key: cord-0835583-p9x52qqz
authors: Chen, Jonathan Hon-Kwan; Yip, Cyril Chik-Yan; Chan, Jasper Fuk-Woo; Poon, Rosana Wing-Shan; To, Kelvin Kai-Wang; Chan, Kwok-Hung; Cheng, Vincent Chi-Chung; Yuen, Kwok-Yung
title: Clinical Performance of the Luminex NxTAG CoV Extended Panel for SARS-CoV-2 Detection in Nasopharyngeal Specimens from COVID-19 Patients in Hong Kong
date: 2020-07-23
journal: J Clin Microbiol
DOI: 10.1128/jcm.00936-20
sha: 07c7ae5f3f97406be23808058b88449244a36f37
doc_id: 835583
cord_uid: p9x52qqz

In December 2019, the coronavirus disease 2019 (COVID-19) pandemic caused by the severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) was first reported in the Hubei province of China and later spread all over the world. There was an urgent need of a high-throughput molecular test for screening the COVID-19 patients in the community. The Luminex NxTAG CoV extended panel is a high-throughput FDA emergency use-authorized molecular diagnostic assay for SARS-CoV-2 detection. This system targets three genes (ORF1ab, N, and E genes) of SARS-CoV-2, the ORF1ab region of SARS-CoV, and the ORF5 region of MERS-CoV. In this study, we evaluated the diagnostic performance of this system with nasopharyngeal swab specimens of 214 suspected COVID-19 patients in Hong Kong. The results were compared with our routine COVID-19 reverse transcription-PCR (RT-PCR) protocol with a LightMix SarbecoV E-gene kit and an in-house RdRp/Hel RT-PCR assay. The NxTAG CoV extended panel demonstrated 97.8% sensitivity and 100% specificity to SARS-CoV-2 in nasopharyngeal specimens. On low-viral load specimens, the sensitivity of the NxTAG panel could still maintain at 85.71%. Strong agreement was observed between the NxTAG panel and the routine COVID-19 RT-PCR protocol (kappa value = 0.98). Overall, the E gene target of the NxTAG panel demonstrated the highest sensitivity among the three SARS-CoV-2 targets, while the N gene targets demonstrated the least. In conclusion, the NxTAG CoV extended panel is simple to use, and it has high diagnostic sensitivity and specificity to SARS-CoV-2 in nasopharyngeal specimens. We recommend this diagnostic system for high-throughput COVID-19 screening in the community.

I n December 2019, the coronavirus disease 2019 (COVID-19) pandemic caused by the severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) was first reported in the Hubei province of China and then spread throughout the country and later over the world (1) (2) (3) (4) (5) . By 3 May 2020, over 3.35 million confirmed cases were reported to the World Health Organization (WHO) from 215 countries or territories, with nearly 238,000 confirmed deaths (https://www.who.int/emergencies/diseases/novel-coronavirus-2019). In responding to this public health emergency, the U.S. Food and Drug Administration (FDA) authorized over 30 in vitro diagnostic commercial assays for SARS-CoV-2 detection under an emergency use authorization (EUA) (https://www.fda.gov/emergency -preparedness-and-response/mcm-legal-regulatory-and-policy-framework/emergency -use-authorization#covidinvitrodev).

The NxTAG CoV extended panel (Luminex Molecular Diagnostics, Toronto, Canada)

is a recently EUA-approved qualitative, multiplex, high-throughput molecular diagnostic test for the detection of SARS-CoV-2 in nasopharyngeal samples. The assay simultaneously targets the SARS-CoV-2 open reading frame 1ab (ORF1ab) region, the nucleoprotein (N) gene, and the envelope protein (E) gene (6) . If any of the three targets show positive, that sample will be considered SARS-CoV-2 detected. The assay also targets the SARS-CoV ORF1ab region and the Middle East respiratory syndrome coronavirus (MERS-CoV) ORF5 region for SARS-CoV and MERS-CoV detection, respectively (7, 8) . The assay is designed for differentiating all of the three most life-threatening coronaviruses, SARS-CoV, MERS-CoV, and SARS-CoV-2, in a single-tube reaction.

