key: cord-0733931-mofs9fpe
authors: Kline, Ahnika; Putnam, Nicole E.; Youn, Jung-Ho; East, Amanda; Das, Sanchita; Frank, Karen M.; Zelazny, Adrian M.
title: Dacron swab and PBS are acceptable alternatives to flocked swab and viral transport media for SARS-CoV-2
date: 2020-09-16
journal: Diagn Microbiol Infect Dis
DOI: 10.1016/j.diagmicrobio.2020.115209
sha: 561e6f4c4dba1a25bfbb04ecdf13a3ffc01696eb
doc_id: 733931
cord_uid: mofs9fpe

Nasopharyngeal flocked swabs placed in viral transport media (VTM) are the preferred collection methodology for respiratory virus testing. Due to the rapid depletion of available reagents and swabs, we have validated an alternative swab placed in phosphate buffered saline (PBS) for use in respiratory virus testing in a SARS-CoV-2 real-time PCR assay and a multiplexed respiratory virus panel. We collected nasopharyngeal (NP) swabs and oropharyngeal (OP) swabs from ten healthy volunteers. Flocked swabs were placed in VTM and alternative swabs in PBS. In this feasibility study, we show that NP collection is better for detection of human material than OP collection, as measured by significantly lower RNase P gene cycle threshold values, and that a dacron polyester swab in PBS shows equivalent detection of SARS-CoV-2 and RSV to a flocked swab in VTM in contrived specimens. Diluted SARS-CoV-2 positive patient specimens are detectable for up to 72 hours at 4 °C.

Following its emergence in late 2019, Severe Acute Respiratory Syndrome-Coronavirus-2 (SARS-CoV-2) has reached pandemic proportions causing the associated Coronavirus Disease 2019 . In order to monitor viral spread through the population, it is critical that testing facilities are able to detect this novel coronavirus. Viral detection has overwhelmingly been taken over by molecular methods, through the use of Polymerase Chain Reaction (PCR) assays and multiplexed syndromic platforms. For this novel coronavirus, clinical microbiology and public health laboratories in the United States have relied on commercially-developed emergency use authorizations (EUAs) as a response to the SARS-CoV-2 pandemic (1). For these EUA assays and patients with respiratory symptoms, it is commonplace to utilize nasopharyngeal (NP) collection with a nylon-tipped flocked swab to promote the collection of cell-associated virus. However, in response to the dramatic increase in global and nationwide testing, there has been a shortage of reagents available to testing laboratories. The current reagents approved for viral testing include flocked swabs placed into viral transport media (VTM), and a shortage could have the undesired effect to halt testing for COVID-19 and other respiratory illnesses (2) . This feasibility study was conducted in response to the nationwide shortage in collection reagents during the pandemic caused by SARS-CoV-2 (3) . In this study, we show that a dacron polyester swab in PBS can be substituted for a flocked swab in VTM for both nasopharyngeal (NP) and oropharyngeal (OP) collection, and that PBS is a suitable media to allow for the detection of both SARS-COV-2 and RSV in contrived specimens and diluted known SARS-CoV-2 positive patient specimens over time. 

In response to the SARS-CoV-2 pandemic, the Centers for Disease Control and Prevention (CDC) 2019 novel Coronavirus (2019-nCoV) Real-Time PCR Diagnostic Panel has received U.S. Food and Drug Administration (FDA) approval and has been adopted by the National Institutes of Health (NIH) Clinical Center, herein referred to as the SARS-CoV-2 RT-PCR assay (1, 2). The SARS-CoV-2 RT-PCR assay is used to screen NP and OP swab specimens in VTM and BAL for SARS-CoV-2 from patients with respiratory symptoms. The SARS-CoV-2 RT-PCR assay implemented at NIH Clinical Center utilizes the easyMAG automated nucleic acid extractor (bioMérieux, Marcy l'Etoile, France). A Taqman assay using two primer/probe sets is used to detect two distinct regions of the N gene (nucleocapsid protein, referred to as N1 and N2) and an internal control to detect the human RNase P (RP1) gene present in all specimen collections. This assay was performed on the ABI 7500 Fast Real-Time PCR System (Thermo Fisher Scientific, Waltham, MA).

