key: cord-0914960-j41c15mw authors: Bullard, Jared; Dust, Kerry; Funk, Duane; Strong, James E; Alexander, David; Garnett, Lauren; Boodman, Carl; Bello, Alexander; Hedley, Adam; Schiffman, Zachary; Doan, Kaylie; Bastien, Nathalie; Li, Yan; Van Caeseele, Paul G; Poliquin, Guillaume title: Predicting infectious SARS-CoV-2 from diagnostic samples date: 2020-05-22 journal: Clin Infect Dis DOI: 10.1093/cid/ciaa638 sha: 8e94da73a982fbf3ce95fe1c280f6a76734925c6 doc_id: 914960 cord_uid: j41c15mw BACKGROUND: RT-PCR has become the primary method to diagnose viral diseases, including SARS-CoV-2. RT-PCR detects RNA, not infectious virus, thus its ability to determine duration of infectivity of patients is limited. Infectivity is a critical determinant in informing public health guidelines/interventions. Our goal was to determine the relationship between E gene SARS-CoV-2 RT-PCR cycle threshold (Ct) values from respiratory samples, symptom onset to test (STT) and infectivity in cell culture. METHODS: In this retrospective cross-sectional study, we took SARS-CoV-2 RT-PCR confirmed positive samples and determined their ability to infect Vero cell lines. RESULTS: Ninety RT-PCR SARS-CoV-2 positive samples were incubated on Vero cells. Twenty-six samples (28.9%) demonstrated viral growth. Median TCID50/ml was 1780 (282-8511). There was no growth in samples with a Ct > 24 or STT > 8 days. Multivariate logistic regression using positive viral culture as a binary predictor variable, STT and Ct demonstrated an odds ratio for positive viral culture of 0.64 (95% CI 0.49-0.84, p<0.001) for every one unit increase in Ct. Area under the receiver operating characteristic curve for Ct vs. positive culture was OR 0.91 (95% CI 0.85-0.97, p<0.001), with 97% specificity obtained at a Ct of >24. CONCLUSIONS: SARS-CoV-2 Vero cell infectivity was only observed for RT-PCR Ct < 24 and STT < 8 days. Infectivity of patients with Ct >24 and duration of symptoms >8 days may be low. This information can inform public health policy and guide clinical, infection control and occupational health decisions. Further studies of larger size are needed. The emergence of SARS-CoV-2, the causative agent of COVID-19, represents a public health emergency of historic proportion. The global containment efforts have had broad societal and economic impacts. Policy decisions to relax public health measures will require a better understanding of duration of infectivity. This information will also impact infection control practices and occupational health. To date, the diagnosis of COVID-19 has relied on the detection of SARS-CoV-2 through molecular detection. While this method is both rapid and highly sensitive, there are important limitations. Several studies describe the persistence of SARS-CoV-2 RNA within different body sites (1, 2) . It is known from other viruses that viral RNA can persist beyond infectivity (3, 4) . As a result, demonstration of in vitro infectiousness on cell lines is a more informative surrogate of viral transmission. The ability of viral culture to inform infectivity is an important aspect of diagnostics but its use is hampered by its difficult and labour-intensive nature. This is further complicated by the need for containment level 3 facilities in the case of SARS-CoV-2. In a recent cohort study of nine patients, no virus could be recovered beyond 7 days post symptom onset (1) . This important study is limited by the small number of patients examined and the fact that all nine cases are linked, therefore the data may represent a unique viral subpopulation. Here we add to the existing body of literature by presenting viral culture results on a larger cross-sectional group of patients, compared to PCR data and time of symptom onset. A c c e p t e d M a n u s c r i p t 5 All samples in this study were obtained to support routine care and surveillance of the public health response in the province of Manitoba, Canada. All suspect COVID-19 cases had SARS-CoV-2 RT-PCR performed on nasopharyngeal (NP) or endotracheal (ETT) samples at Cadham Provincial Laboratory (CPL), the public health laboratory. NP swabs and ETT specimens in viral transport media were stored at 4°C for 24-72 hours until they were tested for the presence of SARS-CoV-2 RNA using real-time RT-PCR targeting a 122nt portion of supplemented with 5% Fetal Bovine Serum (FBS), 1% penicillin/ streptomycin (P/S), 0.5 g/mL Amphotericin B and 1% L-glutamine, were seeded into 96 well plates (Thermo Scientific, 167008) at 70% confluency. Using dilution blocks, patient samples were serially diluted 10-fold from 10 -1 to 10 -8 in MEM supplemented with 2% FBS, 1% Penicillin/Streptomycin, 0.5 g/mL Amphotericin B and 1% L-glutamine. Dilutions were placed onto the Vero cells in triplicate and incubated at 37°C with 5% CO2 for 96 hours. Following incubation of 4 days, cytopathic effect (CPE) was evaluated under a microscope and recorded. TCID50 and TCID50/mL were calculated using the Reed and Muench method previously described (6) Data are presented as mean  standard deviation for normally distributed data and as median PCR and other nucleic amplification (NA) strategies have surpassed viral culture as the gold standard viral diagnostic, because of