Coronavirus disease 2019 (COVID-19) was first detected in Wuhan, Hubei Province, China. The disease is caused by severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) [1, 2].　To date, there have been over 100 million cases in the ongoing SARS-CoV-2 pandemic, with over 2 million deaths globally, according to the COVID-19 dashboard, Center for Systems Science and Engineering, Johns Hopkins University. We cannot accurately diagnose patients with COVID-19 based on the observable symptoms alone because these are nonspecific, such as fever, cough, sputum production, and shortness of breath, and are shared by other respiratory infections [3]. Infiuenza virus is a common cause of respiratory infections. Influenza and SARS-CoV-2 infections present similar symptoms and modes of transmission, including transmission through contact, droplets, and fomites [4].

Alveolar type II cells (AT2 pneumocytes), the primary site of infiuenza replication, especially infiuenza A, have been implicated as targets for SARS-CoV-2 infection [5]. There is scant evidence regarding the interaction between COVID-19 and infiuenza. One case report described that symptoms worsened when patients were co-infected with SARS-CoV-2 and infiuenza A [6]. Another report described rapid respiratory deterioration in four patients infected with SARS-CoV-2 and infiuenza A or B [7]. Moreover, simultaneous infection with SARS-CoV-2 and other respiratory pathogens may infiuence the morbidity and prognosis in patients [8]. It is important to accurately detect SARS-CoV-2 and infiuenza viruses, including possible coinfections in the community [9].

Reverse-transcription PCR (RT-PCR) is highly sensitive and specific and has become the standard test for detecting respiratory viruses, including SARS-CoV-2 [8]. This study aimed to evaluate a multiplex PCR to detect SARS-CoV-2 and infiuenza A and B viruses using the ExiStation™ system (Bioneer, Daejeon, Korea).

1. Clinical specimens

This study was approved by the Institutional Review Board of the Seoul National University Hospital. The study included 1,098 clinical respiratory specimens obtained from the Seoul National University Hospital, Seoul National University Boramae Medical Center, and Green Cross Laboratory. A total of 378 sputum samples and 720 nasopharyngeal/oropharyngeal swabs were collected from patients infected with SARS-CoV-2 or infiuenza. All the sputum samples and 382 nasopharyngeal or oropharyngeal swabs were analyzed for the presence of SARS-CoV-2. The remaining 338 nasopharyngeal or oropharyngeal swabs were tested for infiuenza A and B.

Clinical testing was performed using retrospective clinical samples. We analyzed the samples using the Allplex™ 2019-nCoV Assay (Seegene, Seoul, Korea) and STANDARD M nCoV Real-Time Detection kit (SD Biosensor, Suwon, Korea) for SARS-CoV-2, and Allplex™ Respiratory Panel 1 (Seegene) for infiuenza viruses. All specimens were stored at -70°C until testing with the AccuPower^® RV1 Real-Time RT-PCR Kit (Bioneer) and the AccuPower^® RV1 Multiplex Kit (Bioneer). The samples were not frozen or thawed more than once.

2. AccuPower^® RV1 Real-Time RT-PCR Kit and AccuPower^® RV1 Multiplex Kit

The AccuPower^® RV1 Kits can detect SARS-CoV-2, infiuenza A (Infiuenza A-H1, Infiuenza A-H3N2, and Infiuenza A-pdm09), and infiuenza B with different Ct values. The test was performed using an Exicycler™ 96 (Bioneer) for real-time PCR according to the manufacturer’s instructions. The results were interpreted automatically, and the analyzed data were presented using Bioneer’s software (ExiStation™ and ExiDiagnosis). A positive result entails that the Ct value of the sample was below the cut-off Ct value. A negative result indicated the absence of infection confirmed by amplification of the internal control.

BIONEER’s nucleic acid extraction eluted 400 μL of the clinical sample in a final volume of 100 μL using an automated extraction protocol on ExiStation™ and ExiPrep™ 48 Viral DNA/RNA Kit (Bioneer). The ExiStation™ system consisted of ExiPrep™48Dx (Bioneer) for RNA extraction and Exicycler™96 for real-time RT-PCR.

We used 50 μL aliquots for the AccuPower^® RV1 Real-Time RT-PCR Kit analysis and 10 μL aliquots for the AccuPower^® RV1 Multiplex Kit analysis. The AccuPower^® RV1 Real-Time RT-PCR Kit was optimized for ExiStation™ and the AccuPower^® RV1 Multiplex Kit was optimized for Exicycler™96. To determine clinical sensitivity and specificity, we compared the results of AccuPower^® kits with those of the Allplex™ 2019-nCoV assay and the Standard M nCoV Real-Time Detection Kit for SARS-CoV-2 and those of the Allplex™ Respiratory Panel 1 for infiuenza viruses.