In order to determine the suitability of the dacron polyester swab (Thermo Fisher Scientific, 149601J, Waitham, MA) in PBS at pH 7.2 (KD Medical, RGF-3190, Columbia, MD) compared to the flocked nylon swab (Becton Dickinson, BD B220252, Franklin Lakes, NJ) in VTM (Thermo Fisher Scientific, R12500 MicroTest M4 Tube, Waitham, MA) for NP and OP collection, each swab combination was used for a NP and an OP collection on ten healthy volunteers to give a total of 40 samples. Healthy volunteers consented to have replicate swabs tested for RP1, N1, and N2 gene with the SARS-CoV-2 RT-PCR assay and respiratory virus testing with the BioFire FilmArray Respiratory Pathogen Panel. Collection was performed by trained personnel J o u r n a l P r e -p r o o f Journal Pre-proof wearing N95 masks, eye protection, gloves and disposable gowns. Specimens were transported in a secondary labeled container. Each swab was placed into 3 mL of media (PBS or VTM) and stored on ice for transport to the clinical laboratory for testing. Upon receipt, a portion of each specimen was aliquoted in a biosafety cabinet and stored at 4°C. To test for the adequate collection of human material during collection, one aliquot of each specimen was processed at 24 hours and 72 hours for nucleic acid extraction. NP and OP specimens from both comparison conditions (flocked swab in VTM; dacron swab in PBS) were extracted and subjected to RT-PCR looking for amplification of the RNase P gene.

To test for the ability of the transport medium to adequately preserve the virus prior to detection, 2 aliquots of each NP collection were spiked with a stock of cultured RSV to generate contrived specimens. The RSV stock was stored at -70°C at 10,000 times the limit of detection (LOD) for the BioFire FilmArray Respiratory Panel (bioMérieux, Marcy l'Etoile, France), which was previously defined as 2 Tissue Culture Infectious Dose 50/mL (TCID 50 /mL). Handling of RSV and healthy volunteer specimens was done in a biosafety cabinet while wearing a N95 mask.

The day of collection, the 10,000X-LOD stock was diluted 1:10 in PBS and 1:100 into the aliquot to give a final RSV concentration of 10X-LOD. The stored RSV was diluted once in PBS to 10X-LOD and tested immediately to confirm detectability of the virus. At 24 and 72 hours after collection and storage at 4°C, 300 µL of contrived specimen was tested using the BioFire FilmArray Respiratory Panel. One aliquot of each specimen was tested at 24 hours to account J o u r n a l P r e -p r o o f Journal Pre-proof for potential real-time storage and turn-around of specimens collected one day and tested the next, and the remaining was tested at 72 hours to test the longevity of sample storage.

With the use of a biosafety cabinet and standard precautions, three paired specimens from healthy volunteers were spiked with heat-inactivated SARS-CoV-2 viral particles. While

practices, heat treatment of SARS-CoV-2 renders the virus non-infectious and the heatinactivated form is considered a BSL-1 agent per the manufacturer's instructions. Heatinactivated SARS-CoV-2 (ATCC, VR-1986HK, Manassas, VA) was diluted to a final concentration of 2x10 3 times the established LOD used for the SARS-CoV-2 EUA assay detection of N1 and N2 genes. The LOD was previously determined by our laboratory to be 0.5 genome copies/µL using digital droplet PCR. Media was either the VTM from which a flocked nylon swab from a NP collection had been inserted, or PBS from which a dacron polyester from a NP collection had been inserted. Three aliquots of 250 µL were made of the contrived specimens containing SARS-CoV-2 and were stored at 4°C. Nucleic acid extraction and PCR were perfomed as described in the SARS-CoV-2 RT-PCR assay at time 0, 24 hours, and 72 hours after spiking (1). reactions that had C t values between 15-32 for the N1 gene target and 17-36 for the N2 gene target. This allowed for testing the detection of strong (C t 15-24) and weakly (C t > 30) positive specimens prior to dilution (4). The known SARS-CoV-2 positive samples were identified from patients that had been sampled with the standard collection method: a NP collection from a flocked nylon swab placed into VTM. Each sample was stored at 4°C until it was diluted 1:10 in pooled media from NP collections from healthy volunteers. Media used for dilution was either VTM into which a collected flocked nylon swab had been inserted, or PBS into which a collected dacron polyester swab had been inserted. All diluted samples were stored at 4°C. SARS-CoV-2 PCR was then performed at time 0, 24 hours, and 72 hours to determine whether SARS-CoV-2 remained detectable in media obtained using the new collection method.

Equivalence and stability of RP1 gene detection was measured using a three-way ANOVA with Tukey's multiple comparisons test to compare the effect of variation of three factors: time (24 vs. 72 hrs), collection type (NP vs. OP), and swab/media (flocked swab/VTM vs. dacron swab/PBS). Our goal of equivalence for detection of the N1, N2, and RP1 genes was concordant results within 1 cycle threshold (C t ) value. Similarly, our intent to measure stability when comparing the time points of each collection type was a range within 1 C t value. Final determination of equivalence and stability were based on consistent trends between comparisons. For specimens diluted 1:10 (n = 1 per condition), equivalence and stability were defined as the comparison of paired specimens between VTM and PBS and between time J o u r n a l P r e -p r o o f Journal Pre-proof points, respectively, within 1 C t value. RSV detection through the BioFire FilmArray platform was qualitative in nature. Therefore, equivalence between VTM and PBS and stability between 24 and 72 hours was defined as 90% qualitative concordance in detection of RSV in the same collection type (i.e., NP, OP).

