Xpert xpress sars cov 2

NP swabs for confirmatory molecular testing were collected by a health care professional, placed in viral transport medium (VTM) (virus sampling kit; Yocon Biology Technology Company, Beijing, China), and sent to the reference laboratory. Molecular confirmation was carried out according to the manufacturer’s instructions using Xpert Xpress SARS-CoV-2 or Xpert Xpress SARS-CoV-2/Flu/RSV assays (Cepheid, Sunnyvale, CA), the Aptima SARS-CoV-2 assay on the Panther system (Hologic, Inc., San Diego, USA), or the cobas SARS-CoV-2 test on the 6800 instrument (Roche Diagnostics, Meinheim, Germany) (43 (link)). Where available, cycle threshold (C_T) values were recorded. For consistency, only C_T values derived from the Xpert SARS-CoV-2 FluA/B/RSV assay were used for the C_T stratification analyses, as this platform was used for the majority of samples. Positive and negative RT-PCR results were communicated to the patient, and any positive results were also reported to Public Health for further management and case contact tracing.

Diagnostic confirmation for COVID-19 was performed using RT-PCR on specimens obtained via nasopharyngeal swab, according to the protocol instituted at HIAE. The following RT-PCR kits were utilized: XGEN MASTER COVID-19 (Mobius, Pinhais, Paraná, Brazil), cobas SARS-CoV-2 Test (Roche Molecular Systems, Branchburg, NJ), Xpert Xpress SARS-CoV-2 (Cepheid, Sunnyvale, CA, USA), and Abbott RealTime SARS-CoV-2 (Abbott Molecular, Des Plaines, IL).

The sample used for the GENECUBE^® examination analysis of SARS-CoV-2 was also used for the RT-PCR assays. All assays were performed with the previously described magLEAD conditions [10 (link)] (Fig 1). In addition to the standard protocol with magLEAD purification, a rapid protocol created by Hiromichi Suzuki, TOYOBO Co., Ltd. and Precision System Science Co., Ltd. were evaluated for saliva samples and samples for limit of detection (LOD) analysis. For the rapid protocol, in the preparation of saliva samples, purification and extraction processes were adjusted, and the total process time was shortened to approximately 10 minutes. The comparison of the standard protocol and the rapid protocol for each magLEAD purification processes is summarized in Table 1. The rapid protocol is commercially available in Japan as MagDEA Dx SV 200 for GENECUBE^®.
If discordance was recognized between GENECUBE^® and the reference RT-PCR, an additional evaluation was performed with Xpert^® Xpress SARS-CoV-2 and GeneXpert^® (Cepheid Inc., Sunnyvale, CA, USA) [15 (link)] analyses for anterior nasal samples according to the manufacturer’s instructions documented in the package insert, and with an RT-PCR with LightMix^® Modular SARS-CoV (COVID19) E-gene (Roche Diagnostics KK) [16 (link)] for saliva samples along with re-evaluation with the NIID RT-PCR method.

After the arrival of the UTM samples, purification and RNA extraction were performed with magLEAD 6gC (Precision System Science Co., Ltd., Chiba, Japan) from 200 µL aliquots of UTM for in-house reverse transcription (RT)-PCR on the same day as sample collection. RNA was eluted in 100 µL and stored at − 80 °C after the in-house RT-PCR test. The eluted samples were transferred to Denka Co., Ltd., every week for a reference real-time RT-PCR test on Applied Biosystems QuantStudio 3 (Thermo Fisher Scientific Inc., Waltham, MA, USA) using a QuantiTect Probe RT-PCR Kit (QIAGEN Inc., Germantown, MD, USA) and primer/probe N and N2 set^{8 (link)}.
The presence or absence of SARS-CoV-2 was defined by the results of the reference real-time RT-PCR test. However, if discordance existed between the reference real-time RT-PCR test and the in-house RT-PCR test, a re-evaluation was performed with an Xpert Xpress SARS-CoV-2 and GeneXpert System (Cepheid, Sunnyvale, CA, USA), the results of which provided the final judgment.

PCR testing for SARS-CoV-2 at our institution mainly used pharyngeal rinse water, but we also accepted results from external facilities that had used throat swabs and sputum, if the test had been performed by a certified laboratory. All respiratory specimens were analyzed immediately after collection in the diagnostic virology unit of our hospital by means of a commercial IVD/CE-compliant RT-PCR assay (Xpert^® Xpress SARS-CoV-2, Cepheid GmbH, Krefeld, Germany).
All patients with a positive PCR test result displayed signs of respiratory infection, so the term “COVID-19” is used synonymously for all SARS-CoV-2-positive patients.
Hearts taken from deceased COVID-19 patients were analyzed for SARS-CoV-2 using real time PCR as previously described (22 (link)). Trials to replicate and thus enrich SARS-CoV-2 from patient tissue in permanent monolayer cell cultures followed by real time PCR could also not detect SARS-CoV-2 in heart tissue from our COVID-19 patient cohort. For PCR, nucleic acids were extracted from fresh-frozen organ slices using the EZ1 Virus Mini Kit v2.0 with the EZ1 Advanced XL system (Qiagen, Hilden, Germany). Viral ssRNA was amplified using the SARS-CoV-2 E gene RT-PCR with StepOnePlus Real-Time PCR System (Thermo Fisher Scientific, Schwerte, Germany). Bacteriophage MS2 served as an internal control for extraction and amplification efficacy.