key: cord-0853238-nzd5wqqi
authors: Etievant, S.; Bal, A.; Escurret, V.; Brengel-Pesce, K.; Bouscambert, M.; Cheynet, V.; Generenaz, L.; Oriol, G.; Destras, G.; Billaud, G.; Josset, L.; Frobert, E.; Morfin, F.; Gaymard, A.
title: Sensitivity assessment of SARS-CoV-2 PCR assays developed by WHO referral laboratories
date: 2020-05-04
journal: nan
DOI: 10.1101/2020.05.03.20072207
sha: e4f3edef4a101fe34500d935bb880aeecc5b88f4
doc_id: 853238
cord_uid: nzd5wqqi

The sensitivity of SARS-CoV-2 RT-PCR tests developed by Charite (Germany), HKU (Hong-Kong), China CDC (China), US CDC (United-States), and Institut Pasteur, Paris (France) was assessed on SARS-CoV-2 cell culture supernatants and clinical samples. Although all RT-PCR assays performed well for SARS-CoV-2 detection, RdRp Institut Pasteur (IP2, IP4), N China CDC, and N1 US CDC were found to be the most sensitive.

A new human coronavirus called severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) emerged in China, in December 2019 [1] . SARS-CoV-2 is responsible for the coronavirus disease 2019 (COVID-19) which was declared a pandemic on 12 March 2020 by the World Health Organization (WHO) [2] . As of 16 April, 1 991 562 cases have been reported including 130 885 deaths [3] . A sensitive diagnostic assay is crucial to limit SARS-CoV-2 spreading as it allows to early detect new cases which lead to patient isolation and contact tracing. The first SARS-CoV-2 genome was published on 10 January 2020 [4] enabling the rapid design of a real-time reverse-transcriptase polymerase chain reaction (RT-PCR) assay by Charité (Germany) [5, 6] . This test was the first to be dispatched by WHO [7] and was widely implemented in clinical virology laboratories worldwide [8] .

Since then, WHO has published [9] other approaches developed by referral laboratories including HKU (Hong-Kong) [10, 11] , China CDC (China) [12] , US CDC (United-States) [13] and Institut Pasteur, Paris (France) [14] . These assays target two or three different SARS-CoV-2 gene regions, including RdRp (RNA-dependent RNA polymerase), N (nucleocapsid protein), E (envelope protein), ORF1ab nsp10 (non-structural protein 10), and ORF1b nsp14 (non-structural protein 14) . In the present study, we aimed to compare the sensitivity of these different RT-PCR assays.

Different RNA concentrations obtained by a nine-fold serial dilution of SARS-CoV-2 cell culture supernatants as well as clinical samples were first tested using each RT-PCR assay. Limit of detection (LoD) for the three most sensitive assays was then assessed. Clinical samples with low viral concentration or tested negative were further tested using these three assays.

Thirty-two clinical samples (nasopharyngeal aspirates) were provided by the Hospices Civils de Lyon -University Hospital, France. Eight clinical samples were tested using all PCR assays, and twenty four samples were tested only with the most sensitive assays. Samples were frozen at -80°C before extraction. A positive sample from a patient was cultivated on buffalo green monkey cells in a biosafety level 3 laboratory to collect cell culture supernatants containing SARS-CoV-2. The SARS-CoV-2 culture had an infectious titer of 8.27 log 10 TCID 50 as assessed by the Reed and Muench statistical method [15] .

RNA extraction was performed using the EMAG ® platform (Biomerieux, Marcy-l'Étoile, France), according to manufacturer's instructions. RT-PCR were performed following published instructions [5, 6, [10] [11] [12] [13] [14] which are summarised in table 1 and 2. Since the China CDC protocol does not specify polymerase, thermocycler, volume of RNA extract, and amplification cycles, the same instructions as the HKU assay were applied. RdRp IP2 and IP4 assays from Institut Pasteur, Paris (France) can be multiplexed or used in simplex [14] . Preliminary comparison on SARS-CoV-2 cell culture . CC-BY-NC-ND 4.0 International license It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. (which was not certified by peer review)

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Sensitivity for each RT-PCR assay was first assessed using serial dilutions from 10 -3 to 10 -9 of SARS-CoV-2 cell culture supernatants (one replicate for 10 -3 and 10 -4 , three replicates for 10 -5 and 10 -6 , and five replicates for 10 -7 to 10 -9 ). The E Charité and N2 US CDC assays were positive for all specimens including negative samples and negative controls (water). These false amplifications were explored (details below) but sensitivity of these assays was not further assessed. . CC-BY-NC-ND 4.0 International license It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity.

