological data36 into mariCloud™ service. The hands-on time is one minute per sample, and the analyzer works in continuous-feed and walk-away mode. The mariPOC SARS-CoV-2 test is available as a single pathogen test and as part of syndromic multianalyte tests covering, among others, influenza viruses. The throughput of one mariPOC analyzer can be up to 300 single analyte tests or 100 multianalyte tests in 24 hours. The results are reported in two phases, great majority of the infectious cases in twenty minutes and very low positive and negative cases in 55 minutes or two hours, depending on the test configuration.