This is an Accepted Manuscript for Infection Control & Hospital Epidemiology as part of the Cambridge Coronavirus Collection. DOI: 10.1017/ice.2021.369 Article Type: Original Article Impact of COVID-19 pre-test probability on positive predictive value of high cycle threshold SARS-CoV-2 real-time reverse transcription PCR test results Jonathan B. Gubbay MBBS1,2,3,*, Heather Rilkoff, MPH1, Heather L. Kristjanson, PhD1, Jessica D. Forbes, PhD2, Michelle Murti, MD1,4 , AliReza Eshaghi, PhD1, George Broukhanski, PhD1,2, Antoine Corbeil, MD1,2, Nahuel Fittipaldi PhD1, 2, Jessica P. Hopkins MD1,4,5, Erik Kristjanson, BSc1, Julianne V. Kus, PhD1,2, Liane Macdonald, MD1,4, Anna Majury PhD1,6, Gustavo V Mallo, PhD1, Tony Mazzulli, MD2,7, Roberto G. Melano, PhD1,2, Romy Olsha, MD1, Stephen J. Perusini1, Vanessa Tran, PhD1,2, Vanessa G Allen, MD1,2, Samir N Patel, PhD1,2 1 Public Health Ontario, 661 University Avenue, Toronto, Ontario, Canada M5G 1M1 2 Department of Laboratory Medicine and Pathobiology, University of Toronto, 1 King’s College Circle, Toronto, Ontario M5S 1A8 3 Division of Infectious Diseases, Department of Paediatrics, The Hospital for Sick Children, 555 University Ave, Toronto, Ontario, Canada M5G 1X8. 4 Dalla Lana School of Public Health, University of Toronto, 155 College Street, Toronto, ON, Canada M5T 3M7 5 Department of Health Research Methods, Evidence, and Impact, McMaster University, 1280 Main Street West, Hamilton, Ontario, Canada L8S 4K1 6 Department of Pathology and Molecular Medicine, Queens University, 88 Stuart Street, Kingston, Ontario, Canada K7L 3N6 7 Department of Microbiology, Sinai Health/University Health Network, 600 University Avenue, Toronto, Ontario, Canada M5G 1X5 *Corresponding Author: Jonathan B. Gubbay, Public Health Ontario, 661 University Avenue, Toronto, Ontario, Canada, M5G 1M1, Phone: +1 647-792-3170, Fax: +1 416-235-5800, Email: jonathan.gubbay@oahpp.ca Downloaded from https://www.cambridge.org/core. 17 Aug 2021 at 12:20:50, subject to the Cambridge Core terms of use. Previous Presentation of This Data This manuscript has been posted as a preprint to medRxiv.org - the preprint server for Health Sciences . A direct link is available at: https://www.medrxiv.org/content/10.1101/2021.03.02.21252768v1 Findings were also shared as a “Focus On” informational document on the Public Health Ontario website in September 2020 titled An Overview of Cycle Threshold Values and their Role in SARS-CoV-2 Real-Time PCR Test Interpretation. Abbreviated Title: Positive predictive value of SARS-CoV-2 PCR tests Word count: Abstract 205 words; Manuscript 3000 words Downloaded from https://www.cambridge.org/core. 17 Aug 2021 at 12:20:50, subject to the Cambridge Core terms of use. Abstract Objectives Performance characteristics of SARS-CoV-2 nucleic acid detection assays are understudied within contexts of low pre-test probability, including screening asymptomatic persons without epidemiological links to confirmed cases, or asymptomatic surveillance testing. SARS-CoV-2 detection without symptoms may represent presymptomatic or asymptomatic infection, resolved infection with persistent RNA shedding, or a false positive test. This study assessed positive predictive value of SARS-CoV-2 real-time reverse transcription polymerase chain reaction (rRTPCR) assays by retesting positive specimens from five pre-test probability groups ranging from high to low with an alternate assay. Methods A total of 122 rRT-PCR positive specimens collected from unique patients between March and July 2020 were retested using a laboratory-developed nested RT-PCR assay targeting the RNAdependent RNA polymerase (RdRp) gene followed by Sanger sequencing. Results Significantly fewer (15.6%) positive results in the lowest pre-test probability group (facilities with institution-wide screening having ≤ 3 positive asymptomatic cases) were reproduced with the nested RdRp gene RT-PCR assay than in each of the four groups with higher pre-test probability (individual group range 50·0% to 85·0%). Conclusions Large-scale SARS-CoV-2 screening testing initiatives among low pre-test probability populations should be evaluated thoroughly prior to implementation given the risk of false positives and consequent potential for harm at the individual and population level. Keywords SARS-CoV-2 PCR; cycle threshold; false positive tests; test performance; positive predictive value; asymptomatic PCR testing Downloaded from https://www.cambridge.org/core. 17 Aug 2021 at 12:20:50, subject to the Cambridge Core terms of use. Introduction Widespread laboratory testing