their wider application, higher sensitivity, rapid performance, and ability for field deployment. A major drawback to PCR and other diagnostic approaches (including other NA, serology, antigen detection) is that they all fail to determine virus infectivity: PCR sensitivity is excellent but specificity for detecting replicative virus is poor (13) . Our study utilized a crosssectional approach to correlate COVID-19 symptom onset to specimen collection with SARS-CoV-2 E gene RT-PCR and virus viability as determined by cell culture. These results demonstrate that infectivity (as defined by growth in cell culture), is significantly reduced when RT-PCR Ct values are greater than 24. For every 1 unit increase in Ct, the odds ratio for infectivity decreased by 32%. The high specificity of Ct and STT suggests that Ct values greater than 24, along with duration of symptoms greater than 8 days may be used in combination to determine duration of infectivity in patients. Positive cell culture results in our study were most likely between days one and five. This finding is consistent with existing literature (1, 2) . This study is the first to report a large enough data set that demonstrates a link between in vitro viral growth, Ct value and STT. These results have implications for clinical care, infection prevention and control and public health. These data can be used to efficiently target case finding efforts by better defining the period of maximal transmission risk. This will be of particular importance in the maintenance phase of the response, where case finding efforts to rapidly interrupt chains of transmission will be essential. Isolation of COVID-19 cases in the community is typically recommended for at least ten days after symptom onset. Our data supports this approach. Jurisdictions across Canada and the US are recommending a variety of strategies to discontinue isolation of hospitalized COVID-19 cases (7) (8) (9) (10) (11) (12) . Clinical criteria including 14 days from symptom onset or 72 hours symptom free (whichever is A c c e p t e d M a n u s c r i p t 9 longer) are being used in some while other jurisdictions are using two negative NP RT-PCR results 48 hours apart after 14 days of symptoms. Our data supports the former approach since RT-PCR positivity persists significantly beyond infectivity; the alternative approach may lead to unnecessary isolation, and use of PPE and testing resources. The qualitative reporting of results of SARS-CoV-2 RT-PCR as positive or negative is sufficient for diagnosis but may be supplemented by Ct, a semiquantitative value, as well as time of symptom onset to guide infection control, public health and occupational health decisions. Our study has important limitations. First, our study utilized a single SARS-CoV-2 gene target (E gene). Though other gene targets may offer greater specificity, SARS-CoV-2 E-gene is more consistently used in both laboratory-developed tests (LDT) and commercial assays. The testing criteria in Manitoba had sufficient pre-test probability to make the likelihood of a false positive remote. In addition, the first 71 of 90 samples were confirmed using the described protocol with CDC N1-gene target (14) . Second target confirmation was discontinued at that time based on being satisfied with testing criteria and assay sensitivity to accurately identify true COVID-19 cases. Reagent supply also played a role. Second, the recall bias of symptom onset is possible, but this likely would have been equally distributed between those who were culture positive and negative. Third, the infectivity of certain individual cases and the accuracy of our culture assay may have unique variations. Though some individuals in our cross-sectional study would be considered Virological assessment of hospitalized patients with COVID-19 Temporal dynamics in viral shedding and transmissibility of COVID-19 The dynamic relationship between clinical symptomatology and viral shedding in naturally acquired seasonal and pandemic influenza virus infection Comparison of quantitative reverse transcription-PCR to viral culture for assessment of respiratory syncytial virus shedding Detection of 2019 novel coronavirus (2019-nCoV) by realtime RT-PCR Determination of 50% endpoint titer using a simple formula Coronavirus disease (COVID-19): For health professionals Symptom-Based Strategy to Discontinue Isolation for Persons with COVID-19 Shared Health Manitoba. Provincial COVID-19 resources for health-care providers and staff COVID-19 (coronavirus) public health expertise and reference centre COVID-19 Health Care Resources. Public Health Ontario BC Centre for Disease Control (BCCDC) The Crux of Ebola Diagnostics Research Use Only 2019-Novel Corovnavirus (2019-nCoV) Real-time RT-PCR Primer and Probe Information (CDC N1 gene M a n u s c r i p t 10 In conclusion, the SARS-CoV-2/COVID-19 pandemic represents a dynamic situation where decisions and policy must be guided by evidence. Our study showed no positive viral cultures with a Ct greater than 24 or STT greater than 8 days. The odds of a positive culture were decreased by 32% for each unit increase in Ct. This data, if confirmed, may help guide isolation, contact tracing, and testing guidelines.