key: cord-0829890-i8u0j87l authors: Yip, Cyril Chik-Yan; Sridhar, Siddharth; Cheng, Andrew Kim-Wai; Leung, Kit-Hang; Choi, Garnet Kwan-Yue; Chen, Jonathan Hon-Kwan; Poon, Rosana Wing-Shan; Chan, Kwok-Hung; Wu, Alan Ka-Lun; Chan, Helen Shuk-Ying; Chau, Sandy Ka-Yee; Chung, Tom Wai-Hin; To, Kelvin Kai-Wang; Tsang, Owen Tak-Yin; Hung, Ivan Fan-Ngai; Cheng, Vincent Chi-Chung; Yuen, Kwok- Yung; Chan, Jasper Fuk-Woo title: Evaluation of the commercially available LightMix® Modular E-gene kit using clinical and proficiency testing specimens for SARS-CoV-2 detection date: 2020-05-27 journal: J Clin Virol DOI: 10.1016/j.jcv.2020.104476 sha: 428cf7a2dceee064a713ae941765daf8c26fa430 doc_id: 829890 cord_uid: i8u0j87l BACKGROUND: Rapid and sensitive diagnostic assays for SARS-CoV-2 detection are required for prompt patient management and infection control. The analytical and clinical performances of LightMix® Modular SARS and Wuhan CoV E-gene kit, a widely used commercial assay for SARS-CoV-2 detection, have not been well studied. OBJECTIVE: To evaluate the performance characteristics of the LightMix® E-gene kit in comparison with well-validated in-house developed tests. STUDY DESIGN: Serial dilutions of SARS-CoV-2 culture isolate extracts were used for analytical sensitivity evaluation. A total of 289 clinical specimens from 186 patients with suspected COVID-19 and 8 proficiency testing (PT) samples were used to evaluate the diagnostic performance of the LightMix® E-gene kit against in-house developed COVID-19-RdRp/Hel and COVID-19-N RT-PCR assays. RESULTS: The LightMix® E-gene kit had a limit-of-detection of 1.8 × 10(-1) TCID(50)/mL, which was one log(10) lower than those of the two in-house RT-PCR assays. The LightMix® E-gene kit (149/289 [51.6%]) had higher diagnostic sensitivity than the in-house assays (144/289 [49.8%] for RdRp/Hel and 146/289 [50.5%] for N). All three assays gave correct results for all the PT samples. Cycle threshold (Cp) values of the LightMix® E-gene kit and in-house assays showed excellent correlation. Reproducibility of the Cp values was satisfactory with intra- and inter-assay coefficient of variation values <5%. Importantly, the LightMix® E-gene kit, when used as a stand-alone assay, was equally sensitive as testing algorithms using multiple SARS-CoV-2 assays. CONCLUSIONS: The LightMix® E-gene kit is a rapid and sensitive assay for SARS-CoV-2 diagnostics, with fewer verification requirements compared to laboratory-developed tests Keywords: LightMix E-gene; SARS-CoV-2; COVID-19; diagnostic; evaluation; PCR . In late December 2019, a novel coronavirus, now named severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), was identified in patients with pneumonia in Wuhan, China [1] . SARS-CoV-2 is efficiently transmitted from person to person and has rapidly disseminated globally [2, 3] . The World Health Organization declared Coronavirus Disease 2019 (COVID-19) as a pandemic in early March 2020. As of 23 May 2020, over 4.9 million COVID-19 cases including more than 327,000 deaths attributable to SARS-CoV-2 have been reported globally (https://www.who.int/emergencies/diseases/novel-coronavirus-2019). Rapid, sensitive and specific diagnostic tests for COVID-19 are of paramount importance to facilitate early identification of cases, contact tracing, and isolation [4, 5] . The gold standard for laboratory diagnosis of COVID-19 is by reverse-transcription polymerase chain reaction (RT-PCR) (https://www.who.int/emergencies/diseases/novelcoronavirus-2019/technical-guidance/laboratory-guidance). While a number of in-house and commercial RT-PCR assays for COVID- 19 have been developed in the past few months, the clinical performance of some of these assays has not been well studied. In the present study, we evaluated the performance of a commercially available LightMix ® Modular SARS and Wuhan CoV E-gene kit capable of detecting SARS-CoV-2 against well-validated in-house developed RT-PCR assays targeting RNA-dependent RNA polymerase (RdRp)/Helicase (Hel) and nucleocapsid (N) regions of SARS-CoV-2 [6] using archived clinical specimens and proficiency testing samples. J o u r n a l P r e -p r o o f SARS-CoV-2 was isolated from a nasopharyngeal aspirate specimen of a patient with COVID-19 in Hong Kong as previously described [7] . SARS-CoV-2 stock (1.8×10 7 TCID50/mL) was prepared by one additional passage in VeroE6 cells [8, 9] . For analytical sensitivity evaluation, 10-fold serial dilutions of total nucleic acid (TNA) extracted from the SARS-CoV-2 isolate were used. For analytical