The Effects of “Dry Swab” Incubation on SARS-CoV-2 Molecular Testing

Abstract

Background

Widespread testing of SARS-CoV-2 has resulted in shortages of collection devices and transport media. We evaluated the stability of flocked swabs inoculated with SARS-CoV-2-containing specimen incubated dry (i.e., without transport medium) at room temperature.

Methods

A pool of SARS-CoV-2 positive specimen was used to inoculate flocked swabs. Five swabs were placed immediately into universal transport media (UTM) following inoculation, and tested immediately (day 0). Fifteen of the swabs were placed into sterile 15-mL conical tubes and incubated at room temperature for 1, 2, or 7 days. Following incubation, swabs were hydrated in separate vials of UTM and tested. This protocol was repeated for viral transport media (VTM) and saline. As a comparison, a series of swabs was prepared and tested in parallel, but stored in the corresponding liquid transport media (UTM, VTM, or saline) and incubated at room temperature. Testing was performed at 1, 2, and 7 days postinoculation in duplicate. All molecular testing was performed using the Roche cobas SARS-CoV-2 assay.

Results

All dry swabs tested on days 1, 2, and 7 provided results that were within 2 cycle thresholds (C_Ts) of the average C_T values for swabs hydrated in the same media and tested on day 0. There was no statistical difference in C_T values between swabs incubated in liquid media versus dry swabs incubated at room temperature prior to hydration in liquid media.

Conclusions

The utilization of “dry swabs” may simplify specimen collection, negate the need for liquid transport media, and mitigate safety risks while preserving the accuracy of testing.

Introduction

Shortages in universal transport media (UTM) and viral transport media (VTM) have forced laboratories and manufacturers to explore alternative approaches to specimen collection and transport for the testing of SARS-CoV-2 (1–3). Some laboratories have developed protocols for making UTM or VTM in house while others are distributing modified collection devices with limited amounts of transport media (4). In some instances, providers may be asked to place the specimen collection device/swab into a limited amount of transport media, shake the swab vigorously, and discard it (5). This process has the potential to generate aerosols and infectious waste, resulting in a potential biosafety concern, especially in high-traffic patient care areas. A potential alternative is to transport the specimen collection swabs in a tube without liquid transport media (dry swabs), and hydrate them for analysis in transport media upon laboratory receipt. The transport of dry swabs allows for the relegation of aerosol generating processes to a biological safety cabinet and eliminates the need to distribute aliquots of collection media to collection sites. This may also address the challenge that some transport devices (VTM) require refrigeration after collection, a logistical challenge, especially at outdoor drive-through specimen collection sites. Little is known about the stability of SARS-CoV-2 on a dry swab, or the impact on testing accuracy. We evaluated the stability of dry swabs for the testing of SARS-CoV-2 when hydrated in UTM, CDC VTM, and 0.9% saline following various lag times after collection at room temperature incubation.

Impact Statement

The work herein is an initial evaluation of a novel strategy for the collection and transport of swab specimens submitted for SARS-CoV-2 testing. Although the work is preliminary in nature, by highlighting the stability of SARS-CoV-2 nucleic acid on dry swab specimens, the acceptability of this specimen collection and transport technique can be further investigated. This strategy negates the need for liquid transport media and, if adopted, could address significant transport media shortage challenges affecting the widespread availability of SARS-CoV-2 testing.

Methods

Preparation of Positive Swabs

This quality improvement project was reviewed by the local Washington University Institutional Review Board and was determined to not be human participant research. All swabs used in this study were flocked swabs (COPAN). To achieve uniform inoculation, a single pool of SARS-CoV-2 positive UTM from previously tested nasopharyngeal swab specimens was used to inoculate flocked swabs. The pool was tested by the Roche cobas SARS-CoV-2 assay to confirm positivity prior to specimen inoculation. Sixty (60) swabs were inoculated by submersing them in the UTM pool for 3–5 s. Each swab was then immediately placed in a sterile 15-mL conical tube. Swabs were incubated and tested as outlined next. On the day of testing, dry swabs were eluted in 1 mL of liquid media by submerging for 3–5 seconds without vortexing. Swabs were left in the liquid media until immediately prior to testing liquid media. Liquid media was tested within 12 h of dry swab inoculation in accordance with the manufacturer’s instructions for the cobas SARS-CoV-2 assay.

