Real‐life evaluation of a rapid extraction‐free SARS‐CoV‐2 RT‐PCR assay (COVID‐19 PCR Fast‐L) for the diagnosis of COVID‐19

Abstract

To the Editor, Timely and rapid diagnosis of severe acute respiratory syndrome coronavirus 2 (SARS‐CoV‐2) infection by reverse transcription‐ polymerase chain reaction (RT‐PCR) is paramount to the control of the coronavirus disease 2019 (COVID‐19) pandemic. Attempts have been made to shorten molecular testing turnaround by skipping nucleic acid extraction, thus performing RT‐PCR directly on heat‐ treated respiratory specimens. A number of either “in‐house”‐ developed or commercial (i.e., Cepheid Xpert Xpress SARS‐CoV‐2 assay) RT‐PCR or loop‐mediated amplification protocols have been developed and found to display clinical sensitivities ranging from 75% to 98% when compared to RT‐PCR preceded by viral RNA extraction. Here, we conducted a real‐life evaluation of the performance of a commercially‐available free‐extraction RT‐PCR, the Ascires COVID‐19 PCR Fast‐L (Sistemas Genómicos®), which is multiplexed to amplify two conserved sequences within ORF‐1ab/1a (FAM and CY5 channels), one of which lies within the RdRP gene (FAM channel) and returns qualitative results in less than 1 h. In this assay, nasopharyngeal (NP) specimens are transferred to 1ml of transport/extraction buffer containing proteinase K provided by the manufacturer, placed in a dry bath at 60°C for 5min, then at 98°C for 2min, and finally on ice for cooling. Target amplification is carried out using the AriaMx Real‐Time PCR System (Agilent®), and results are analyzed and interpreted automatically by The AriaMx Software version 1.5. Thermal cycling conditions are shown in the footnote of Table 1. The assay includes an internal heterologous DNA control (HEX probe). According to the manufacturer, the limit of detection (LOD) of the assay is approximately 4000 copies/ml (95% confidence interval [CI]). This prospective study enrolled 662 patients between November 23 and December 10, 2020, attended at the Emergency Department of the Hospital Clínico Universitario of Valencia (HCU) with clinical suspicion of COVID‐19. The study was approved by the HCU INCLIVA Research Ethics Committee. NP specimens were collected by trained nurses and transferred to 1ml of transport/ extraction buffer, as stated above. Samples were immediately delivered to the Microbiology Service of HCU, where they were kept at 4°C until processed. Specimens were split into two aliquots, one of which was processed as per routine by using the Applied BiosystemsTM MagMAXTM Viral/Pathogen II Nucleic Acid Isolation Kits coupled with the Thermo ScientificTM KingFisher Flex automated instrument followed by RT‐PCR employing the TaqPath COVID‐19 Combo Kit (Thermo Fisher Scientific). According to the manufacturer, the LOD of the assay is 250 copies/ml (for the N gene target). The other aliquot was analyzed by the COVID‐19 PCR Fast‐L assay within 24 h upon receipt. The SARS‐CoV‐2 RNA (AMPLIRUN® TOTAL SARS‐CoV‐2 RNA Control; Vircell S.A) was used as standard material for estimation of viral loads (in copies/ml) in NP. Out of 662 NP samples, 68 (10.3%) and 582 (87.9%) returned positive and negative results, respectively, by both assays, and 12 (1.8%) yielded discordant results (TaqPath positive/PCR Fast‐L negative), thus resulting in an excellent concordance between the assays, with a Kappa index of 0.90 (95% CI: 0.85–0.96). Overall positive percent agreement (PPA) and negative percent agreement across the assays were 85% (95% CI: 75.5%–91.2%) and 100% (95% CI: 99.3%–100%), respectively. As shown in Figure 1, RT‐PCR Ct values were significantly lower (p < 0.001) in samples returning