Dacron swab and PBS are acceptable alternatives to flocked swab and viral transport media for SARS-CoV-2

Abstract

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 polymerase chain reaction assay and a multiplexed respiratory virus panel. We collected nasopharyngeal (NP) swabs and oropharyngeal (OP) swabs from 10 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 h at 4 °C.

Keywords: 2019-nCOV; Alternative reagents; COVID-19; SARS-CoV-2.

Fig. 1

Dacron polyester swab in PBS shows equivalent performance and stability up to 72 h compared to a flocked nylon swab in VTM. Nasopharyngeal and oropharyngeal collections were obtained using a flocked nylon swab placed into VTM (red, circle) or a Dacron polyester swab and placed into PBS (blue, square). C_t values for detection of the human RNase P (RP1) gene were measured by RT-PCR from 10 healthy volunteers at 24 h (light red, light blue) or 72 h (dark red, dark blue). Each of 8 collection condition and time point combinations was tested on 10 healthy volunteers, with paired comparisons shown between flocked swab/VTM and Dacron swab/PBS collection methods. A 3-way ANOVA with Tukey's multiple-comparisons test was performed to compare C_t values between NP and OP collection types in each swab/media combination at 24 and 72 h. Multiple comparisons show that at both 24 and 72 h, the C_t values between NP-VTM and OP-VTM and NP-PBS and OP-PBS differ significantly, with all 4 comparisons giving P < 0.0001. Thus, the collection type (NP versus OP) led to a significant source of variation (P < 0.0001).

Fig. 2

PBS and VTM show equivalent detection of heat-inactivated SARS-CoV-2 and human material. Nasopharyngeal collections were obtained using a flocked swab placed into VTM (solid symbols) or a Dacron polyester swab and placed into PBS (open symbols). C_t values for detection of the N1 (green) and N2 (blue) nucleocapsid gene segments and internal control RP1 (magenta) gene were measured at time 0 (n = 3), 24 h (n = 5), and 72 h (n = 5) from media spiked with heat-inactivated SARS-CoV-2. One sample in the VTM 72-h condition failed the nucleic acid extraction step. Each symbol represents the C_t value from 1 sample, with VTM and PBS paired symbols connected by lines, represented as time 0 (gray dashed line), 24 h (black dotted line), and 72 h (black solid line).

Fig. 3

PBS shows similar detection of diluted positive SARS-CoV-2 patient specimens compared to VTM using the SARS-CoV-2 real-time PCR assay. Ten known positive SARS-CoV-2 patient specimens with varying C_t values were diluted 1:10 into VTM from a flocked swab NP collection (green circles) or PBS from a Dacron swab NP collection (blue squares). Paired C_t values from each diluted sample are shown for the N1 and N2 genes at 0 h and 72 h after dilution.