The copyright holder for this preprint this version posted May 4, 2020. .

The limit of detection (LoD) is defined as the lowest amount of viral genome that can be detected with a 95% hit rate. Probit analysis was applied by including five additional replicates of each dilution of the cell culture supernatants for the three most sensitive referral laboratories. Due to limited quantity of clinical samples we tested only one target of each referral laboratories: N China CDC, N1 US CDC, and RdRp IP2. LoD of N3 US CDC was not determined as this assay is not specific for SARS-CoV-2 detection and was removed from the new version of the US CDC assay [13] . We chose to determine LoD for RdRp IP2 and not IP4 as IP2 detected more replicates at the 10 -9 dilution on cell culture supernatants.

The 95% hit rate obtained was 1.36 log 10 Figure 2 ) but no significant differences (Ct difference <2) were observed. All negative samples were tested negative using these three assays.

Since E Charité and N2 US CDC assays were positive for all specimens and replicates including negative samples and controls, we tested four additional negative clinical samples, water, and one additional clinical sample tested positive for each target. Amplicon size was analysed using Agilent DNA 1000 kit (Agilent technologies; supplementary figure 1).

For E Charité, negative samples showed two amplicons, one at 84 base pairs (bp) and one at 121 bp whereas the positive sample only had one amplicon at 121 bp, which is close to the expected size of a specific amplification (table 1) . Thus, the false positive amplification obtained using E Charité might derive from a contamination (amplicon size at 121bp) but could also be associated with an aspecific amplification (amplicon size at 84bp). Using the N2 US CDC assay, negative samples showed one amplicon at 73 bp which is close to the expected size of a specific amplification (table 1) . Thus, the false positive amplification obtained using N2 US CDC might be due to a contamination. Sequencing of these amplicon products should be performed for further investigation.

The present study is the first to compare the sensitivity of five RT-PCR-based methods developed by referral laboratories. N China CDC, N1 US CDC, and RdRp IP2 and IP4 were found to be the most sensitive assays on SARS-CoV-2 cell culture supernatants and clinical respiratory samples. Vogels et al. compared performances of SARS-CoV-2 PCR assays developed by the same referral laboratories except those from Institut Pasteur. Using RNA-spiked mock samples, they found that ORF HKU was . CC-BY-NC-ND 4.0 International license It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity.

The copyright holder for this preprint this version posted May 4, 2020. . one of the most sensitive assays [16] . Herein, ORF HKU was more sensitive than RdRp Charité but slightly less sensitive than other assays such as N1 US CDC or N HKU. Although RdRp Charité performed well for the lowest dilutions, it was nevertheless found to be less sensitive than others, a result in line with those of Vogels et al. [16] . It is worth noting that the Charité assay was the first to be published at the early stage of the pandemic [9] and has been widely used worldwide [8] . This assay was initially designed for the diagnosis of SARS-related CoVs and then optimised for SARS-CoV-2 detection [5] . Thanks to this assay, an important number of COVID-19 diagnoses were made, which contributed to limit the spread of the outbreak. In line with the present results, it was reported that RdRp IP2 and RdRp IP4 sensitivity was similar when used in multiplex [14] , suggesting that the Institut Pasteur assay should preferentially be used in multiplex. As previously reported [16], we identified probable primer contamination using N2 US CDC and E Charité which prevented us from further evaluation of the sensitivity. Although not observed herein, the amplification of non-specific products for ORF1 and N China CDC, and N2 and N3 US CDC has also been reported [17] .

The sensitivity of other RT-PCR tests recently developed [7] should be explored in further studies.

Furthermore, the specificity of each assay was not evaluated in the present study and should be determined. However, we chose to extensively assess sensitivity as a sensitive test is critical for early detection of new COVID-19 cases. The data presented herein are of prime importance to facilitate the equipment choice for all diagnostic laboratories, as well as for the development of marketed tests.

Sensitive tests should be widely implemented to limit the spread of the current outbreak and prepare for the post-epidemic phase and future seasonal epidemics. . CC-BY-NC-ND 4.0 International license It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. (which was not certified by peer review)

The copyright holder for this preprint this version posted May 4, 2020. . . CC-BY-NC-ND 4.0 International license It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. (which was not certified by peer review)

The copyright holder for this preprint this version posted May 4, 2020. 

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