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

Jamil N Kanji,Mathew Diggle,Kanti Pabbaraju,Jia Hu,Byron M Berenger,Clayton Macdonald,Abhaya Prasad,Graham Tipples,Nathan Zelyas,Muhammad Naeem Khan

doi:10.1186/s12985-021-01489-0

Abstract

BackgroundCOVID-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.MethodsSARS-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.ResultsDuring 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.ConclusionsStudies 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.

Highlights

COVID-19 is diagnosed via detection of SARS-CoV-2 RNA using real time reverse-transcriptase polymerase chain reaction
Test results and patient demographics were extracted from the laboratory information system to identify patients between January 21 and April 18, 2020, with an initial negative SARS-CoV-2 result followed by a positive result on repeat testing within 14 days hereon defined as discordant test results [11]
Repeat testing of swab 1 for each of the 49 patients using a combination of three alternate assays revealed five FN results (Table 2)

Summary

Introduction

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. Initially available nucleic acid tests (NATs) may lack data on the frequency of false negative results which can unnecessarily lead to repeated testing. Studies of false-negative (FN) results from respiratory samples for SARS-CoV-2 are variable demonstrating FN rates (FNRs) ranging from 1 to 30% [1, 2]. Current guidance from the World Health Organization (WHO) and others calls for repeat testing (including sampling of the lower respiratory tract) in individuals

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