for SARS-CoV-2, the cause of the COVID-19 pandemic, has led to the observation of positive real-time reverse transcription PCR (rRT-PCR) test results in persons without symptoms. This may represent active presymptomatic (patients who later develop symptoms) or asymptomatic (patients who never develop symptoms prior to or following testing) infections, resolved infections with persistent viral RNA shedding, or false positive laboratory tests.1 The likelihood of a false positive rRT-PCR result increases as pre-test probability of the condition it is designed to detect decreases. Examples of low pre-test probability scenarios include asymptomatic groups with no known exposure to COVID-19 cases and communities with low prevalence of COVID-19. Further, a positive rRT-PCR result nearing the assay limit of detection (LOD) has a greater likelihood of being false positive. 2 False positive results can be attributable to pre-analytical errors (e.g., specimen contamination or aliquoting errors), analytical errors (e.g., quality assurance failures, reagent contamination, or non-specific assay signal), or post-analytical errors (e.g., improper result interpretation or transcription). False positive results can have unintended consequences on individual wellbeing and the public health response including outbreak declaration and modelling, case reporting, and resource allocation.3 The cycle threshold (Ct) value, an indirect measure of viral load, and its application to test interpretation has become an important tool public health tool. Together with available clinical and epidemiological factors, the Ct value can help determine appropriate public health follow-up (e.g., contact tracing and/or outbreak declaration) for asymptomatic patients. 4 However, multiple studies have shown that Ct values overlap between symptomatic, presymptomatic, and asymptomatic cases, and that time from initial infection to testing is the most significant determinant of Ct value.5-7 Presymptomatic persons may have comparable viral loads to symptomatic individuals and may be just as likely to infect others, hence their identification has implications for public health management.8,9 Ontario, Canada, (population ~14.7 million), identified the country’s first COVID-19 case in a patient who presented to hospital on January 23, 2020 10,11. The first pandemic wave peaked in April 2020 and was characterized by disproportionate impact on congregate settings including residents in long-term care (LTC), retirement homes and some workplaces.12 During the first Downloaded from https://www.cambridge.org/core. 17 Aug 2021 at 12:20:50, subject to the Cambridge Core terms of use. wave, asymptomatic screening programs and policies were implemented within some LTC, acute care (e.g., hospitals), and workplace settings. This widespread testing brought into focus the interpretation and implications of positive SARS-CoV-2 rRT-PCR results with high Ct values, since many of these settings had both low prevalence and low pre-test probability of COVID-19. This study evaluates the relative burden of false positive testing outcomes when testing persons in low pre-test probability settings by exploring the likelihood of a reproducible positive test result upon retesting specimens having high rRT-PCR Ct values, stratified by pre-test probability. Methods Public Health Ontario (PHO) Laboratory, the Ontario provincial public health and reference laboratory, conducts approximately 25% of the province’s SARS-CoV-2 testing. Specimens are submitted from acute care, community, institutional, occupational settings, and from outbreaks. Specimen data were obtained from the PHO laboratory information system (LIS). A total of 122 specimens from unique patients aged 10 to 99 years (median 53.5 years) who underwent clinical testing between mid-March and July 2020 were included in the analysis. All specimens included were initially positive by rRT-PCR with Ct value ≥ 35 using either (i) a laboratory-developed test (LDT) targeting the envelope (E) gene, or (ii) a commercial assay targeting the E and open reading frame 1ab (ORF1ab) genes (cobas® SARS-CoV-2, Roche Diagnostics, Germany).13 Interpretation of results for the LDT E gene rRT-PCR assay was based on prior validation data, which determined a LOD of 192 copies/ml of primary sample (95% CI 16 to 2,392 copies/ml of specimen), corresponding to Ct values between 34·8 and 38·7. Based on these data, LDT Ct results ≤38·0 are reported as detected, ≥40·0 are reported as not detected, and between 38·1 and 39·9 are reported as indeterminate.14 Indeterminate results