COVID-19: A PCR-defined pandemic

Abstract

The numbers of coronavirus disease 2019 (COVID-19) cases are increasing steadily in many parts of the world, and the global and devastating impact of the current pandemic on all aspects of our life is evident. The number of positive molecular diagnostic tests, which are largely based on real-time (RT) PCR assays that detect genetic material of the causative agent severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), still forms the basis for reporting both symptomatic and asymptomatic cases worldwide. These figures are also used to calculate the basic reproduction number, defined as R-zero (R0), a value relating to the average number of people an infected individual will infect. Asymptomatic carriers of the virus, including those super-spreading the virus, are not routinely captured in common testing strategies, which rather concentrate on symptomatic individuals, returnees from high-risk areas, and other high-risk groups (Nikolai et al., 2020Nikolai L.A. Meyer C.G. Kremsner P.G. Velavan T.P. Asymptomatic SARS Coronavirus 2 infection: invisible yet invincible.Int J Infect Dis. 2020; 100: 112-116Abstract Full Text Full Text PDF PubMed Scopus (129) Google Scholar). Clearly, any ascription of positive results to a COVID-19 diagnosis requires the occurrence of clinical symptoms and further evaluation and confirmation by physicians, including the appraisal of distinct laboratory parameters. In diagnostic SARS-CoV-2 assays, RT-PCR is based on the detection of the amount of distinct genetic fragments of the virus in an individual. The amount of gene fragments is routinely determined semi-quantitatively through the cycle threshold (Ct) value, which corresponds to the number of PCR amplification cycles in the diagnostic assays required to yield positive results. The Ct value increases with a decreasing viral load, and a low Ct value indicates a high viral load (Velavan and Meyer, 2020Velavan T.P. Meyer C.G. Reply to: asymptomatic infection by SARS 2 coronavirus: invisible but invincible.Int J Infect Dis. 2020; Google Scholar). In ambiguous cases, true quantitative assays can be applied. When assessing impending public health risks, as in the current pandemic, and when imposing local or countrywide lockdown measures, it is important to establish whether infection and infectivity can be determined based only on positive results of individual and mass diagnostic PCR tests. While the presence of RNA fragments identified by qualitative SARS-CoV-2 PCR tests certainly indicates prior contact with the viral genome or parts of it, no reliable statement can be made with regard to the actual infectivity of an individual. During the clinical course of COVID-19, the release of virus particles from somatic cells and shedding of the virus does not necessarily imply that the virus is contagious. Questions to gauge the reliability and dependability of PCR tests and their significance and impact on the clinical presentation have arisen, due to the finding of positive tests results during the phase of physical recovery from COVID-19 in patients who have already been discharged from hospital on the basis of several negative test results (Lu et al., 2020Lu J. Peng J. Xiong Q. Liu Z. Lin H. Tan X. et al.Clinical, immunological and virological characterization of COVID-19 patients that test re-positive for SARS-CoV-2 by RT-PCR.EBioMedicine. 2020; 59102960Abstract Full Text Full Text PDF PubMed Scopus (110) Google Scholar). Observations so far suggest a mean incubation period of 5 days and median incubation period of 4–5 days (Lauer et al., 2020Lauer S.A. Grantz K.H. Bi Q. Jones F.K. Zheng Q. Meredith H.R. et al.The Incubation period of coronavirus disease 2019 (COVID-19) from publicly reported confirmed cases: estimation and application.Ann Intern Med. 2020; 172: 577-582Crossref PubMed Scopus (3247) Google Scholar, McAloon et al., 2020McAloon C. Collins A. Hunt K. Barber A. Byrne A.W. Butler F. et al.Incubation period of COVID-19: a rapid systematic review and meta-analysis of observational research.BMJ Open. 2020; 10e039652Crossref PubMed Scopus (235) Google Scholar) from exposure to the onset of symptoms. Viral RNA is detectable in the airways 2–3 days before the onset of symptoms, peaks at the onset of symptoms, and decreases over the following 7–8 days in most patients (Rhee et al., 2020Rhee C. Kanjilal S. Baker M. Klompas M. Duration of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infectivity: when is it safe to discontinue isolation?.Clin Infect Dis. 2020; Crossref Scopus (126) Google Scholar, Team, 2020Team C.-I. Clinical and virologic characteristics of the first 12 patients with coronavirus disease 2019 (COVID-19) in the United States.Nat Med. 2020; 26: 861-868Crossref PubMed Scopus (234) Google Scholar, To et al., 2020To K.K. Tsang O.T. Leung W.S. Tam A.R. Wu T.C. Lung D.C. et al.Temporal profiles of viral load in posterior oropharyngeal saliva samples and serum antibody responses during infection by SARS-CoV-2: an observational cohort study.Lancet Infect Dis. 2020; 20: 565-574Abstract Full Text Full Text PDF PubMed Scopus (2049) Google Scholar, Wolfel et al., 2020Wolfel R. Corman V.M. Guggemos W. Seilmaier M. Zange S. Muller M.A. et al.Virological assessment of hospitalized patients with COVID-2019.Nature. 