key: cord-282421-yialyuav
authors: Alcoba-Florez, Julia; Gil-Campesino, Helena; de Artola, Diego García-Martínez; González-Montelongo, Rafaela; Valenzuela-Fernández, Agustín; Ciuffreda, Laura; Flores, Carlos
title: Sensitivity of different RT-qPCR solutions for SARS-CoV-2 detection
date: 2020-08-01
journal: Int J Infect Dis
DOI: 10.1016/j.ijid.2020.07.058
sha: 
doc_id: 282421
cord_uid: yialyuav

Abstract Objectives The ongoing COVID-19 pandemic continues imposing a demand for diagnostic screening. In anticipation that the recurrence of outbreaks and the measures for lifting the lockdown worldwide may cause supply chain issues over the coming months, we assessed the sensitivity of a number of one-step retrotranscription and quantitative PCR (RT-qPCR) solutions to detect SARS-CoV-2. Methods We evaluated six different RT-qPCR alternatives for SARS-CoV-2/COVID-19 diagnosis based on standard RNA extractions. That of best sensitivity was also assessed with direct nasopharyngeal swab viral transmission medium (VTM) heating, overcoming the RNA extraction step. Results We found a wide variability in the sensitivity of RT-qPCR solutions that associated with a range of false negatives from as low as 2% (0.3-7.9%) to as much as 39.8% (30.2-50.2). Direct preheating of VTM combined with the best solution provided a sensitivity of 72.5% (62.5-81.0), in the range of some of the solutions based on standard RNA extractions. Conclusions We evidenced sensitivity limitations of currently used RT-qPCR solutions. Our results will help to calibrate the impact of false negative diagnoses of COVID-19, and to detect and control new SARS-CoV-2 outbreaks and community transmissions.

. Given the high sensitivity compared to serological testing (Cassaniti et al. 2020) , standard diagnosis continues to rely on RNA extractions from respiratory or oral samples followed by one-step reverse transcription and real-time quantitative PCR (RT-qPCR) that entail one or several primer-probe sets for targeting SARS-CoV-2 sequences . While it has been shown that protocol modifications aiming to overcome supply chain issues and accelerate diagnosis affect assay sensitivity (Alcoba-Florez et al. 2020; Esbin et al. 2020) , differences in target priming efficiencies and RT-qPCR kit components are also expected to account for dissimilarities in false negative results (Nalla et al. 2020 ).

Here we aimed to evaluate the sensitivity of six different RT-qPCR solutions, including five marketed kits and one based on the World Health Organization diagnostic assays with the best sensitivity Vogels et al. 2020) , using RNA extractions from nasopharyngeal swab viral transmission medium (VTM). The alternative with the best sensitivity was also assessed by a direct preheating of VTM samples to skip the RNA extraction step that was described elsewhere (Alcoba-Florez et al. 2020).

The study was conducted at the University Hospital Nuestra Señora de Candelaria (Santa Cruz de Tenerife, Spain) from March to June 2020. We evaluated six different RT-qPCR solutions ( Table 1) , four based on three viral targets and two based on one viral target. 

Since all samples were COVID-19 positive for at least one solution/viral target, results with threshold cycle (Ct) values above 40 or those that remained undetected during the 45 cycles of the experiments were considered FN observations (Figure 1, Table 1 ).

Attending to individual targets, we found that the most sensitive solution was the Table 1 ).

RT-qPCR for selected target genes of SARS-CoV-2 has been key in the global response to the pandemic. Given the rapid spread of the virus at this time, it is likely that the RT-qPCR assays will continue to be a central tool for controlling COVID-19. However, as happened in the past due to supply chain issues, policy decisions and laboratory testing capacities (Alcoba-Florez et al. 2020) , it is predictable that the diagnosis of COVID-19 will continue relying on a variety of solutions among laboratories and countries (Vogels et al. 2020) .

Our results evidenced a wide variability in the sensitivity of RT-qPCR solutions for SARS-CoV-2 detection which associated with a proportion of FN ranging from as low as 2% (0.3-7.9%) to as much as 39.8% (30.2-50.2). Given that the same patient nasopharyngeal samples were assayed for the different solutions, well-known factors affecting SARS-CoV-2 sensitivity (stage of infection and type of specimen) (Pan et al. J o u r n a l P r e -p r o o f 2020; Wölfel et al. 2020) were suitably controlled in the study since all solutions were equally affected. Thus, we are confident that the differences in sensitivity among solutions were due to their different components (i.e. primers-sets, buffers, enzymes and reagent contents in general). These findings will help to assess the impact of the selected solution on FN diagnoses of COVID-19 (Ramdas et al. 2020) and to choose a solution that minimize misdiagnoses of an active SARS-CoV-2 infection.

JAF and CF designed the study. JAF, HGC, and DGM participated in data acquisition.

JAF, LC and CF performed the analyses and data interpretation. LC, AVF, RGM and CF wrote the draft of the manuscript. All authors contributed in the critical revision and final approval of the manuscript. Corman et al. (2020) . 

Fast SARS-CoV-2 detection by RT-qPCR in preheated nasopharyngeal swab samples

Members of the San Matteo Pavia COVID-19 Task Force. Performance of VivaDiag COVID-19 IgM/IgG Rapid Test is inadequate for diagnosis of COVID-19 in acute patients referring to emergency room department

Detection of 2019 novel coronavirus (2019-nCoV) by real-time RT-PCR

Overcoming the bottleneck to widespread testing: A rapid review of nucleic acid testing approaches for COVID-19 detection

Comparative Performance of SARS-CoV-2 Detection Assays Using Seven Different Primer-Probe Sets and One Assay Kit

Viral load of SARS-CoV-2 in clinical samples

Report from the American Society for Microbiology COVID-19 International Summit

Test, re-test, re-test': using inaccurate tests to greatly increase the accuracy of COVID-19 testing

Analytical sensitivity and efficiency comparisons of SARS-COV-2 qRT-PCR primer-probe sets

Virological assessment of hospitalized patients with COVID-2019

We deeply acknowledge the University Hospital Nuestra Señora de Candelaria board of directors and the executive team for their strong support and assistance in accessing diverse resources used in the study.

The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

This research was funded by Cabildo Insular de Tenerife [grant number