Rapid Adaptation and Continuous Performance Evaluation of SARS-CoV-2 Envelope Gene (E-gene) Real-Time RT-PCR Assays to Support the Hospital Surge in Test Demand

We describe timely adaption of both published WHO E-gene protocol and commercially available LightMix Modular E-gene assay to the test platform (ABI 7900 Fast real-time analyzer and TaqMan Fast One-step Virus Master Mix) available in an accredited tertiary hospital laboratory with on-going evaluation to ensure provision of quality service within time constraint. The LightMix Modular E-gene was slightly more sensitive when compared to the WHO E-gene, both analytically and diagnostically. The assay was recommended for screening of SARS-CoV-2 infection. With the availability of technically competent staff through continuous training, provision of round the clock service is feasible despite the test is of high complexity. The shorten turn-around-time of about 4 hours per batch could support surges in testing demand, which is essential to control the current SARS-CoV-2 pandemic, to prevent potential overwhelming of the healthcare system and to optimize utilization of the isolation beds.


Background
The severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) was rst reported in Wuhan, China, in the end of December 2019 [1]. The genome published on January 11th 2020 (GenBank accession number MN908947) shows a high similarity to the SARS-CoV-1 which was epidemic in Hong Kong in 2003 [2]. Early February 2020, there were 42,708 con rmed infections and 1017 fatalities in China [3]. To control disease spread and prevent outbreak which could overwhelm the healthcare system, rapid implementation and provision of quality assured molecular diagnostic service is crucial.
Owing to its immediate availability, the LightMix Modular E-gene assay (Tib-Molbiol, Germany) designed for use with the LightCycler 480 system and the LightCycler multiplex RNA virus master (both are from Roche Diagnostics, Germany) was implemented in our laboratory for emergency use on 1st February 2020. An alternative E-gene protocol published by Corman et al [4] is recommended by World Health Organization (WHO) [5] and is widely used for screening of SARS-CoV-2 in Europe [6,7]. Here we reported the timely adaption of published protocol and commercial assay to our routine testing platform with on-going performance evaluation to ensure provision of quality service by a National Association of Testing Authorities (NATA, Australia) accredited laboratory.

Objective
The aim of this study was to adapt and compare the performance of the WHO E-gene and the LightMix Egene using ABI 7900 Fast real-time analyzer and TaqMan Fast Virus One-step Master Mix (both are from Applied Biosystems, USA) for screening of the SARS-CoV-2 in respiratory samples.

Nucelic acid extraction
Nucleic acids were extracted with the MagMax™ 96 viral RNA isolation kit using the MagMax high throughput nucleic acid processor (both are from Applied Biosystems, USA). Sample input volume was 200 µl and elution volume was 50 µl.

Adapted LightMix E-gene and WHO E-gene set up
Final reaction volume was 20 µl with 5 µl of the 4 x TaqMan Fast Virus One-step Master Mix and 10 µl of template. The LightMix E-gene assay contained 0.5 µl of the E-gene parameter speci c reagent. For the WHO E-gene assay, the primers (E_Sarbeco_F and E_Sarbeco_R) and BHQ1 probe (E_Sarbeco_P1) sequences (Tib-Molbiol, Germany) and their nal concentrations recommended by Corman et al were adopted. Thermal cycling conditions with the ABI 7900 Fast real-time analyzer were 55 °C for 5 min, 95 °C for 20 s, followed by 40 cycles of 95 °C for 3 s and 60 °C for 30 s with data acquisition for the LightMix Egene but 58 °C for the WHO E-gene. For both assays, RNA extracted from positive samples and nucleasefree water were used as positive and no template control respectively.

Analytical performance
The limits of detection (LoD) of both assays were assessed with the Exact Diagnostics SARS-CoV-2 standard (Exact Diagnostics, USA) which was diluted to 200, 100, 50 and 25 copies per mL. Each dilution was extracted with the MagMax system and tested 5 times with each assay. Four pooled samples (Pool 1 to Pool 4, Table 1) were prepared from the ZeptoMetrix respiratory veri cation panel 2 (ZeptoMetrix, USA) to assess their analytical speci cities. MERS-CoV RNA from the 2016 Quality Control and Molecular Diagnostics (QCMD) panel and SARS-CoV-1 RNA provided by Public Health Laboratory Services Branch (PHLSB) of Hong Kong were also tested. Precision was evaluated with the RNA control with cycle threshold (Ct ) ~ 28 which was tested in ve separate runs.

Accuracy
Both assays were assessed with the QCMD Coronavirus outbreak preparedness (CVOP) external quality assurance (EQA) pilot scheme issued in end of April 2020. The samples were extracted with the MagMax system followed by testing with both protocols.

Diagnostic performance
Ninety respiratory samples from non-duplicated patients (3 sputum; 62 pooled nasopharyngeal swab and throat swab; 23 pooled nasopharyngeal aspirate and throat swab; and 2 deep throat saliva), were tested by both protocols. All E-gene positive cases were further con rmed by PHLSB which is one of the WHO designated reference laboratories. Using the WHO E-gene as the reference method, diagnostic sensitivity and speci city of the LightMix E-gene were evaluated. Discrepancies were resolved with i) the Xpert Xpress SARS-CoV-2 assay (Cepheid, USA); ii) results of con rmation by PHLSB; iii) clinical information and availability of additional SARS-CoV-2 test results.

Results
When tested with the Exact Diagnostics standard, the LightMix E-gene and WHO E-gene could detect down to 4 and 8 copies/reaction (equivalent to 100 and 200 copies/mL) respectively. These were within the con dence interval reported by the manufacturer and by Corman et al. Both assays did not cross-react with MERS-CoV RNA and the various respiratory pathogens listed in Table 1 but could detect SARS-CoV-1 RNA.
Both assays were also precise with CV less than 3% when tested with the positive control in ve separate runs.
When assessed with the eight EQA samples, both assays gave correct results with comparable Ct values. The borderline positive sample was detected by both assays and reproducible results (ΔCt = 0.14) were also obtained for the duplicate samples in the scheme.
For the clinical samples, correlation of the results by both assays was shown in Table 2: There were 3 samples with discordant results (Table 2). After resolving discrepancies, they can be categorized as true positive or false positive (Table 3) with diagnostic sensitivity and speci city listed in Table 4:   Table 4). To avoid unnecessary quarantine, contact tracing and other public health issues, con rmation of any positive result by this assay is essential and we send these samples to PHLSB for con rmation.
Variation in instrument and reagents might affect performance of published protocols. Konrad et al reported that a relative high proportion of non-speci c signal is found in the Corman et al protocol when an alternative one-step RT-PCR system was used [9]. This was not observed in our adapted protocol. Recently Okamato et al [10] and Yip et al [11] also reported that the LightMix E-gene assay was sensitive when compared to their corresponding reference method. After the rapid adaptation and implementation of the LightMix E-gene within 48 hours in early February, we continuously evaluated the assays to ensure test quality. The turn-around-time (TAT) is approximately 4 hours and we now provide 5 batches of run daily with testing capacity of about 350. This shorten TAT greatly facilitates isolation bed utilization.

Conclusion
In conclusion, the LightMix Modular E-gene assay adapted for use with our testing platform is sensitive and suitable for initial screening. Continuous training to maintain a team of competent staff familiar with conventional molecular testing is crucial in particular when novel pathogen emerges and sample-to-answer automated system is not available.