The human internal control gene RP1 was detectable in all collection conditions, with exception of one failed extraction of a NP collection (VTM/flocked swab) at 72 hours (Supplemental Table 1 ). The dacron swab in PBS showed equivalent performance in the detection of the RP1 gene when compared to the currently used flocked swab in VTM for NP and OP collection methods at 24 and 72 hours (Figure 1) . Moreover, the signal was comparable between 24 and 72 hour time points, indicating that the viral target was stable in both VTM and PBS when stored at 4°C (Figure 1 ). The human RP1 gene was not measured at 0 hours, and thus stability cannot be determined between 0 and 24 hours. These data show that C t values are influenced by the collection method (NP vs. OP). Data here show that a lower C t value is attainable with the NP collection when compared to OP collection, regardless of the swab/media used for specimen storage or the time point tested. 

Here, the feasibility of using PBS for viral detection was determined by using contrived specimens spiked with live RSV virus at 10X-LOD. A 10X RSV-LOD dilution in PBS was tested immediately and was detected by the BioFire FilmArray Respiratory Pathogen Panel (data not shown). At 24 hours, RSV was detected in 9 of 10 spiked VTM samples and 10 out of 10 spiked PBS samples (Table 1) . Additionally, at 72 hours RSV was detected in 9 out of 10 spiked VTM samples and all 10 spiked PBS samples (Table 1) . Thus, RSV was detected in all spiked PBS media, but was not detected in two specimens of spiked VTM. These data suggest that PBS is both equivalent in performance to VTM and stable between 24 and 72 hours, as it meets the definition of 90% qualititative concordance between media and time points. 

Heat-inactivated SARS-CoV-2 viral particles were detected in spiked healthy volunteer media following NP collection at time 0, 24 hours, and 72 hours after spiking (Supplemental Table 2 ; Figure 2 ). One sample at 72 hours in VTM had no amplification for any of the targets, and thus no C t value for N1, N2, or RP1, consistent with an extraction failure. In response to this failed extraction, two additional samples were spiked and extracted for PCR at 24 and 72 hours, to give a final n = 3 (time 0) and n = 5 (24 hours, 72 hours). Two paired N2 gene C t values had a difference of 1.80 and 1.85, and two paired RP1 data points differed by 2.83 and 3.99 (Supplemental Table 2 ). However, other absolute differences were consistently closer to or less than 1 C t value (N1 n = 12; N2 n = 10; RP1 n = 10), demonstrating that paired specimens were similarly detected in VTM and PBS (Supplemental Table 2 ; Figure 2 ). Accordingly, equivalence standards were met for the all gene targets (N1, N2, RP1) in the SARS-CoV-2 real-time PCR assay. To compare stability, each target C t value was compared between time 0, 24 hours, and 72 hours. Curiously, the N1 target appeared to have a decreasing C t value over time, and this trend was consistent between VTM and PBS (Supplemental Table 2 ). However the N2 and RP1 gene targets had stable C t values over the time points, ranging within roughly 1 C t value (Supplemental Table 2 ).

Journal Pre-proof For unknown reasons, the N1 gene appears to trend toward a lower C t count over time being used. While we found that substituting PBS for VTM is useful for molecular detection using PCR, it may not be appropriate for viral culture if that were required.

These results come at a critical moment. SARS-CoV-2 testing must continue using reagents that allow for equivalent testing performance, and we have shown here that commonly stocked laboratory items, including dacron polyester swabs and PBS, may be substituted for viral detection after appropriate equivalence studies are conducted. 

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Verification procedure for commercial tests with Emergency Use Authorization for the detection of SARS-CoV-2

Nasal swab sampling for SARS-CoV-2: A convenient alternative in time of nasopharyngeal swab shortage

Comparison of Copan Eswab and FLOQswab for COVID-19 PCR diagnosis: Working around a supply shortage

The Detection of SARS-CoV-2 using the Cepheid Xpert Xpress SARS-CoV-2 and Roche 2 cobas SARS-CoV-2 Assays

Comparison of Abbott ID Now, Diasorin Simplexa, and CDC FDA EUA methods for the detection of SARS-CoV-2 from nasopharyngeal and nasal swabs from individuals diagnosed with COVID-19

Evaluation of saline, phosphate buffered saline and minimum essential medium as potential alternatives to viral transport media for SARS-CoV-2 testing

Memoli for supplying us with live RSV virus for this experiment. We thank the healthy volunteers for allowing repeat NP and OP specimen collection. We also acknowledge the laboratory technologists, staff, nurses, and physicians working diligently in response to the SARS-CoV-2 pandemic.