may be due to low viral target quantity approaching the assay LOD, failed viral RNA extraction, or nonspecific reactivity (false signal). When important to clinical or public health management, repeat testing is recommended. Specimens tested with the cobas® SARS-CoV-2 rRT-PCR assay were reported as detected or not detected - the manufacturer does not include an indeterminate range. Although the Ct value for detected specimens is provided by the instrument, the maximum number of cycles of PCR Downloaded from https://www.cambridge.org/core. 17 Aug 2021 at 12:20:50, subject to the Cambridge Core terms of use. amplification used in the assay is proprietary. Any specimen with a Ct value provided for E and/or ORF1ab target is considered SARS-CoV-2 detected. As determined by PHO Laboratory’s verification, the E gene 95% LOD of the cobas® SARS-CoV-2 rRT-PCR was -3.9985 log copies /ml (95% CI -3.1696 to -4.8265). This was similar to the ORF1ab gene 95% LOD -4.1175 log copies/ml (95% CI 3.5875 to -4.6475), and several logs lower than the LDT E gene LOD. To be included in the study, specimens had to have a high Ct value of ≥35 on either the LDT or cobas® rRT-PCR assay E gene target. An E gene Ct value of ≥35 was chosen as a conservative estimate of lack of infectivity based on other studies using different assays reporting that a Ct of >34 indicates an individual is not likely to be infectious at the time of diagnostic testing. 7,9,15 Specimens were further classified into five groups of differing pre-test probability of COVID-19 based on the presence of symptoms, prior laboratory detection of SARS-CoV-2, and epidemiological links to other positive cases, Information was collected from the PHO laboratory requisition. Table 1 describes the groups, ordered from highest pre-test probability (Group 1) to lowest pre-test probability (Group 5). Groups 1-4 were tested throughout the study period (March - June 2020), whereas Group 5 was tested beyond the peak of the first pandemic wave (May - July 2020). Groups 4 and 5 only included asymptomatic cases, the former from facilities with outbreaks of ≥10 positive cases, and the latter from facilities that underwent institution-wide screening or outbreak investigations with ≤ 3 positive cases. The study dataset was produced by manually reviewing a line list of positive specimens of appropriate Ct values available at PHO Laboratory, Toronto, which met inclusion criteria. Group 1, representing the highest pre-test probability group, consisted of persons who had a confirmed infection with a prior positive result at a Ct value <30. Group 5, which consisted of asymptomatic positive cases in facilities undergoing screening with three or fewer positive cases identified, was defined as the lowest pre-test probability group due to the asymptomatic status and lower likelihood of exposure to COVID-19. No Group 5 facilities were in outbreak at the time of screening, which was confirmed by review of the provincial public health information system for the reporting and surveillance of communicable diseases. Group 5 was thus chosen as the reference group when conducting statistical analyses. Decreasing levels of pre-test probability were attributed from Group 2 to Group 4. Specimens included in this study were retested with a LDT end-point nested RT-PCR assay targeting the RNA dependent RNA polymerase (RdRp) gene, followed by Sanger sequencing of Downloaded from https://www.cambridge.org/core. 17 Aug 2021 at 12:20:50, subject to the Cambridge Core terms of use. amplicons with expected size of 192 base pairs. This assay was adapted from a previously published Middle East Respiratory Syndrome Coronavirus (MERS-CoV) nested PCR: an outer primer and newly designed inner primers targeting SARS-CoV-2 were used for both amplification and sequencing (Table 2).16 The LOD determined during validation was similar to that of the E gene LDT rRT-PCR, at 256 copies/ml of primary specimen (95% CI 37.92 to 1733 copies/ml). This was chosen as the confirmatory assay in this study as it was previously developed and validated at PHO laboratory and used to confirm Ontario’s early cases of SARSCoV-2 infection. In addition, it targeted a different gene than the SARS-CoV-2 rRT-PCR assays outlined above, with reproducibility of detection across multiple gene targets more likely to represent a true positive result. The proportion of specimens detected in each group by the RdRp gene nested PCR assay relative to Group 5 (reference) was calculated using Fisher’s exact test with Bonferroni correction to adjust for multiple comparisons. Per group median and range of Ct values were compared using Wilcoxon rank-sum test. Results were considered significant at a level of 0·05. All analyses were conducted using SAS Enterprise Guide 8.3. 