Dry Swab Incubation

Twenty (20) swabs were used for testing stability of viral detection in universal transport medium. Five were placed immediately into UTM (COPAN), following inoculation and tested immediately (day 0). The remaining swabs were placed into sterile 15-mL conical tubes and incubated at room temperature. On the day of testing, each dry swab was eluted in 1 mL of UTM by submerging for 3–5 seconds without vortexing. Swabs were left in liquid UTM prior to testing. Liquid UTM was tested within 12 h of dry swab inoculation in accordance with the manufacturer instructions. This entailed pipetting a 700 µL aliquot of liquid media into a secondary tube and loading the secondary tube into the automated Roche cobas 6800 instrument.

Testing was performed in sets of 5 at 3 different time points (day 1, day 2, and day 7). This protocol was repeated for VTM (Hardy) and saline (Becton Dickinson) (5 replicates at day 0, day 1, day 2, and day 7).

Liquid Media Incubation

To compare dry swab incubation to incubation in liquid transport media, additional swabs were inoculated into the pooled positive specimen, immediately hydrated in liquid media (UTM, VTM, or saline) and incubated at room temperature. Testing was performed on the liquid media at 1, 2, and 7 days postinoculation. This protocol was performed in duplicate for each liquid media.

Molecular Testing

All molecular testing was performed using the cobas SARS-CoV-2 assay (Roche). The cobas SARS-CoV-2 assay targets ORF1ab gene and E gene sequences of SARS-CoV-2. Both targets were utilized for comparison of C_T results.

Viral Culture

Viral culture was used to determine the viability of SARS-CoV-2 following each incubation. Culture was performed on the initial SARS-CoV-2 pool used to inoculate swabs. Culture was also performed in triplicate from each media used to hydrate swabs following dry incubation at the day 0, day 1, and day 2 time points. Samples were serially diluted in DMEM supplemented with 2% FBS, then added to Vero cell monolayers in 96-well plates and incubated for 1 h at 37 °C. All plates included serial dilutions of a positive control sample with known amount of virus to ensure successful detected live virus. Cells were then overlaid with 1% (w/v) methylcellulose in MEM supplemented with 2% FBS. Plates were incubated for 30 h before fixation using 4% PFA in PBS for 1 h at room temperature. Cells were washed then sequentially incubated with anti-SARS-CoV-2 CR3022 antibody (1 μg/mL) and an HRP-conjugated goat antihuman IgG (Sigma) in PBS supplemented with 0.1% (w/v) saponin (Sigma) and 0.1% BSA (6). TrueBlue peroxidase substrate (KPL) was used to develop the plates before counting the foci on a BioSpot analyzer (Cellular Technology Limited).

Results

Testing of Dry Swabs

The positive pool used to inoculate swabs was positive with C_T results of 19.4 and 19.9 for the ORF1ab and E gene, respectively. Testing of inoculated samples revealed qualitative positive percentage agreement by molecular testing of 100% for each set of 5 replicates at day 0, 1, 2, and 7 (Fig. 1). All dry swabs tested at days 1, 2, and 7 were within 2 C_T of the average C_T values for swabs hydrated in the same media and tested on day 0. No statistically significant difference was observed across all dry swab replicates based on one-way ANOVA with Bonferroni correction between groups within each gene target. The stability in liquid media was similar, with all day 1, 2, and 7 replicates within 2 C_T of the average day 0 result (Fig. 2). Similar to dry swab replicates, no statistically significant difference was observed across all liquid media replicates between groups within each gene target. There was no statistical difference in C_T results between swabs incubated in liquid media versus dry swabs incubated at room temperature prior to inoculation into liquid media. All dry swabs tested at days 1, 2, and 7 were within 2 C_T of the average C_T values for swabs tested following the same incubation time in liquid media.

Fig. 1.

C_T values for dry swabs tested at days 1, 2, and 7 on the cobas SARS-CoV-2 assay. Dry swabs were hydrated in the indicated media (UTM, VTM, or saline) at days 0, 1, 2, and 7 following inoculation into a pooled positive specimen. C_T values for the ORF1ab gene (closed symbols) and E gene (open symbols) are shown as an average of 5 replicates. The standard deviation is indicated by the error bars. No statistically significant difference was observed (one-way ANOVA with Bonferroni correction) between groups within each gene target.

Fig. 2.

C_T values for swabs incubated in liquid media and tested at days 1, 2, and 7 on the cobas SARS-CoV-2 assay. Swabs were immediately hydrated in the indicated media (UTM, VTM, or saline) and incubated for 1, 2, and 7 days following inoculation into a pooled positive specimen. Lines connect specimen tested from the same aliquot of liquid media. C_T values for the ORF1ab gene (A) and E gene (B) are shown for both replicates. No statistically significant difference was observed (one-way ANOVA with Bonferroni correction) between groups within each gene target.