specificity evaluation, TNA extracted from a clinical specimen with human coronavirus HKU1 (HCoV-HKU1) and 17 culture isolates of other human coronaviruses and respiratory viruses were used [6, 10] concentrations of SARS-CoV-2 or negative for SARS-CoV-2 were also evaluated. TNA extraction was performed using NucliSENS easyMAG extraction system (BioMerieux, Marcy-l'Étoile, France) according to the manufacturer's instructions and as previously described [11] [12] [13] . Briefly, 250 µL of each respiratory tract specimen, rectal swab and stool specimen were subjected to extraction with an elution volume of 55 µL; and 100 µL of each plasma specimen were subjected to extraction with an elution volume of 25 µL. The extracts were stored at -80C until use. of nuclease-free water and 5 µL of TNA as the template. The thermal cycling condition was 45°C for 10 min, 95°C for 5 min, followed by 45 cycles of 95°C for 5 s and 55°C for 30 s. Fisher's exact test was used to compare the performance of the assays. Spearman's correlation was used to assess the relation between the Cp values of different assays. The Cp values obtained from the three assays were compared using ANOVA Friedman test with Dunn's multiple comparisons test (a Cp value of 41 was assigned to specimens that tested negative in the J o u r n a l P r e -p r o o f real-time RT-PCR assay). Statistical analysis was performed using GraphPad Prism 8. P<0.05 was considered statistically significant. To determine the analytical sensitivity of the LightMix ® E-gene assay, the limit of detection (LOD) was evaluated by using TNA extracted from the SARS-CoV-2 isolate. Serial 10fold dilutions of SARS-CoV-2 TNA extracted from the viral culture isolate were prepared and tested in triplicate for each concentration in two independent runs. The LOD of the E-gene assay was 1.8 x 10 -1 TCID50/mL (Table 1) . To investigate whether the LightMix ® E-gene assay would non-specifically amplify other human-pathogenic coronaviruses and respiratory viruses, we tested TNA extracted from the clinical respiratory specimen with HCoV-HKU1, and TNAs extracted from the 17 culture isolates of SARS-CoV, MERS-CoV, HCoV-OC43, HCoV-NL63, HCoV-229E, influenza A ((H1N1)pdm09 and H3N2) viruses, influenza B virus, influenza C virus, parainfluenza virus types 1 -4, respiratory syncytial virus, human metapneumovirus, human rhinovirus and human adenovirus. The LightMix ® E-gene assay did not cross react with these respiratory viruses, except SARS-CoV. Different concentrations of TNA extracted from the SARS-CoV-2 isolate were used to evaluate intra-and inter-assay variations by the LightMix ® E-gene assay. Each concentration was tested in triplicate in two independent runs. The total imprecision (% CV) values ranged from 0.72% to 1.54% (Table 2) . We found that adding in-house tests to the LightMix ® E-gene assay in any combination did not result in a significant increase in sensitivity (Table 3 ). There was no significant difference in the detection rate between the LightMix ® E-gene assay and our in-house assays. The sensitivity of these assays did not differ significantly for both respiratory and non-respiratory specimens (Table 4 ). For the specimens with discordant results, their mean cycle threshold (Cp) value was 36.7, J o u r n a l P r e -p r o o f which represented very low viral load in these specimens. Among the eight proficiency testing samples from QCMD, all three assays could give 100% correct results. A good agreement in performance of the LightMix ® E-gene assay compared to the in-house assays was revealed by a strong correlation (Spearman's ρ >0.97; P<0.0001) (Fig. 1) . The Cp values obtained from the 3 different assays were also examined. The median Cp value of the LightMix ® E-gene assay (29.3) was significantly lower than those of COVID-19-RdRp/Hel assay (30.9; P<0.0001) and COVID-19-N assay (31.3; P<0.0001) (Fig. 2) . An increasing number of in-house and commercial assays on COVID-19 diagnosis have been described in the past 5 months [14] [15] [16] [17] [18] [19] [20] [21] [22] [23] [24] [25] [26] . The commercially available LightMix ® Modular SARS and Wuhan CoV E-gene kit is widely used in clinical laboratories, but its performance has not been thoroughly evaluated with clinical specimens. In the present study, we compared the performance of the LightMix ® E-gene kit with two previously established and validated in-house COVID-19 RT-PCR assays [6] using a variety of clinical specimens and proficiency testing samples. According to the manufacturer instructions