Jamil N Kanji1,2, Nathan Zelyas3,4, Clayton MacDonald5, Kanti Pabbaraju6, Muhammad Naeem Khan7, Abhaya Prasad7, Jia Hu8,9, Mathew Diggle3,4, Byron M Berenger6,10, Graham Tipples3,11,12. 1. Public Health Laboratory, Alberta Precision Laboratories, University of Alberta Hospital, 8440 - 112 Street, Edmonton, AB, T6G 2B7, Canada. jamil.kanji@ahs.ca. 2. Division of Infectious Diseases, Department of Medicine, University of Alberta, Room 1NW-29, 16940 - 87 Avenue NW, Edmonton, AB, T5R 4H5, Canada. jamil.kanji@ahs.ca. 3. Public Health Laboratory, Alberta Precision Laboratories, University of Alberta Hospital, 8440 - 112 Street, Edmonton, AB, T6G 2B7, Canada. 4. Department of Laboratory Medicine and Pathology, University of Alberta, University of Alberta Hospital, 8440 - 112 Street, Edmonton, AB, T6G 2B7, Canada. 5. Division of Medical Microbiology and Infection Control, Vancouver Coastal Health Vancouver General Hospital, 899 W 12th Avenue, Vancouver, BC, V5Z 1M9, Canada. 6. Public Health Laboratory, Alberta Precision Laboratories, Foothills Hospital, 1403 - 29 Street NW, Calgary, AB, T2N 2T9, Canada. 7. Health Protection and Communicable Disease Control, Public Health, Alberta Health Services, Coronation Plaza, 14310 - 111 Avenue NW, Edmonton, AB, T5M 3Z7, Canada. 8. Medical Officer of Health (MOH), Public Health, Alberta Health Services, 1213 - 4 Street SW, Calgary, AB, T2R 0X7, Canada. 9. Department of Community Health Sciences, Cumming School of Medicine, University of Calgary, 1403 - 29 Street NW, Calgary, AB, T2N 2T9, Canada. 10. Department of Pathology and Laboratory Medicine, University of Calgary, 3535 Research Road NW, Calgary, AB, T2L 2K8, Canada. 11. Department of Medical Microbiology and Immunology, University of Alberta, 8440 - 112 Street, Edmonton, AB, T6G 2B7, Canada. 12. Li Ka Shing Institute of Virology, University of Alberta, 6-010 Katz Group Centre for Pharmacy and Research, Edmonton, AB, T6G 2E1, Canada.
Abstract
BACKGROUND: COVID-19 is diagnosed via detection of SARS-CoV-2 RNA using real time reverse-transcriptase polymerase chain reaction (rtRT-PCR). Performance of many SARS-CoV-2 rtRT-PCR assays is not entirely known due to the lack of a gold standard. We sought to evaluate the false negative rate (FNR) and sensitivity of our laboratory-developed SARS-CoV-2 rtRT-PCR targeting the envelope (E) and RNA-dependent RNA-polymerase (RdRp) genes. METHODS: SARS-CoV-2 rtRT-PCR results at the Public Health Laboratory (Alberta, Canada) from January 21 to April 18, 2020 were reviewed to identify patients with an initial negative rtRT-PCR followed by a positive result on repeat testing within 14 days (defined as discordant results). Negative samples from these discordant specimens were re-tested using three alternate rtRT-PCR assays (targeting the E gene and N1/N2 regions of the nucleocapsid genes) to assess for false negative (FN) results. RESULTS: During the time period specified, 95,919 patients (100,001 samples) were tested for SARS-CoV-2. Of these, 49 patients were found to have discordant results including 49 positive and 52 negative swabs. Repeat testing of 52 negative swabs found five FNs (from five separate patients). Assuming 100% specificity of the diagnostic assay, the FNR and sensitivity in this group of patients with discordant testing was 9.3% (95% CI 1.5-17.0%) and 90.7% (95% CI 82.6-98.9%) respectively. CONCLUSIONS: Studies to understand the FNR of routinely used assays are important to confirm adequate clinical performance. In this study, most FN results were due to low amounts of SARS-CoV-2 virus concentrations in patients with multiple specimens collected during different stages of infection. Post-test clinical evaluation of each patient is advised to ensure that rtRT-PCR results are not the only factor in excluding COVID-19.
BACKGROUND:COVID-19 is diagnosed via detection of SARS-CoV-2 RNA using real time reverse-transcriptase polymerase chain reaction (rtRT-PCR). Performance of many SARS-CoV-2 rtRT-PCR assays is not entirely known due to the lack of a gold standard. We sought to evaluate the false negative rate (FNR) and sensitivity of our laboratory-developed SARS-CoV-2 rtRT-PCR targeting the envelope (E) and RNA-dependent RNA-polymerase (RdRp) genes. METHODS:SARS-CoV-2 rtRT-PCR results at the Public Health Laboratory (Alberta, Canada) from January 21 to April 18, 2020 were reviewed to identify patients with an initial negative rtRT-PCR followed by a positive result on repeat testing within 14 days (defined as discordant results). Negative samples from these discordant specimens were re-tested using three alternate rtRT-PCR assays (targeting the E gene and N1/N2 regions of the nucleocapsid genes) to assess for false negative (FN) results. RESULTS: During the time period specified, 95,919 patients (100,001 samples) were tested for SARS-CoV-2. Of these, 49 patients were found to have discordant results including 49 positive and 52 negative swabs. Repeat testing of 52 negative swabs found five FNs (from five separate patients). Assuming 100% specificity of the diagnostic assay, the FNR and sensitivity in this group of patients with discordant testing was 9.3% (95% CI 1.5-17.0%) and 90.7% (95% CI 82.6-98.9%) respectively. CONCLUSIONS: Studies to understand the FNR of routinely used assays are important to confirm adequate clinical performance. In this study, most FN results were due to low amounts of SARS-CoV-2 virus concentrations in patients with multiple specimens collected during different stages of infection. Post-test clinical evaluation of each patient is advised to ensure that rtRT-PCR results are not the only factor in excluding COVID-19.
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