In this study, we evaluated the clinical performance of the new NxTAG CoV extended panel with nasopharyngeal specimens of COVID-19 patients in Hong Kong. The diagnostic performance of the NxTAG CoV extended panel was compared with our routine COVID-19 testing protocol using the LightMix SarbecoV E-gene assay (TIB-Molbiol, Berlin, Germany) as the screening assay and our in-house RT-PCR confirmatory assay targeting the RNA-dependent RNA polymerase/helicase (RdRp/Hel) gene of SARS-CoV-2 (9).

Sample collection and ethical approval. A total of 214 archived nucleic acid extracts isolated from 214 nasopharyngeal swab (NPS) specimens of 91 confirmed COVID-19-positive and 123 COVID-19negative patients staying in Queen Mary Hospital were included. Some of these specimens had been evaluated previously using the LightMix SarbecoV E-gene kit and the validated in-house COVID-19-RdRp/ Hel RT-PCR assay (10) . The 214 patients selected in this study had a median age of 51 years (interquartile range [IQR], 31 to 69), were admitted to Queen Mary Hospital through the Accident and Emergency Unit between 5 March and 26 April 2020, and developed COVID-19 clinical symptoms or had COVID-19 patient contact history or travel history.

Another four nasopharyngeal samples with different human coronaviruses (HCoV-HKU1, HCoV-NL63, HCoV-OC43, andHCoV-229E), one cell lysate of SARS-CoV, and two MERS-CoV external quality control samples from Quality Control for Molecular Diagnostics (QCMD) were also included in this evaluation for specificity analysis of the NxTAG CoV extended panel. Eight external quality assurance program (EQAP) specimens for COVID-19 of the QCMD 2020 Coronavirus Outbreak Preparedness EQA pilot panel were also included. This study was approved by the Institutional Review Board of The University of Hong Kong/Hospital Authority Hong Kong West Hospital Cluster (UW20-224).

Nucleic acid extraction. Total nucleic acid (TNA) extraction of the 214 clinical specimens was performed using the eMAG extraction system (bioMérieux, Marcy l'Étoile, France) according to the manufacturer's instructions and as previously described (11) . In brief, 250 l of NPS specimen was used for extraction, and the elution volume was set to 55 l. The MS-2 internal control provided by the NxTAG CoV extended panel was spiked into each sample for monitoring the extraction and amplification steps. The TNA extracts were either used for routine COVID-19 RT-PCR protocol immediately and were then stored at -80°C for this evaluation. The same extracted product of each specimen was used for all the RT-PCRs.

Routine COVID-19 RT-PCR screening and confirmation protocol. For routine diagnosis of COVID-19 in Queen Mary Hospital from January 2020 until the time of writing, the LightMix SarbecoV E-gene assay (TIB Molbiol, Berlin, Germany) and the LightCycler multiplex RNA virus master kit (Roche Diagnostics, Mannheim, Germany) were used as the screening assays (10, 12) . This RT-PCR aimed to target the E gene of SARS-CoV-2, but the assay cross-reacted with the E gene of SARS-CoV or bat SARS-like coronaviruses (Sarbecovirus) as mentioned in the manufacturer's instructions. Briefly, each 20-l RT-PCR mixture contained 4 l of 5ϫ reverse transcriptase quantitative PCR (RT-qPCR) mix, 0.1 l of 200ϫ RT enzyme solution, 0.5 l of LightMix primer and probe reagent mix, 5.4 l of nuclease-free H 2 O, and 10 l of template. The thermal cycling condition was 5 min at 55°C for reverse transcription, 5 min at 95°C for denaturation, and 45 cycles of 5 s at 95°C, 15 s at 60°C, and 15s at 72°C. According to the manufacturer's instructions, samples with a cycle threshold (C T ) value of Ͻ39.0 will be regarded as SARS-CoV-2 or SARS-CoV or bat SARS-like coronaviruses (Sarbecovirus) detected.

Specimens showing positive in the LightMix SarbecoV E-gene RT-PCR assay will be further confirmed by another laboratory-developed SARS-CoV-2 RNA-dependent RNA polymerase/helicase (RdRp/Hel) real-time RT-PCR assay in the LightCycler 480 real-time PCR system (Roche, Basel, Switzerland) as previously described (9) . The assay has been demonstrated to have an analytical sensitivity of 1.8 50% tissue culture infective dose (TCID 50 /ml) and has high specificity to the SARS-CoV-2 RdRp/Hel gene region.