17 The PHO Ethics Review Board determined that this project was exempt from research ethics committee review, as it describes analyses that were completed at PHO Laboratory as part of routine clinical respiratory testing during the first wave of the COVID-19 pandemic in Ontario and were therefore considered public health practice, not research. Results Table 3 describes the results of the specimens overall and by group. After retesting specimens using the RdRp gene nested PCR assay with Sanger sequencing, results varied according to pretest probability. Overall, 66/122 (54·1%) specimens had RdRp gene detected. Highest pre-test probability Groups (1 and 2) had the highest proportion of reproducible positive results (18/23; 78·3%, and 17/20; 85·0%, respectively), and all Groups (1-4) had significantly more positives reproducible on the RdRp assay compared to Group 5. Across all groups, there was a significant difference (p<0·01) in E gene Ct values among specimens that were reproducible on the RdRp gene nested PCR (median Ct 36·2, range 35·0 to 40·6) compared to those that were not reproducible (median Ct 37·5, range 35·2-39·8). SARS-CoV-2 was detected in the RdRp gene Downloaded from https://www.cambridge.org/core. 17 Aug 2021 at 12:20:50, subject to the Cambridge Core terms of use. nested PCR in 55/73 (75.3%) specimens initially tested using the cobas® rRT-PCR assay, whereas it was only detected in 11/49 (22.4%) specimens tested by the E gene LDT rRT-PCR assay (Table 3). Discussion This study was conducted to ascertain the impact of different COVID-19 pre-test probabilities on the likelihood that high Ct rRT-PCR results (Ct ≥35) will be reproducibly positive on a laboratory-developed nested PCR and Sanger assay targeting a different SARS-CoV-2 gene. We documented a much lower rate of reproducible high Ct positive tests among patients in the lowest pre-test probability group of asymptomatic persons included (i.e., within an institution with three or fewer positive patients identified through screening). Among this group, only five (15·6%) of 32 specimens were also positive by RdRp nested PCR. This is in contrast to the higher pre-test probability groups (i.e., symptomatic, exposed to a case, and/or in a facility with ≥10 cases), where 50% to 85% of E gene rRT-PCR positive specimens remained positive by RdRp nested PCR. Although we documented a significant difference in the E gene Ct values among specimens that were reproducible in the RdRp gene nested RT-PCR (median Ct 36·2, range 35·0 to 40·6) compared to those that could not be confirmed (median Ct 37·5, range 35·2-39·8), the absolute difference is too small to be used to inform clinical or public health decisions on cases, and likely depends on the assay(s) used. Lack of detection with the RdRp gene nested PCR assay does not necessarily imply a false positive E gene rRT-PCR result and does not definitively infer false positivity at the individual level. In general, specimens with Ct values well below the assay cut-off for positivity (e.g., Ct values <35 with the positivity cut-off set at Ct 38.0) are less likely to be false positive. If the result is near the assay positivity cut-off, repeat testing of the same specimen may yield a negative result, as assay performance near the LOD is not consistently reproducible. Furthermore, different assays will perform differently on the same specimen with virus quantity near their assay LOD. However, when applied at a group level, these results provide an Downloaded from https://www.cambridge.org/core. 17 Aug 2021 at 12:20:50, subject to the Cambridge Core terms of use. indication of the potential relative contribution of false positive test results that may occur in different settings characterized by pre-test probability. In general, the positive predictive value (PPV) of COVID-19 PCR assays is excellent among patients with high pre-test probability, approaching 100%.2 However, when testing asymptomatic patients with low pre-test probability in low prevalence settings, the PPV is inherently different. For example, if community prevalence of SARS-CoV-2 is 1% with a rRTPCR test sensitivity of 90% and specificity of 99%, the PPV of a positive test is only 47.6%. If prevalence were to increase to 5% or 10%, then the PPV increases significantly to 82.6% and 90.9%, respectively. Serosurveys in Ontario using residual convenience specimens found a low adjusted