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

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.

Superiority of MALDI-TOF Mass Spectrometry over Real-Time PCR for SARS-CoV-2 RNA Detection

“…Our data analysis is concordant with other studies reporting false-negative results from RT-PCR tests ranging up to 30% [4]. Several studies have already showed that the diagnostic efficacy between RT-PCR test kits for SARS-CoV-2 nucleic acid detection may differ significantly depending on the probes and primers used [12,13].…”

“…According to the WHO and Chinese Center for Disease Control and Prevention (CDC), real-time PCR (RT-PCR) is considered the 'gold standard' method for detection of SARS-CoV-2 infection [3]. Nevertheless, studies have shown that RT-PCR is an error-prone method showing false-negative results rates from respiratory samples for SARS-CoV-2 ranging from 1% to 30% [4]. These diagnostic errors are related to a number of preanalytical and analytical factors, including the source of the sample, delays or inadequate storage conditions before arrival to the laboratory, possible errors of RNA extractions, as well as the different quality and sensitivity of detection kits [5].…”

“…These diagnostic errors are related to a number of preanalytical and analytical factors, including the source of the sample, delays or inadequate storage conditions before arrival to the laboratory, possible errors of RNA extractions, as well as the different quality and sensitivity of detection kits [5]. Additionally, insufficient viral load, the incubation period of the disease, and the presence of mutations that allow virus to escape from the detection may also lead to the risk of obtaining false-negative results [4]. Therefore, the improvement of the COVID-19 diagnosis is urgently needed for obtaining true negative results, allowing one to exit quarantine and avoid virus transmission and recurrence.…”

Structural basis of fitness of emerging SARS-COV-2 variants and considerations for screening, testing and surveillance strategy to contain their threat

“…46, 48, 51, 54, 55 While three studies on hospital-based testing mentioning the interval to reassessment 57, 58, 63 showed no such linearity between reassessment interval and false negative rate (Figure 6C, Table 6), repeat RT-PCR testing of nasal swabs after a median interval of 7.5 days reported detection of highest fraction of initial false negative RT-PCR results. 58 Plot of RTPCR false negative detection on reassessment against adjusted interval between initial RT-PCR and reassessment combining population-based screening 42-44 and symptom-based testing studies 46, 48, 51, 54, 55 showed a peak between 7-8 days. It should be noted that although desirable, the interval between symptom onset and initial RT-…”

What is the role of bronchoalveolar lavage in the diagnosis of COVID-19?

“…After the initial phase of this pandemic, in fact, some author argued for low sensitivity of RT-PCR performed on NPS [2], suggesting that repeating the test, or using other samples, may reduce the number of false negatives [4], which another study estimated to be as high as 9.3% [5]; noteworthy, other reports found even lower sensitivity for NPS [6,7]. Beyond these data, the overarching question still remains unsolved: how much can we rely on a single negative NPS to rule out COVID-19, if clinical suspicion remains high?…”