key: cord-0763548-1mceq6v0
authors: Kinloch, Natalie N; Ritchie, Gordon; Brumme, Chanson J; Dong, Winnie; Dong, Weiyan; Lawson, Tanya; Jones, R Brad; Montaner, Julio S G; Leung, Victor; Romney, Marc G; Stefanovic, Aleksandra; Matic, Nancy; Lowe, Christopher F; Brumme, Zabrina L
title: Suboptimal biological sampling as a probable cause of false-negative COVID-19 diagnostic test results
date: 2020-06-28
journal: J Infect Dis
DOI: 10.1093/infdis/jiaa370
sha: 284aed341739978fb3e8ca559dc5501f2c238336
doc_id: 763548
cord_uid: 1mceq6v0

False-negative SARS-CoV-2 test results can negatively impact the clinical and public health response to COVID-19. We used droplet digital PCR (ddPCR) to demonstrate that human DNA levels, a stable molecular marker of sampling quality, were significantly lower in samples from 40 confirmed or suspected COVID-19 cases that yielded negative diagnostic test results (i.e. suspected false-negative test results) compared to a representative pool of 87 specimens submitted for COVID-19 testing. Our results support suboptimal biological sampling as a contributor to false-negative COVID-19 test results and underscore the importance of proper training and technique in the collection of nasopharyngeal specimens.

A c c e p t e d M a n u s c r i p t

Accurate COVID-19 diagnosis is critical to a successful clinical and public health response. Current COVID-19 tests detect one or more targets in the SARS-CoV-2 RNA genome, usually by real-time reverse transcriptase (RT)-PCR, and nasopharyngeal swabs have been the preferred sample for testing to date [1] . While PCR-based tests are highly sensitive, falsenegative COVID-19 test results do occur [2, 3] , though reported rates vary. A recent large retrospective study estimated the clinical sensitivity of SARS-CoV-2 molecular assays to be between 58% and 96% [4] , while another reported a 67% SARS-CoV-2 RNA detectability rate in respiratory samples taken within 7 days of hospitalization for COVID-19 [5] . Various factors other than molecular technology contribute to test sensitivity, including the timing of sample collection with respect to infection stage [6, 7] as well as specimen storage and transport [2] .

Improper specimen collection could also contribute to false-negative COVID-19 test results.

Although nasopharyngeal swabs are routinely ordered for respiratory viruses, the collection of a high quality specimen requires training and expertise as it involves insertion of the swab to posterior nasopharynx, a depth of roughly 7cm, followed by rotation and withdrawal of the swab [8] . To investigate suboptimal sample collection as a possible cause of false-negative test results, we quantified human DNA levels recovered on nasopharyngeal swabs submitted to a single laboratory for COVID-19 testing, hypothesizing that human DNA could serve as a stable molecular marker of specimen collection quality.

The St. Paul's Hospital (SPH) Virology laboratory is one of five provincially designated SARS-CoV-2 diagnostic laboratories in British Columbia, Canada. COVID-19 testing on A c c e p t e d M a n u s c r i p t nasopharyngeal swabs (Copan UTM® collection kit or BD universal viral transport system) was performed by total nucleic acid extraction from 500μL medium on the Roche MagNA Pure 96 followed by real-time reverse-transcriptase (RT)-PCR using the Roche LightMix® 2019-nCoV real-time RT-PCR assay, which uses E-Sarbeco primers/probes [9] , or using the Roche cobas® SARS-CoV-2 test.

Between March to May 2020 we identified 40 suspected false-negative nasopharyngeal swab test results from presumed or confirmed COVID-19 cases for which >1mL medium remained for re-testing. These included 23 negative samples from individuals who recorded a positive test within ±12 days of the negative test (where the median time elapsed between negative and positive tests was 4 days, with an interquartile range of 1-6 days) and 17 samples from individuals who tested negative but for whom there was high clinical suspicion of infection by the treating physician with no alternate diagnosis established. A convenience sample of 87 consecutively submitted nasopharyngeal swabs served as a comparison dataset. Remnant specimens were stored at -20°C until re-testing. To standardize nucleic acid extraction across all specimens and to maximize viral RNA recovery, 1mL of medium was extracted on the BioMérieux EasyMag and eluted in 35μL buffer. SARS-CoV-2 detection in suspect falsenegative samples was re-attempted using a nested RT-PCR and sequencing protocol targeting conserved regions in ORF-1a and Spike [10] , where the lower limit of detection of this assay was estimated in-house using serial dilutions of synthetic SARS-CoV-2 RNA standards (Exact Diagnostics).

Human DNA levels were quantified using droplet digital PCR (ddPCR), a technique where each sample is fractionated into 20,000 nanolitre-sized water-in-oil droplets prior to PCR amplification with sequence-specific primers and fluorescent probes, and where Poisson detection sensitivity compared to the original real-time RT-PCR assay, we re-tested the 40 suspected false-negative specimens by nested RT-PCR. All suspect false-negative samples however again tested negative. This indicated that the original negative results were not likely attributable to suboptimal real-time RT-PCR assay performance, but rather suggested that SARS-CoV-2 RNA was exceedingly low or absent in these samples.

We then investigated whether human DNA recovered on the nasopharyngeal swab could serve as a molecular marker of specimen collection quality, reasoning that DNA (by virtue of its stability) would be well-preserved in remnant clinical specimens. We employed a sensitive, multiplexed ddPCR protocol for absolute human RPP30 gene copy number quantification [12] .