monthly seroprevalence of 1·1% among specimens received in June, July and again in August, 2020.18 This provides further evidence that of low community prevalence for SARSCoV-2 in Ontario during the study period. Analysis of results from over 100,000 SARS-CoV-2 tests conducted at PHO Laboratory for asymptomatic screening programs (including long-term care homes, retirement homes, childcare settings, hospitals, settings with migrant workers, and correctional institutions) during the same period as this study identified a positivity rate of 0·2%, (unpublished data). Nearly 70% of positive tests had Ct values ≥35, suggesting that true positivity is likely to be lower, given the potential for false-positive high Ct results in these low-prevalence settings. Limitations of this study include small sample size and use of a non-randomized sampling method that may limit generalizability of findings. All specimens in Groups 2 to 5 were the first specimen submitted to PHO for that individual. It is possible an earlier specimen could have tested positive elsewhere. This would increase the pre-test probability of that specimen regardless of the group to which the individual’s sample was assigned. For similar reasons, it is possible that not all positive cases from individual institutions were captured in our study if some testing for additional cases was done elsewhere or individuals declined testing. To substantiate that the low-prevalence institutional settings had <3 cases, the public health database was checked for outbreak-related cases associated with these settings within a three-week period. It was assumed that the database was correctly updated and an outbreak declared if the number of cases identified by the asymptomatic screening program became greater than three. Downloaded from https://www.cambridge.org/core. 17 Aug 2021 at 12:20:50, subject to the Cambridge Core terms of use. PHO Laboratory obtains clinical information on cases (e.g. symptoms, contact with COVID-19 cases) from the specimen requisition submitted to PHO Laboratory, which may not always be accurate and could not be validated. This may have resulted in case misclassification. Median age varied across patient groups, from 38 to 68.6 years (Table 3). It is known that attack rates varied in different age groups during the first wave of the pandemic, so this may have impacted pre-test probability in the different groups in our study. Groups 1-4 were tested throughout the study period (March - June 2020), whereas Group 5 was tested beyond the peak of the first pandemic wave (May - July 2020). This introduces a potential bias to the study as the pre-test probability was inherently lower in Group 5 independent of the clinical setting we attempted to evaluate, as testing was conducted in this group when disease prevalence was lower in the community. Specimens included in this study were stored at -80˚C for weeks to months prior to conducting the RdRp gene nested RT-PCR. RNA degradation during storage and freeze-thaw is possible and was more likely to affect specimens that were close to the LOD, resulting in a negative RdRp RT-PCR in a specimen that was true rRT-PCR positive at the time of initial testing. Specimen inclusion was based on E gene Ct value at the time of rRT-PCR. Determination of Ct values for LDTs rely on interpretation by the reporting technologist, introducing variability in Ct value assignment. This introduces a risk of reporter bias influencing the specimens included in this study. In addition, the cobas® SARS-CoV-2 rRT-PCR assay has a formal LOD that is several logs lower than that of the E gene LDT and the nested RdRp assay. This may introduce selection bias, as only a subset of specimens were tested with this assay, and misclassification bias if the secondary test is less sensitive than the index test. At PHO laboratory we have not observed a difference in positivity between the cobas® assay and the E gene LDT assay suggesting that their LODs are closer than formally documented (unpublished data). However, we did observe a higher rate of reproducibility among specimens originally tested by the Roche cobas® assay in this study (Table 3). This was likely partly due to 27/49 (55%) LDT-positive specimens included in the study arising from Group 5 patients, the lowest pre-test probability group. Downloaded from https://www.cambridge.org/core. 17 Aug 2021 at 12:20:50, subject to the Cambridge Core terms of use. Despite these limitations, the results presented here are an important step towards quantifying the magnitude of false-positive test results in low-prevalence settings, which will increasingly become the norm in many countries with increased vaccination, and use of widespread testing, including broad testing in low pre-test probability populations. Currently, there exists few studies that have attempted to ascertain prevalence through probability-based population-level surveillance studies, rather than initiating a study in an area known to have low prevalence. 