False negative rate of COVID-19 PCR testing: a discordant testing analysis

• Published in: Virology journal Vol. 18; no. 1; p. 13
• Main Authors: Kanji, Jamil N., Zelyas, Nathan, MacDonald, Clayton, Pabbaraju, Kanti, Khan, Muhammad Naeem, Prasad, Abhaya, Hu, Jia, Diggle, Mathew, Berenger, Byron M., Tipples, Graham
• Format: Journal Article
• Language: English
• Published: England BioMed Central Ltd 09.01.2021; BioMed Central; BMC
• Subjects: Adult; Aged; Aged, 80 and over; Alberta; Canada; clinical examination; Coronaviruses; COVID-19; COVID-19 - diagnosis; COVID-19 - virology; COVID-19 infection; COVID-19 Nucleic Acid Testing - statistics & numerical data; Discordant testing; DNA-directed RNA polymerase; Evaluation; False negative rate; False Negative Reactions; False negative results; Female; Genes; Health aspects; Humans; Infections; Male; Medical laboratories; Middle Aged; Molecular Diagnostic Techniques - statistics & numerical data; nucleocapsid; Nucleocapsids; Pandemics; Patients; Polymerase chain reaction; Public health; Real-Time Polymerase Chain Reaction; reverse transcriptase polymerase chain reaction; Ribonucleic acid; RNA; SARS-CoV-2; SARS-CoV-2 - isolation & purification; Sensitivity and Specificity; Severe acute respiratory syndrome coronavirus 2; Short Report; Viral envelope proteins; Viral proteins; virology; viruses; Canada; Alberta Canada; Alberta; COVID-19; Discordant testing; SARS-CoV-2; False negative rate
• ISSN: 1743-422X; 1743-422X
• DOI: 10.1186/s12985-021-01489-0

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. 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. 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. 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.