Luminex NxTAG CoV extended panel. A total of 35 l of TNA of each sample stored at -80°C was added to the preplated NxTAG lyophilized bead reagents to resuspend the reaction reagents. RT-PCR was then performed according to the NxTAG CoV extended panel product insert instructions. The RT-PCR amplification was performed on an Eppendorf thermocycler (Eppendorf AG, Hamburg, Germany) with the following cycling parameters: one reverse transcription step at 42°C for 20 min; one template denaturation step at 95°C for 2 min and 30 s; 15 first amplification cycles at 95°C for 20 s, 65°C for 60 s, and 72°C for 10 s; 24 nested amplification cycles at 95°C for 20 s, 58°C for 60 s, and 72°C for 10 s; and a hybridization step at 37°C for 45 min. The reaction plate was then transferred to the 37°C preheated MagPix heater plate of the MagPix instrument (Luminex Corporation, Austin, TX), and the signal acquisition was performed using the xPONENT and SYNCT software (Luminex Molecular Diagnostics, Toronto, Canada). Each running batch could handle up to 94 clinical specimens plus the positive and negative experiment controls. The total turnaround time was around 4 h.

Statistical analysis. McNemar's test was used to compare the performance of the assays. A P value of Ͻ0.05 was considered statistically significant. Computation was performed using MedCalc software version 14.12.0 (MedCalc Software, Ostend, Belgium). The area under the curve (AUC) calculated from the receiver operating characteristic (ROC) curve was compared between the two methods for performance evaluation.

Analytical For the other 123 COVID-19-negative samples, all of them (123/123; 100%) were reported negative by the NxTAG CoV extended panel assay, demonstrating a diagnostic specificity of 100% (95% CI, 97.1% to 100%). The positive predictive value was 100% (95% CI, 95.94% to 100%), and the negative predictive value was 98.40% (95% CI, 94.34% to 99.81%). The AUC of the NxTAG panel was 0.99 (95% CI, 0.96 to 1.00) ( Table 1) .

We further divided the 91 positive samples into two groups with a cutoff at LightMix C T 35.00, which was roughly equal to 200 copies/ml according to the QCMD EQAP expected results. There were 77 samples found to have a LightMix C T value of Ͻ35.00, while another 14 samples had a LightMix C T value of Ն35. We observed that the diagnostic sensitivity for the sample group with C T of Ͻ35.00 reached 100%, while the sensitivity of the sample group with C T of Ն35.00 remained at 85.71% (Table 2) .

Although the NxTAG panel simultaneously detected the ORF1ab, N, and E genes of SARS-CoV-2, we observed different diagnostic sensitivities for different gene targets. 

From January to April 2020, the number of requests for the routine COVID-19 RT-PCR protocol per day in Queen Mary Hospital, Hong Kong, ranged from 65 to 140. A high-throughput diagnostic system was necessary to accommodate the daily service need.

In this study, we evaluated the clinical performance of the NxTAG CoV extended panel, which is a new high-throughput FDA EUA diagnostic assay detecting not only SARS-CoV-2, but also MERS-CoV and SARS-CoV. Our results demonstrated that the assay differentiated SARS-CoV-2 well from the other two lethal coronaviruses simultaneously in the same reaction. However, due to the limited availability of SARS-CoV and MERS-CoV clinical specimens in Hong Kong, full evaluation of SARS-CoV and MERS-CoV detection of the NxTAG assay will need further analysis. The NxTAG assay included a single-tube nested RT-PCR amplification and provided ultrasensitive target detection and sealed tube amplification and detection minimized the risk of cross-contamination. From our study, we found that the three targets of the NxTAG panel were highly specific through testing. This NxTAG panel differentiated SARS-CoV-2 from the closely related SARS-CoV, MERS-CoV, and other human coronaviruses. The QCMD EQAP results also demonstrated that the NxTAG panel detected SARS-CoV-2-positive specimens with viral load reaching 2.30 log 10 copies/ml (200 copies/ml).

