Copan eNAT Transport System To Address Challenges in COVID-19 Diagnostics in Regions with Limited Testing Access

Abstract

Community-based health care clinics and hospital outreach services have the potential to expand coronavirus disease 2019 (COVID-19) diagnostics to rural areas. However, reduced specimen stability during extended transport, the absence of a cold chain to centralized laboratories, and biosafety concerns surrounding specimen handling have limited this expansion. In the following study, we evaluated eNAT (Copan Italia, Brescia, Italy) as an alternative transport system to address the biosafety and stability challenges associated with expanding COVID-19 diagnostics to rural and remote regions. In this study, we demonstrated that high-titer severe acute respiratory virus syndrome coronavirus 2 (SARS-CoV-2) lysate placed into eNAT medium cannot be propagated in cell culture, supporting viral inactivation. To account for off-site testing in these settings, we assessed the stability of contrived nasopharyngeal (NP) specimens stored for up to 14 days in various transport media (eNAT, eSwab, viral transport medium [VTM], saline, and phosphate-buffered saline [PBS]) at 4°C, 22 to 25°C, and 35°C. The molecular detection of SARS-CoV-2 was unaffected by sample storage temperature over the 2 weeks when stored in eNAT or PBS (change in cycle threshold, ≤1). In contrast, variable stability was observed across test conditions for other transport media. As eNAT can inactivate SARS-CoV-2, it may support COVID-19 diagnostics at the point of care. Evaluation of compatibility of eNAT with Cepheid Xpert Xpress SARS-CoV-2 assay demonstrated diagnostic accuracy and sensitivity equivalent to those of VTM. Taken together, these findings suggest that the implementation of eNAT as a collection device can expand COVID-19 testing to areas with limited health care access.

Keywords: COVID-19; Cepheid Xpert Xpress SARS-CoV-2; SARS-CoV-2; SARS-CoV-2 inactivation; SARS-CoV-2 specimen stability; eNAT; limited testing access; low-resource settings; point of care; rural healthcare.

FIG 1

eNAT inactivation of SARS-CoV-2. To determine the efficacy of eNAT for inactivating SARS-CoV-2, infectious SARS-CoV-2 was quantified by viral plaque assay on Vero E6 cells following incubation with eNAT or DMEM for 10 min at room temperature. Analysis of eNAT inactivation was performed using two sample types. (A) Swabs were inoculated with infectious SARS-CoV-2 stock (∼100 μl; stock, 2.3 × 10⁷ PFU/ml) or DMEM and were placed into transport tubes containing 1 ml of eNAT or DMEM. (B) Equal volumes of SARS-CoV-2 stock (2.3 × 10⁷ PFU/ml) or DMEM were combined with eNAT or DMEM (final volume of 200 μl) in the absence of a swab. Bars are representative of experimental triplicates (means ± standard deviations). Data displayed are from a single representative experiment of three independent experiments. The dotted line indicates the limit of detection of 500 PFU/ml due to eNAT lysis of the Vero E6 cells at the lowest dilution (10⁻¹). Abbreviations: ND, not detected; LoD, limit of detection; DMEM, Dulbecco’s modified Eagle medium.

FIG 2

Effect of alternative transport medium storage time and temperature on the molecular detection of SARS-CoV-2 nucleic acid in contrived nasopharyngeal swab specimens. Transport medium was stored at 4°C (A), 22 to 25°C (B), or 35°C (C) for 14 days. SARS-CoV-2 C_T values for each sample were determined at baseline and days 1, 3, 7, and 14. The change in C_T value from baseline (ΔC_T) was calculated and plotted. Bars are representative of experimental triplicates and are presented as mean ΔC_T values ± standard deviations from the baseline. Loss in sample sensitivity from the baseline is plotted as a positive value. ΔC_T values of >1 are considered a significant change from baseline. Abbreviations: NaCl, 0.9% saline; PBS, phosphate-buffered saline; VTM, viral transport medium.