Aptima sars cov 2 assay

Samples testing positive for SARS-CoV-2 at sites across the province were referred to the ProvLab for VOC testing. Assays used to test for the presence of SARS-CoV-2 included the following rRT-PCR assays: E gene laboratory-developed test (19 (link)), cobas SARS-CoV-2 (Roche Molecular Systems), Xpert Xpress SARS-CoV-2 (Cepheid), Simplexa COVID-19 Direct (Diasorin Molecular), Allplex 2019-nCoV Assay (Seegene), BD SARS-CoV-2 Reagents for the BD Max System (Becton, Dickinson and Company), Aptima SARS-CoV-2 Assay (Hologic), and an E/N gene laboratory-developed test (unpublished data). Point-of-care testing was also performed using the ID NOW COVID-19 test (Abbott Laboratories) and Panbio rapid antigen test (Abbott Laboratories).

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.

The Aptima SARS-CoV-2 assay on the Panther instrument (Hologic, Inc., San Diego, CA, USA) targets two parts of the ORF1 ab region of the SARS-CoV-2 genome and one internal control. This test is based on End-Point Transcription-Mediated Amplification (EP-TMA), which is a binary test for the presence or absence of SARS-CoV-2 [11 (link),12 (link)]. After amplification, chemiluminescent probes hybridize to amplicons and emit light measured by a luminometer in relative light units (RLUs). For valid samples, the RLU value will range between approximately 250 (internal control only) and 1250 RLUs with a positive internal control, and two positive targets (of 500 RLUs each). Assay results are determined by a cutoff based on the total RLU and the kinetic curve type.

Our laboratory began testing on 13 March 2020 with initial capacity for around 150 PCR tests per day, before increasing to around 500 tests per day in late April during wave 1 and up to 1000 tests per day during wave 2 (online supplemental figure 1).
Testing commenced during the first wave on 13 March 2020 was limited to cases requiring admission or inpatients who had symptoms of fever or cough, as per national recommendation; guidance suggested cases which did not require admission should not be tested. For wave 2, all cases admitted to the hospital were screened and underwent universal interval screening at varying time points. Staff testing for symptomatic healthcare workers (HCWs) was also introduced towards the end of wave 1. Comparative analysis was therefore restricted to SARS-CoV-2 RNA-positive cases requiring admission. Cases without laboratory confirmation of SARS-CoV-2 infection were not included.
Assays used for the detection of SARS-CoV-2 RNA include PCR testing using Aus Diagnostics or by the Hologic Aptima SARS-CoV-2 Assay. Nucleic acid was first extracted using the QIAGEN QIAsymphony SP system and a QIAsymphony DSP Virus/Pathogen Mini Kit (catalogue number 937036) with the off-board lysis protocol.

Testing of HCW for SARS-CoV-2 was performed using routine diagnostic procedures in the virology laboratory of the University Hospital of Lyon (Hospices Civils de Lyon, HCL) and included: transcription mediated amplification (TMA) (Aptima SARS-CoV-2 Assay, Hologic, Marlborough, US), loop-mediated isothermal amplification (LAMP) (SARS-CoV-2 ID NOW, Abbott, Sligo, Ireland) and RT-qPCR with different kits (Cobas 6800 SARS-CoV-2 assay, Roche, Basel, Switzerland or Panther Fusion SARS-CoV-2 assay, Hologic). To determine the prevalence of SARS-CoV-2 variants of concern (VOC) in HCW, available positive samples with quantification cycle (Cq) < 28 were sequenced using COVIDSeq (Illumina) as previously described [12 (link)]. Libraries were sequenced to 1 M paired-end reads (2 × 100 bp) and data were analysed using the in-house seqmet bioinformatic pipeline (available at https://github.com/genepii/seqmet). Clades and lineages were determined on samples with genome coverage > 90% using Nextclade and PangoLEARN, respectively.

Clinical samples collected through nasopharyngeal swabs were shipped to the referral laboratory (Microbiology Services, Metropolitan Clinical Laboratory, Hospital Universitari Germans Trias i Pujol), where they were stored at 4°C before chemical inactivation using lysis buffer. SARS-CoV-2 infection was diagnosed by using either the Novaplex SARS-CoV-2/FluA/FluB/RSV RT-PCR Assay (Seegene Inc., https://www.seegene.com) or Aptima SARS-CoV-2 assay (Hologic, https://www.hologic.com) according to the manufacturers’ instructions. Panbio Ag-RDT kits (Abbott, https://www.abbott.com) were used in situ at primary healthcare centers according to the manufacturer’s instructions.

VALCOR samples were tested with the initial panel at UZ Leuven using the TaqPath COVID-19 assay as a comparator (11 (link), 12 (link)). The initial panel testing was performed, between 1 April 2020 and 15 January 2021. Nucleic acids were extracted using the MagMax Viral Pathogen II extraction kit (ThermoFisher Scientific) on the KingFisher High Throughput platform. The target genes of Taqpath COVID-19 are N gene, ORF1ab gene, and S gene. The results were reported as log copies/mL based on the quantification cycle (Cq, also called Ct) value of the N gene. Detection of two or more target genes was considered a positive result. Samples were reported negative when all viral gene targets were Cq > 37 and the MS2 internal control was Cq < 27.5. VL was estimated using a standard curve and semi-quantitatively reported (14 (link)). In case of discordance between the initial panel testing and the TaqPath COVID-19 assay, a third assay, either Abbott Alinity m system (IL, USA), Aptima SARS-CoV-2 assay (Hologic Panther system, MA, USA) (12 (link)), or Xpert Xpress SARS-CoV-2 assay (Cepheid GeneXpert system, CA, USA), was deployed as an adjudicator.