19,20 Examples of targeted low-prevalence studies include examination of potential SARS-CoV-2 wastewater detection, and serosurveillance studies in low prevalence areas.21,22 The results of this study have implications for informing future testing approaches, including the utility of conducting broad screening with PCR-based tests in settings with low pre-test probability. For example, in Ontario, this work has been used to inform recent public health approaches, resulting in discontinuation of unnecessary public health management, such as case isolation, contact tracing, and outbreak declaration, for asymptomatic SARS-CoV-2 rRT-PCR positive persons with low pre-test probability who are negative on retesting. 2 Downloaded from https://www.cambridge.org/core. 17 Aug 2021 at 12:20:50, subject to the Cambridge Core terms of use. In conclusion, SARS-CoV-2 Ct values can be of use when interpreting positive laboratory results derived from patients with low pre-test probability, in particular asymptomatic persons with no epidemiological link to a confirmed COVID-19 case and/or low community COVID-19 prevalence. Health care providers, public health professionals, policymakers and the public will benefit from ongoing education to understand that false positive tests will occur when testing asymptomatic individuals during periods of low community prevalence of SARS-CoV-2. These false positive tests and unnecessary public health actions likely outweigh the benefits from the low numbers of true cases detected among these populations. Once high levels of vaccination coverage are achieved and low test positivity is observed among persons with clinical indications for testing (symptomatic persons or asymptomatic contacts of confirmed cases), cessation of screening of asymptomatic persons without epidemiological risk factors for SARS-CoV-2 infection should be considered after conducting a risk assessment at the jurisdictional level. Acknowledgements. We gratefully acknowledge the staff of Virus Detection and Molecular Diagnostics, Public Health Ontario laboratory, for diagnostic testing of SARS-CoV-2 specimens. Financial Support. This work was funded by Public Health Ontario. Potential conflicts of interest. All authors declare no competing interests. Downloaded from https://www.cambridge.org/core. 17 Aug 2021 at 12:20:50, subject to the Cambridge Core terms of use. REFERENCES 1. Management of Cases and Contacts of COVID-19 in Ontario; May 6, 2021 (version 12.0). Ontario Ministry of Health. Toronto, ON: Queen’s Printer for Ontario; 2020. https://www.health.gov.on.ca/en/pro/programs/publichealth/coronavirus/docs/contact_mngmt /management_cases_contacts.pdf . Published May 6, 2021. Accessed July 7, 2021. 2. Real-Time PCR Cycle Threshold (Ct) Values: An Overview of Cycle Threshold Values and their role in SARS-Cov-2 Real-Time PCR Test Interpretation. Toronto, ON: Queen's Printer for Ontario; 2020. 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Euro Surveill. 2012 Dec 6;17(49):20334. 17. SAS Institute Inc. SAS® Enterprise Guide 8.2: User’s Guide. Cary, NC: SAS Institute Inc. 2019. 18. PHO’s COVID-19 Serosurveillance Initiative. Public Health Ontario website. https://www.publichealthontario.ca/en/data-and-analysis/infectious-disease/covid-19-datasurveillance/covid-19-serosurveillance. Published 2020. Accessed November 9, 2020. 19. Gudbjartsson DF, Helgason A, Jonsson H, et al. Spread of SARS-CoV-2 in the Icelandic population. N Engl J Med. 2020; 382: 2302–15 20. Vodičar PM, Valenčak AO, Zupan B, et al. Low prevalence of active COVID-19 in Slovenia: a nationwide population study on a probability-based sample. Microbiol Infect. 2020 Nov;26(11):1514-1519. 21. Randazzo W, Truchado P, Cuevas-Ferrando E, et al. SARS-CoV-2 RNA in wastewater anticipated COVID-19 occurrence in a low prevalence area. Water Res. 2020; 181: 115942 22. Bendavid E, Mulaney B, Sood N, et al. COVID-19 Antibody Seroprevalence in Santa Clara County, California. Int J Epidemiol. 