key: cord-0948487-shdg4l3r authors: Wolters, Femke; de Bovenkamp, Jeroen van; den Bosch, Bart van; den Brink, Sharon van; Broeders, Maaike; Chung, Ngoc Hoa; Favié, Barbara; Goderski, Gabriel; Kuijpers, Judith; Overdevest, Ilse; Rahamat-Langedoen, Janette; Wijsman, Lisa; Melchers, Willem JG; Meijer, Adam title: Multi-center evaluation of cepheid xpert® xpress SARS-CoV-2 point-of-care test during the SARS-CoV-2 pandemic date: 2020-05-11 journal: J Clin Virol DOI: 10.1016/j.jcv.2020.104426 sha: b774f598b90bc04d00ce3c0fcb997b0d3d33b826 doc_id: 948487 cord_uid: shdg4l3r BACKGROUND: With the outbreak of SARS-CoV-2, rapid diagnostics are paramount to contain the current pandemic. The routinely used realtime RT-PCR is sensitive, specific and able to process large batches of samples. However, turnaround time is long and in cases where fast obtained results are critical, molecular point of care tests (POCT) can be an alternative. Here we report on a multicenter evaluation of the Cepheid Xpert Xpress SARS-CoV-2 point-of-care test. STUDY DESIGN: The Xpert Xpress SARS-CoV-2 assay was evaluated against the routine in-house real-time RT-PCR assays in three medical microbiology laboratories in The Netherlands. A sensitivity and specificity panel was tested consisting of a dilution series of SARS-CoV-2 and ten samples containing SARS-CoV-2 and a range of other seasonal respiratory viruses. Additionally, 58 samples of patients positive for SARS-CoV-2 with different viral loads and 30 tested negative samples in all three Dutch laboratories using an in-house RT-PCR, were evaluated using Cepheids Xpert Xpress SARS-CoV-2 cartridges. RESULTS: Xpert Xpress SARS-CoV-2 point of care test showed equal performance compared to routine in-house testing with a limit of detection (LOD) of 8.26 copies/mL. Other seasonal respiratory viruses were not detected. In clinical samples Xpert Xpress SARS-CoV-2 reaches an agreement of 100 % compared to all in-house RT-PCRs CONCLUSION: Cepheids GeneXpert Xpert Xpress SARS-CoV-2 is a valuable addition for laboratories in situations where rapid and accurate diagnostics are of the essence. 1 In December 2019 the emergence of a novel coronavirus was reported in Wuhan, China (1) . Since its emergence, the disease COVID-19 caused by coronavirus SARS-CoV-2 has spread rapidly with at the end of April worldwide more than 3 million cases and deaths reaching 200.000(2). Clinical symptoms range from mild upper respiratory tract symptoms to severe bilateral pneumonia, with large numbers of patients being admitted to hospital, putting tremendous pressure on health care systems (3) (4) (5) . The diagnosis of COVID-19 is based on a combination of clinical symptoms with or without radiological imaging, confirmed by SARS-CoV-2 PCR (6) . Currently, either an RT-PCR targeting the envelope (Egene) in combination with polymerase (RdRp-gene) is being used as described by Corman et al. (7) or targeting the nucleoprotein (N-gene) as described by the US Centers for Disease Control and Prevention (8) . However Preparations for the specificity and sensitivity quality assessment panels for SARS-CoV-2 were done at the RIVM. A sensitivity panel was created by 10-fold diluting the inactivated SARS-CoV-2 stock in MEM with Hanks' salts from 10-4 -10-10. For each dilution HEp2 cells were added at a concentration of 10,000 cells per ml. Details of the panel are listed in Table 2 . Concentrations of copies per sample were determined using dPCR performed on positive-strain genomic RNA. The RdRp-PCR also detects negative-strain genomic RNA and the E-gene PCR additionally detects sub-genome messengers. Upper respiratory tract samples of patients tested for SARS-CoV-2 between January 2020 and March 2020 were included in the evaluation. Samples were primarily taken by nasopharyngeal or mid- (table 3) . Both the sensitivity and specificity panels were distributed blinded. They were shipped frozen on dry ice to ensure the same number of freeze thaw cycles for all three laboratories. At all three laboratories the panel specimens were processed as clinical specimens in the routine diagnostic procedure using the locally implemented RT-PCR for E-gene and/or RdRp-gene and/or N-gene (Table 4) None Specimens selected by lab 1 had lower viral load than those selected by the other two laboratories, likely because of the slight difference in sensitivity of the in-house assays ( Figure 2&4 ). When retesting these samples in the in-house RT-PCR they were negative. In this study we show that the Xpert Xpress SARS-CoV-2 is a random-access system suitable for Point-of-Care testing that is highly specific to and sensitive for the detection of SARS-CoV-2, compared to in-house testing, and furthermore has a run-time of 45-50 minutes with hands on time limited to 2-3 minutes. Current routine molecular diagnostics for SARS-CoV-2 are able to perform high throughput processing, however turnaround time is relatively high which hinders patient management and infection control policies. In clinical samples Xpert Xpress SARS-CoV-2 reaches an agreement of 100% compared to all inhouse RT-PCRs and the assay outperforms routinely used diagnostic platforms in the sensitivity panel. The two samples with E-gene and N2-gene only results in Xpert Xpress SARS-CoV-2 were retested in in-house RT-PCR, thus freeze-thawing, these turned out to be negative. This indicates that viral loads of these sample are at the limit of detection which for the Xpert Xpress SARS-CoV-2 was found to be around 8.26 cp/mL and that multiple freeze-thaw steps of samples understandably has a significant impact on detection. In our evaluation, the LOD was lower (8.26 cp/ml) compared to the LOD provided by the manufacturer (250cp/mL). Additionally, a recent evaluation of multiple molecular point of care tests showed an LOD of 100cp/mL (11) (12) (13) . This discrepancy is most likely due to the different methods used for determining input concentrations. Digital PCR used to determine the number of copies per mL was performed on positive-strain genomic RNA. The RdRp PCR also detects negative strain genomic RNA and the Egene PCR additionally detects subgenome messenger RNA which is why the true number of target templates for the diagnostic PCR in the sensitivity panel is probably higher. This could explain the differences in LOD. Xpert Xpress SARS-CoV-2 is for use on nasopharynx swabs/nasal wash samples in Cepheid viral transport medium. The current evaluation on clinical samples was done using both UTM and GLY medium, as choice of transport medium is limited during the current pandemic, which shows reliable results. Equally so in the sensitivity and specificity panel using MEM with Hanks' salts. Due to shortages in reagents and plastics many laboratories in The Netherlands have switched to use the E-gene RT-PCR only (14) . Accordingly, we suggest that in countries where SARS-CoV-2 is China Novel Coronavirus I, Research T. 2020. A Novel Coronavirus from Patients with Pneumonia in China Singapore Novel Coronavirus Outbreak Research T. 2020. 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