Overall, we observed significantly lower human DNA levels in the suspected false-negative nasopharyngeal swab samples compared to a panel of consecutive samples submitted for testing during the same period, though overlap between groups was still substantial (Figure 1, p<0.001) .

Specifically, suspected false negative specimens harbored a median 3,409 (Interquartile range We chose human DNA as a molecular marker of sampling quality because of its stability.

RNAseP RNA-specific primer/probe set, in part to assess sample quality [11] . To investigate the relationship between human cells as measured by RPP30 gene copy number using ddPCR, and 

Our results underscore the importance of proper training and technique in the collection of high quality nasopharyngeal specimens. They also highlight the potential utility of including a molecular marker of sampling quality in SARS-CoV-2 diagnostic assays that could serve as an endogenous control. While the major commercial assays (e.g. Roche cobas® SARS-CoV-2;

A c c e p t e d M a n u s c r i p t https://www.fda.gov/media/136049) include an internal RNA control for nucleic acid extraction and RT-PCR amplification, these do not provide a measure of biological sampling quality. While the US-CDC 2019-nCoV real-time RT-PCR diagnostic panel does feature a human RNAseP RNA-specific primer/probe set, in part to assess sample quality [11] , our findings suggest that the interpretation criteria for this control may be too liberal. Specifically, the US-CDC's instructions for use, issued on 15-Mar-2020, state that failure to detect RNAseP within 40 PCR cycles can indicate insufficient biological material in the sample or other assay problems. In our retrospective test panel of 91 remnant nasopharyngeal nucleic acid extracts however, the 90th percentile Ct value for RNAseP was 25.9 (range 19.65 to 27.77; see results). The observation that even the lowest decile of samples in terms of RNAseP RNA levels (possibly representing those for which sampling was the least robust) still amplified well before Ct<40 suggests that this threshold may be insufficient to identify suboptimally-collected samples.

Some limitations of our study merit mention. Our use of a convenience sample of 87 consecutively submitted nasopharyngeal swabs may not represent an ideal control group, as there is no guarantee that these samples were collected using appropriate or consistent technique.

Indeed, the wide range of human DNA levels observed in this group, and the substantial overlap with the suspected false negative group, corroborate this notion. This limitation however should only serve to reduce our study's statistical power. Moreover, approximately 40% of our suspected false negative tests derived from patients with high clinical suspicion of SARS-CoV-2 infection but whose diagnosis was never confirmed. As diagnoses may not have been made in a consistent manner across treating physicians, these samples may be less likely to represent false negative results. However, our observation that human DNA levels in both subcategories of the false-negative group were significantly lower than in the comparison group suggests that this A c c e p t e d M a n u s c r i p t limitation may be minimal. It is also important to note that our study was not designed to identify a threshold of human DNA (or RNA) that could define a properly-collected SARS-CoV-2 nasopharyngeal swab, and that future studies attempting to do so would need to consider that recovery efficiency (and thus total yield) of different types of nucleic acid may differ by extraction platform (e.g. human DNA levels recovered in the present study differed between BioMérieux and MagNA Pure platforms, see Results), and possibly by swab type. It is also important to note that, although human DNA levels can serve as a surrogate marker of the amount of biological material collected, sampling the correct anatomical location is also critical, particularly for nasopharyngeal swabs.

Our observations strongly support suboptimal biological sampling, but not PCR sensitivity for SARS-CoV-2 RNA detection, as a contributing cause of false-negative COVID-19 test results. M a n u s c r i p t A c c e p t e d M a n u s c r i p t Human DNA levels (RPP30 gene target) were measured using ddPCR in nasopharyngeal extracts as a molecular marker of biological sampling quality. "Suspect false-negatives" included 23 negative samples from individuals who recorded a positive test within ±12 days of the negative test (grey) and 17 samples from individuals with high clinical suspicion of being infected but never molecularly confirmed (white). The comparison dataset was a consecutive set of 87 samples submitted for testing in April 2020 to the same laboratory (black). P-values report the significance level between the comparison dataset and the "suspect false-negative" group as a whole (black), between the comparison dataset and the negative samples from individuals who reported a positive test within ±12 days (grey) and between the comparison dataset and the negative samples from individuals with high clinical suspicion (white).

A c c e p t e d M a n u s c r i p t Figure 1 

Interim Guidelines for Collecting, Handling, and Testing Clinical Specimens from Persons for Coronavirus Disease

False Negatives and Reinfections: the Challenges of SARS-CoV-2 RT-PCR Testing

False Negative Tests for SARS-CoV-2 Infection -Challenges and Implications

Clinical Performance of SARS-CoV-2 Molecular Testing

Antibody responses to SARS-CoV-2 in patients of novel coronavirus disease

Virological assessment of hospitalized patients with COVID-2019

Variation in False-Negative Rate of Reverse Transcriptase Polymerase Chain Reaction-Based SARS-CoV-2 Tests by Time Since Exposure

How to Obtain a Nasopharyngeal Swab Specimen

Detection of 2019 novel coronavirus (2019-nCoV) by real-time RT-PCR

Detection of second case of 2019-nCoV infection in Japan

CDC 2019-Novel Coronavirus (2019-nCoV) Real-Time RT-PCR Diagnostic Panel Instructions for use

A quantitative approach for measuring the reservoir of latent HIV-1 proviruses

We thank Dr. Christopher Sherlock for helpful discussions. We thank the laboratory teams at the A c c e p t e d M a n u s c r i p t