2021 May 17;50(2):410-419 Downloaded from https://www.cambridge.org/core. 17 Aug 2021 at 12:20:50, subject to the Cambridge Core terms of use. Table 1: Study Patient Categories and Definitions Groupa 1b 2 3c 4 5 Category Confirmed cases with second positive specimen of high Ct value Symptomatic patient with high Ct positive specimen Asymptomatic - exposure to probable or confirmed case Asymptomatic - facility with ≥10 positive cases Facility with institution-wide screening, with ≤3 positive cases, all asymptomatic Definition Persons who initially tested positive with a low Ct value (<30) and had a subsequent test with a high Ct value (≥35) Having a positive test with high Ct value (Ct ≥35) and at least one symptom as noted in the PHO LIS Indicated as asymptomatic in the PHO LIS. Tested due to exposure to probable or confirmed case OR residing at same address as another positive case Indicated as asymptomatic in the PHO LIS and tested as part of an outbreak with at least 10 positive cases Tested as part of an outbreak or screening investigation having three or fewer asymptomatic positive tests and no symptomatic positive cases in PHO LIS Ct-cycle threshold, PHO-Public Health Ontario, LIS-Laboratory Information System a Group 1 represents patients with highest pre-test probability and Group 5 represents those with lowest pre-test probability. b 20 patients were symptomatic, two were asymptomatic at time of first test, and one did not have symptom information available at time of first test c Group 3 contains specimens from institutional outbreaks (as well as non-outbreaks), and thus some specimens could also be classified in the Group 4 (facility ≥10 positive cases) category Downloaded from https://www.cambridge.org/core. 17 Aug 2021 at 12:20:50, subject to the Cambridge Core terms of use. Pre-test probability High Low TABLE 2: SARS-COV-2 Laboratory Developed RdRp Nested PCR Primers in Use at PHO Laboratory * Primer position aligned with SARS-CoV-2 RdRp nested PCR Sequence 5’ to 3’ primers SARS-CoV-2 (NCBI Reference Sequence: NC_045512.2) Nested PCR outer primers TGCCATTAGTGCAAAGAATAGAGC 1507815101bp GCATGGCTCTATCACATTTAGG 1531915298bp Nested PCR inner primers GCACCGTAGCTGGTGTCTCT 1510415123bp AATCCCAACCCATAAGGTGA 1529515276bp RdRp = RNA dependent RNA polymerase *Protocol was adapted from Corman et al., 2012 (ref. 16) Downloaded from https://www.cambridge.org/core. 17 Aug 2021 at 12:20:50, subject to the Cambridge Core terms of use. TABLE 3: Initial E Gene PCR and RdRp PCR Results Stratified by Patient Category PATIENTS Median Groupa Age N (years) (Range) 1 23e 2 20f 3 15g 4 32 h 5 32i Total 122j 52 (14-99) 68·5 (2694) 38 (10-93) 57·5 (1597) 46 (17-95) 53·5 (1099) DETECTED BY RdRp PCR Median Ct (Range) on initial 36·6 (35·0-38·3) 36·1 (35·4-38·0) 37·5 (35·4-40·6) 36·9 (35·2-39·8) 36·9 (35·0-40·6) N (%) on initial E gene 18 (78·3) N (%) 17 (85·0) 10 (66·7) 16h (50·0) 5 (15·6) 66 (54·1) (Range) on P- initial E gene valueb PCR 36·7 (35·0-38·3) 5 (21·7) 38·1 (35·9-38·4) <0·0001 36·9 (35·03-38·3) 3 (15·0) 36·3 (35·6-37·4) <0·0001 36·0 (35·4-37·2) 5 (33·3) 37·5 (36·0-38·0) 0·0078 36·6 (35·4-40·6) 16 (50·0) 37·7 (35·5-38·3) 0·035 36·2 (35·6-37·5) 27 (84·3) 37·0 (35·2-39·8) (ref)c 36·2 (35·0-40·6) 56 (45·9) 37·5 (35·2-39·8) Refer to Table 1 for Group definitions P-values compare proportion detected in each Group to Group 5, as the reference group. Downloaded from https://www.cambridge.org/core. 17 Aug 2021 at 12:20:50, subject to the Cambridge Core terms of use. Median Ct PCR a b PCR Median Ct (Range) E gene PCR 36·9 (35·0-38·4) NOT DETECTED BY RdRp <0·0001 d c Represents reference group to which other groups are compared d P-value compares Groups 1 to 4 combined to Group 5, as the reference group e Among the 23 positives, 14/17 and 4/6 detected by the Roche and LDT assay, respectively, were confirmed in the RdRp assay f Among the 20 positives, 13/14 and 4/6 detected by the Roche and LDT assay, respectively, were confirmed in the RdRp assay g Among the 15 positives, 10/14 and 0/1 detected by the Roche and LDT assay, respectively, were confirmed in the RdRp assay h Among the 32 positives, 15/23 and 1/9 detected by the Roche and LDT assay, respectively, were confirmed in the RdRp assay i Among the 32 positives, 3/5 and 2/27 detected by the Roche and LDT assay, respectively, were confirmed in the RdRp assay j Among all positive specimens, 55/73 (75.3%) and 11/49 (22.4%) tested by the Roche and LDT assay, respectively, were confirmed in the RdRp assay Ct = cycle threshold; E gene PCR = envelope gene real-time reverse-transcription PCR; RdRp PCR = RNA dependent RNA polymerase gene end-point PCR with Sanger sequencing. Downloaded from https://www.cambridge.org/core. 17 Aug 2021 at 12:20:50, subject to the Cambridge Core terms of use.