for the LightMix ® E-gene assay, this assay can detect not only SARS-CoV-2, but other sarbecoviruses including SARS-CoV and bat SARSrelated viruses. In our analytical specificity evaluation, the LightMix ® E-gene assay detected SARS-CoV but not other common human-pathogenic respiratory viruses and coronaviruses, while our in-house COVID-19-RdRp/Hel and COVID-19-N assays were specific for SARS-CoV-2 without cross-reactivity with SARS-CoV. The LightMix ® E-gene assay was likely intentionally designed to cross-react with SARS-CoV because of the scarce information on the pandemic [14, 15] . It would therefore be a reasonable strategy to use the sensitive LightMix ® Egene assay as the first line screening assay for suspected COVID-19 cases, followed by confirmation by sequencing or another RT-PCR assay specific to SARS-CoV-2 (https://www.who.int/publications-detail/laboratory-testing-for-2019-novel-coronavirus-insuspected-human-cases-20200117). The LightMix ® E-gene assay was highly sensitive for SARS-CoV-2 RNA detection, with LOD of 1.8×10 -1 TCID50/mL, which is one log10 TCID50/mL lower than our previously described COVID-19-RdRp/Hel and COVID-19-N assays (1.8 TCID50/mL) [6] . The median Cp value of the LightMix ® E-gene assay was also significantly lower than the in-house assays. This might be due to the higher volume of specimen template used in the LightMix ® E-gene assay than the inhouse assays. Another possibility is that the LightMix ® E-gene assay and our in-house assays were performed using different PCR reagents and thermocycling conditions. Nevertheless, no significant difference in the sensitivity was noted among these three assays for both respiratory and non-respiratory specimens. Reproducibility of the Cp values was satisfactory with the intraand inter-assay coefficient of variation values of <5% [27] [28] [29] . The Cp values of the LightMix ® E-gene/COVID-19-RdRp/Hel and E-gene/COVID-19-N assays showed excellent correlation. All three assays performed well in the proficiency testing samples from QCMD. These findings suggested that the LightMix ® E-gene assay and our in-house assays showed excellent diagnostic performance for SARS-CoV-2 detection. Healthcare facilities including our hospital use RT-PCR negativity as a criterion for hospital discharge. However, the false-negative rate of RT-PCR assays may rise during the convalescent phase of illness as the patient's viral load drops. Thus, it remains controversial as to how many RT-PCR assays targeting different gene regions should be used to test convalescent phase patients. Our results showed that the LightMix ® E-gene assay performed well as a standalone test with similar sensitivity as other testing algorithms using multiple tests for follow-up clinical specimens. This feature is reassuring and obviates the need for testing follow-up specimens especially in areas where diagnostic kits remain limited. In addition to the analytical and clinical performance, the turnaround time and cost are also essential factors affecting the choice of diagnostic assays, especially when there is a large number of clinical specimens from patients with suspected COVID-19 during this pandemic. The sample-to-extract time was the same among the three assays because the same extraction method was used, while the PCR running time of the LightMix ® E-gene assay (66 min) was slightly shorter than our in-house COVID-19-RdRp/Hel and COVID-19-N assays (72 min). For the reagent cost including the PCR reagents and primers/probes, our in-house assays (US$2 per reaction) were much lower than the LightMix ® E-gene assay (US$10 per reaction). For clinical laboratories without the necessary expertise in the development of in-house assays, the LightMix ® E-gene kit might be an alternative commercially available diagnostic option. In conclusion, the LightMix ® Modular SARS and Wuhan CoV E-gene kit is a rapid and highly sensitive assay for screening the suspected cases in clinical microbiology laboratories. Further confirmation can be achieved by performing another assay specific to SARS-CoV-2, such as our in-house COVID-19-RdRp/Hel and COVID-19-N assays with lower cost. This study was approved by Institutional Review Board of the University of Hong Kong/Hospital Authority Hong Kong West Cluster. Data records were de-identified and completely anonymous, so informed consent was waived. The datasets used and/or analyzed during the current study are available from the corresponding author on reasonable request. 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