Viral Culture Results

Positive and negative controls for all viral cultures yielded acceptable results. The only specimen positive by culture was the original pool used to prepare the swabs. Culture results for all dry swab replicates from the day 0, 1, and 2 specimens hydrated in UTM, VTM, and saline were negative. This was despite moderately low C_T values, (between 23 and 26 for both assay targets). Culture results from liquid controls were also negative.

Discussion

The ability to obtain robust C_T values from dry swabs stored for a week at room temperature despite culture negativity has important implications for SARS-CoV-2 testing. Specimen collection has the potential to be simplified by removing the need for liquid transport media. Liquid media may not need to be included with specimen collection kits and providers would not have to hydrate swabs at the point of collection. This latter step may generating unnecessary waste and aerosols depending on swab type and media container used, therefore being able to perform hydration and testing away from specimen collection has important safety ramifications. Since many communities may have limited amounts of specimen collection sites, simplification of specimen collection may allow better accessibility to patients. Since transport from limited collection sites may be prolonged, the data indicating that dry swabs are stable for at least a week are encouraging.

Our data indicate that rather than being utilized for specimen transport, liquid media could be used to simply hydrate swabs and release them of virus prior to testing following receipt at the testing laboratory. This allows these steps to be performed in a controlled environment within a biosafety cabinet. If the liquid media is being used to only hydrate the swab it may not need to include all the components necessary for long term stability. Our data showing a similar result from swabs hydrated in saline compared to UTM and VTM supports this. For assays utilizing their specific elution or lysis/inactivation buffer, there may be potential to directly inoculate the dry swab into assay specific buffer, bypassing the need for liquid media altogether and potentially alleviating any dilution of target that may occur in the process of eluting the swab in liquid media.

The use of dry swab specimens has important safety benefits as well. While specimens maintained robust C_Ts even following 7 days of dry incubation, virus was not cultivable. The inability to culture virus may have been secondary to dilution brought about by our experimental technique, though the robust C_T values suggest the inability to culture was in part related to viral stability. This suggests a dry swab approach may have an ability to maintain diagnostic accuracy while minimizing exposure of laboratory workers to viable virus (7).

Our study has several limitations. We only evaluated stability around one specific C_T value, so although our C_T values did not show significant change this relationship may differ at higher or lower values. Furthermore, we did not evaluate extremes in temperature or atmosphere or other conditions that may affect stability, nor did we evaluate swabs other than the commonly used flocked versions. Finally, our swab specimens were prepared by preparation from a positive patient pool previously incubated in UTM. This technique was needed in order to assure uniform inoculation of swabs for comparator purposes. However the stability of patient specimens collected directly from the nasopharynx or oropharynx may differ and warrant additional investigation. Any of these variables could dramatically affect the performance of testing from “dry swabs” and require additional study prior to implementing this method.

Herein we describe a novel technique for the collection and transport of specimens for SARS-SoV-2 testing. The utilization of “dry swabs” has the potential to simplify specimen collection, negate the need for liquid transport media, and mitigate safety risks, all while preserving the accuracy of testing.

 

Author Contributions: All authors confirmed they have contributed to the intellectual content of this paper and have met the following 4 requirements: (a) significant contributions to the conception and design, acquisition of data, or analysis and interpretation of data; (b) drafting or revising the article for intellectual content; (c) final approval of the published article; and (d) agreement to be accountable for all aspects of the article thus ensuring that questions related to the accuracy or integrity of any part of the article are appropriately investigated and resolved.

Authors’ Disclosures or Potential Conflicts of Interest: Upon manuscript submission, all authors completed the author disclosure form. Disclosures and/or potential conflicts of interest: Employment or Leadership: None declared. Consultant or Advisory Role: C.-A.D. Burnham, Thermo Fisher, Cepheid, Bio Rad Laboratories; N.W. Anderson, Diasorin Molecular. Stock Ownership: None declared. Honoraria: C.-A.D. Burnham, Cepheid, Bio Fire; N.W. Anderson, BioMerieux. Research Funding: B.T. McCune, NIH F32 AI138392. Expert Testimony: None declared. Patents: None declared.

Role of Sponsor: The funding organizations played no role in the design of study, choice of enrolled patients, review and interpretation of data, preparation of manuscript, or final approval of manuscript.