2020; 581: 465-469Crossref PubMed Scopus (4045) Google Scholar, Zou et al., 2020Zou L. Ruan F. Huang M. Liang L. Huang H. Hong Z. et al.SARS-CoV-2 viral load in upper respiratory specimens of infected patients.N Engl J Med. 2020; 382: 1177-1179Crossref PubMed Scopus (3028) Google Scholar). Viral load kinetics and the duration of viral shedding are vital elements in determining the SARS-CoV-2 infectivity. A systematic review and meta-analysis has confirmed that SARS-CoV-2 viral shedding may be longer and is proportional to the severity of illness. However, the viability of the virus is short and not beyond 9 days of illness (Cevik et al., 2020Cevik M TM, Lloyd O, Maraolo AE, Schafers J, Ho A. SARS-CoV-2, SARS-CoV, and MERS-CoV viral load dynamics, duration of viral shedding, and infectiousness: a systematic review and meta-analysis. The Lancet Microbe 2020.Google Scholar). Studies evaluating the duration of SARS-CoV-2 infectivity based on cell culture and/or secondary infection rates clearly imply that the virus cannot be cultured from respiratory samples after day 8 of clinical disease (Bullard et al., 2020Bullard J. Dust K. Funk D. Strong J.E. Alexander D. Garnett L. et al.Predicting infectious SARS-CoV-2 from diagnostic samples.Clin Infect Dis. 2020; (May 22: ciaa638)https://doi.org/10.1093/cid/ciaa638Crossref PubMed Scopus (647) Google Scholar, Cevik et al., 2020Cevik M TM, Lloyd O, Maraolo AE, Schafers J, Ho A. SARS-CoV-2, SARS-CoV, and MERS-CoV viral load dynamics, duration of viral shedding, and infectiousness: a systematic review and meta-analysis. The Lancet Microbe 2020.Google Scholar, La Scola et al., 2020La Scola B. Le Bideau M. Andreani J. Hoang V.T. Grimaldier C. Colson P. et al.Viral RNA load as determined by cell culture as a management tool for discharge of SARS-CoV-2 patients from infectious disease wards.Eur J Clin Microbiol Infect Dis. 2020; 39: 1059-1061Crossref PubMed Scopus (525) Google Scholar). A detailed virological analysis of COVID-19 cases confirmed that the virus can only be cultured from respiratory samples during the first week of symptoms, but not after day 8, in spite of persisting high virus loads as determined by quantitative RT-PCR (Wolfel et al., 2020Wolfel R. Corman V.M. Guggemos W. Seilmaier M. Zange S. Muller M.A. et al.Virological assessment of hospitalized patients with COVID-2019.Nature. 2020; 581: 465-469Crossref PubMed Scopus (4045) Google Scholar). In addition, the US Centers for Disease Control and Prevention collected data from adults in various age groups and with varying disease severity, and indicated that the virus could not be cultured more than 10 days after the onset of symptoms (CDC, 2020aCDC. Centers for Disease Control and Prevention: duration of isolation and precautions for adults with COVID-19. https://www.cdc.gov/coronavirus/2019-ncov/hcp/duration-isolation.html (Accessed 30 October 2020).Google Scholar). Furthermore, virus culture has been found to be unfeasible in cases with a Ct value exceeding 33 (La Scola et al., 2020La Scola B. Le Bideau M. Andreani J. Hoang V.T. Grimaldier C. Colson P. et al.Viral RNA load as determined by cell culture as a management tool for discharge of SARS-CoV-2 patients from infectious disease wards.Eur J Clin Microbiol Infect Dis. 2020; 39: 1059-1061Crossref PubMed Scopus (525) Google Scholar, Rhee et al., 2020Rhee C. Kanjilal S. Baker M. Klompas M. Duration of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infectivity: when is it safe to discontinue isolation?.Clin Infect Dis. 2020; Crossref Scopus (126) Google Scholar). Of note, the US CDC reasonably recommends a symptom-based decision for returning from isolation, specifically rejecting the exclusively test-based strategy, unless it would result in an earlier decision. A prospective cohort study involving the first 100 COVID-19 patients in Singapore also showed that attempts to culture the virus failed in all PCR-positive samples with a Ct value >30 (Young et al., 2020Young B.E. Ong S.W.X. Kalimuddin S. Low J.G. Tan S.Y. Loh J. et al.Epidemiologic features and clinical course of patients infected with SARS-CoV-2 in Singapore.JAMA. 2020; 323: 1488-1494Crossref PubMed Scopus (1323) Google Scholar). A study by the Korean Centers for Disease Control and Prevention, including 285 patients who had recovered from COVID-19 clinically and had been discharged from hospital, showed that these individuals tested positive again by PCR an average of 45 days after the onset of the first symptoms (CDC, 2020aCDC. Centers for Disease Control and Prevention: duration of isolation and precautions for adults with COVID-19. https://www.cdc.gov/coronavirus/2019-ncov/hcp/duration-isolation.html (Accessed 30 October 2020).Google Scholar; CDC Korea, 2020bCDC K. Korean CDC: findings from investigation and analysis of re-positive cases. https://www.cdc.go.kr/board/board.es?mid=&bid=0030&act=view&list_no=367267&nPage=1 (Accessed 30 October 2020).Google Scholar). Ct values are lowest shortly after symptom onset and correlate significantly with time elapsed since the onset of symptoms (Salvatore et al., 2020Salvatore P.P. Dawson P. Wadhwa A. Rabold E.M. Buono S. Dietrich E.A. et al.Epidemiological correlates of PCR cycle threshold values in the detection of SARS-CoV-2.Clin Infect Dis. 2020; Crossref Scopus (32) Google Scholar). Defining the duration of infectivity of SARS-CoV-2 has major implications in determining incidences. Several studies now suggest that persistent positive RT-PCRs do not necessarily indicate the presence of replication-competent viruses (Alexandersen et al., 2020Alexandersen S. Chamings A. Bhatta T.R. SARS-CoV-2 genomic and subgenomic RNAs in diagnostic samples are not an indicator of active replication.medRxiv. 2020; (2020.06.01.20119750)Google Scholar, Rhee et al., 2020Rhee C. Kanjilal S. Baker M. Klompas M. Duration of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infectivity: when is it safe to discontinue isolation?.Clin Infect Dis. 2020; Crossref Scopus (126) Google Scholar). In fact, sustained RNA detection does not indicate sustained infectivity, and SARS-CoV-2 genomic and sub-genomic RNAs in diagnostic samples are not an indicator of active virus replication (Alexandersen et al., 2020Alexandersen S. Chamings A. Bhatta T.R. SARS-CoV-2 genomic and subgenomic RNAs in diagnostic samples are not an indicator of active replication.medRxiv. 2020; (2020.06.01.20119750)Google Scholar). It has also been shown that the contagiousness in patients with mild or moderate COVID-19 decreases rapidly to near zero approximately 10 days after symptom onset (Rhee et al., 2020Rhee C. Kanjilal S. Baker M. Klompas M. Duration of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infectivity: when is it safe to discontinue isolation?.Clin Infect Dis. 2020; Crossref Scopus (126) Google Scholar). Early testing for SARS-CoV-2 in individuals with symptoms is important to determine infectivity based on low Ct values, and early isolation practices to effectively interrupt SARS-CoV-2 transmission should be commenced when first symptoms appear. However, testing individuals 7 days after the onset of symptoms, which is more likely to be done in low- and middle-income countries, but also occurs in developed countries, only contributes to the assessment of the case numbers. Thus, the veracity of the testing strategy with regard to an estimation of infectivity is questionable. It has to be considered whether only laboratory diagnoses of SARS-CoV-2 by RT-PCR are sufficient to allow the assessment of infectivity. The availability of SARS-CoV-2 antigen testing offers advantages over the diagnosis of SARS-CoV-2 by RT-PCR in terms of reliability. Rapid antigen testing works best in cases of high viral load, in pre-symptomatic and early symptomatic cases up to 5 days after the onset of symptoms. Rapid antigen testing is sensitive enough for cases with a high viral load or low RT-PCR cycle threshold (Ct <25). The European Centre for Disease Prevention and Control (ECDC, 2020ECDC Options for the use of rapid antigen tests for COVID-19 in the EU/EEA and the UK. European Center for Disease Prevention and Control.2020https://www.ecdc.europa.eu/sites/default/files/documents/Options-use-of-rapid-antigen-tests-for-COVID-19.pdfGoogle Scholar) agrees on antigen testing with a World Health Organization performance requirement of >80% sensitivity and >97% specificity. Compared to diagnoses of SARS-CoV-2 by RT-PCR, rapid antigen testing can help to reduce further transmission through early detection, allowing a rapid start of contact-tracing (ECDC, 2020ECDC Options for the use of rapid antigen tests for COVID-19 in the EU/EEA and the UK. European Center for Disease Prevention and Control.2020https://www.ecdc.europa.eu/sites/default/files/documents/Options-use-of-rapid-antigen-tests-for-COVID-19.pdfGoogle Scholar). As there is a strong correlation between PCR results and the feasibility of virus culture with respect to Ct values, guidelines could be implemented in order to provide sound recommendations, e.g., inclusion of antigen tests or determination of the RNA copy number, on the self-isolation of health-care workers, quarantined individuals from high-risk areas, and others. Clinical parameters, as well as prolonged infectivity in immunocompromised individuals, need to be taken into account. Such evidence-based guidelines might exert an enormous societal impact. Both authors have an academic interest and contributed equally. TPV is a member of the Pan African Network for Rapid Research, Response, and Preparedness for Infectious Diseases Epidemics consortium (PANDORA-ID-NET RIA2016E-1609).