For clinical specimens, we tested 214 NPS specimens with the NxTAG CoV extended panel and compared their results with our routine COVID-19 RT-PCR protocol, which was a combination use of the LightMix SarbecoV E-gene assay and the in-house RdRp/Hel RT-PCR assay. The NxTAG panel demonstrated good diagnostic performance with 97.80% sensitivity and 100% specificity for SARS-CoV-2 detection. Out of the 214 clinical samples, 212 of them showed concordant results between the two methods. Our study demonstrated that the LightMix C T value of 35.00 was roughly equal to 200 copies/ml viral load by using the QCMD EQAP samples. Therefore, we showed that all three gene targets (ORF1ab, N, and E) used in the NxTAG panel performed well when the viral loads of samples were higher than 200 copies/ml. When handling samples with viral loads lower than 200 copies/ml, we observed that the sensitivity of the gene targets in the NxTAG panel dropped significantly. The E gene target of the NxTAG panel was found to be the most sensitive gene target in the assay, while the N gene target was demonstrated to be the least sensitive target among the three targets in the assay for low-viral load COVID-19 samples. As the E gene of SARS-CoV-2 has been reported to have higher gene expression efficiency (13) , this can explain the better performance of the E gene among the 3 gene targets in the NxTAG assay. The low sensitivity of the N gene target may be due to the incomplete optimization of the target amplification in the assay.

In addition to diagnostic performance, sample throughput and ease of use are also important factors to consider during this pandemic. Our routine COVID-19 RT-PCR protocol included a nucleic acid extraction step, a screening RT-PCR step with the commercial LightMix E-gene screening RT-PCR, and a confirmation RT-PCR with our in-house RdRp/Hel RT-PCR. The average processing time for a COVID-19-positive specimen would be around 4.5 to 5 h. Since the whole process required two times of master-mix preparation and template inoculation steps, it was not user-friendly to laboratory workers, especially when they were handling over 90 samples at a time. Multiplexing the screening E gene and confirmatory RdRp/Hel gene RT-PCR would be useful to shorten the processing time. However, it would require extra optimizations and evaluations. The Luminex MagPix system has been widely installed around the world, and the NxTAG assay is designed for high-throughput pathogen detection. The processing time for a complete NxTAG assay run is 4 h, and the manual handling procedures are simple (14) . As the throughput of the system can be up to 96 samples per batch, it is suitable for routine screening of COVID-19-suspected cases in regional hospitals during the COVID-19 pandemic. However, the NxTAG assay used quite large amounts of TNA (35 l) for each reaction, and repeat testing or further confirmation work may require extra nucleic acid extraction.

In conclusion, the NxTAG CoV extended panel demonstrated diagnostic performance comparable to the other WHO-recommended commercially available COVID-19 RT-PCR assays. The NxTAG system is simple to handle and supports high-throughput sample screening. This system will be a useful tool for large-scale screening of COVID-19 cases.

China Novel Coronavirus Investigating and Research Team. 2020. A novel coronavirus from patients with pneumonia in China

Washington State 2019-nCoV Case Investigation Team. 2020. First case of 2019 novel coronavirus in the United States

Epidemiologic characteristics of early cases with 2019 novel coronavirus (2019-nCoV) disease in Korea

A familial cluster of pneumonia associated with the 2019 novel coronavirus indicating person-to-person transmission: a study of a family cluster

Genomic characterization of a novel SARS-CoV-2

SARS coronavirus replicase proteins in pathogenesis

MERS-CoV: understanding the latest human coronavirus threat

Improved molecular diagnosis of COVID-19 by the novel, highly sensitive and specific COVID-19-RdRp/Hel real-time reverse transcription-PCR assay validated in vitro and with clinical specimens

Evaluation of the commercially available LightMix modular E-gene kit using clinical and proficiency testing specimens for SARS-CoV-2 detection

Differential cell line susceptibility to the emerging novel human betacoronavirus 2c EMC/ 2012: implications for disease pathogenesis and clinical manifestation

Detection of 2019 novel coronavirus (2019-nCoV) by real-time RT-PCR

From SARS and MERS CoVs to SARS-CoV-2: moving toward more biased codon usage in viral structural and nonstructural genes

Clinical evaluation of the new high-throughput Luminex NxTAG respiratory pathogen panel assay for multiplex respiratory pathogen detection

We acknowledge the kind generosity of Luminex for providing the NxTAG CoV extended panel kits used in this study. Luminex had no role in the design and conduct of the study, in the collection, analysis and interpretation of data, or in the preparation, review, or approval of the manuscript.We declare no conflict of interest.