3. Sequencing

The samples that exhibited discrepancies in their results were confirmed by sequencing. We extracted the total RNA from the samples according to the manufacturer’s instructions using the ExiStation™ and ExiPrep™ 48 Viral DNA/RNA Kit. We referred to the WHO protocol for the PCR conditions and primer sequences used for amplification [9]．Primer sequences for infiuenza were as follows: infiuenza type A M30F2/08: 5′-ATG AGY CTT YTA ACC GAG GTC GAA ACG-3′ and infiuenza type A M264R3/08: 5′-TGG ACA AAN CGT CTA CGC TGC AG-3′ for the M gene; infiuenza type B Victoria lineage Bvf224: 5′-ACA TAC CCT CGG CAA GAG TTT C-3′; infiuenza type B Victoria lineage Bvr507: 5′-TGC TGT TTT GTT GTC GTT TT-3′; infiuenza type B Yamagata lineage BYf226: 5′-ACA CCT TCT GCG AAA GCT TCA-3′; and infiuenza type B Yamagata lineage BYf613: 5′-CAT AGA GGT TCT TCA TTT GGG TT-3′ for the HA gene. Sequencing was performed using an ABI 3730xl DNA Analyzer (Applied Biosystems, Foster City, CA, USA).

4. Analytical sensitivity and specificity

We determined the analytical sensitivity of SARS-CoV-2 using 3-fold serially diluted virus samples. For sputum, 900, 300, 100, 33.3, 11.1, and 3.7 copies/reaction, and for swabs, 300, 100, 33.3, 11.1, 3.7, and 1.23 copies/reaction, were tested in 72 replicates for six concentrations. Similarly, influenza A and B were diluted 3-fold from 600 copies/reaction to 0.27 copies/reaction and tested in 72 replicates for six concentrations.

SARS-CoV-2 was obtained from SeraCare Life Sciences (Milford, MA, USA) and infiuenza viruses from Zeptometrix (Buffalo, NY, USA). Protocols for analytical sensitivity followed the Clinical and Laboratory Standards Institute guideline EP17-A2 [10]. Analytical sensitivity was estimated via probit regression analysis using R Studio (version 3.6.1).

The analytical specificities of the AccuPower^® kits were tested using 40 different pathogens. DNA or RNA was extracted from each reference panel and assayed using the same protocols used for clinical samples.

5. Statistical analysis

We assessed significant differences between the two methods using 2×2 contingency tables and the VassarStats website (http://vassarstats.net/) and performed agreement statistics (kappa calculation) to compare the detection sensitivity of the AccuPower^® kits and the comparative methods. Clinical sensitivity, specificity, and kappa statistics were reported with 95% confidence intervals (CIs).

1. Comparison of the AccuPower^® Kits and the confirmation results

Out of the 760 samples analyzed for SARS-CoV-2, 384 (190 sputum and 194 swab samples) were found to be positive, and 376 (188 sputum and 188 swab samples) were negative. Out of the samples tested for infiuenza, 81 were positive, and 95 were negative (Table 1).

Table 1 Comparison of the results of the AccuPower^® RV1 Real-Time RT-PCR Kit and the confirmation tests for the detection of SARS-CoV-2 and influenza viruses

Virus	Specimen type	Agreement
		Positive			Negative
		%	No.	95% CI	%	No.	95% CI
SARS-CoV-2	Sputum	100	190/190	98.02–100	100	188/188	98.00–100
	Swab	100	194/194	98.06–100	100	188/188	98.00–100
Influenza A	Swab	100	81/81	95.47–100	100	81/81	95.47–100
Influenza B	Swab	100	81/81	95.47–100	100	95/95	96.11–100

Abbreviation: CI, confidence interval.

For SARS-CoV-2, the evaluation was conducted at three institutions. The Green Cross Laboratory results were confirmed using the Allplex™ 2019-nCoV Assay and Seoul National University Hospital and Boramae Medical Center results using the STANDARD M nCoV Real-Time Detection Kit. The SARS-CoV-2 results analyzed with AccuPower^® RV1 Kits were compared with the confirmed results from each institution (Table 2). Both the positive and negative agreement between the results from AccuPower^® RV1 kits and the confirmation results for SARS-CoV-2 was 100%. The kappa values of the two methods were 1.0. Both the positive and negative agreement values of infiuenza A and infiuenza B between the results from AccuPower^® RV1 Real-Time RT-PCR Kit and Allplex™ Respiratory Panel 1 were 100%. However, the positive and negative agreement between infiuenza A and B between the results from AccuPower^® RV1 Multiplex Kit and Allplex™ Respiratory Panel 1 was 98.77%. The kappa value was 0.99.

Table 2 Comparison of AccuPower^® RV1 Real-Time RT-PCR Kit, AccuPower^® RV1 Multiplex Kit and reference method

Virus	Specimen type	Agreement
		No.		AccuPower® RV1 Real-Time RT-PCR Kit			No.		AccuPower® RV1 Multiplex Kit
		Positive	Negative	Sensitivity (95% CI)	Specificity (95% CI)	Kappa (95% CI)	Positive	Negative	Sensitivity (95% CI)	Specificity (95% CI)	Kappa (95% CI)
SARS-CoV-2	Sputum	190/190	188/188	100 (98.02–100)	100 (98.00–100)	1 (0.99–1.00)	190/190	188/188	100 (98.02–100)	100 (98.00–100)	1 (0.99–1.00)
	Swab	194/194	188/188	100 (98.06–100)	100 (98.00–100)	1 (0.99–1.00)	194/194	188/188	100 (98.06–100)	100 (98.00–100)	1 (0.99–1.00)
Influenza A	Swab	80/81	81/81	100 (95.47–100)	100 (95.47–100)	1 (0.98–1.00)	80/81	81/81	98.77 (93.33–99.78)	100 (95.47–100)	0.99 (0.97–1.00)
Influenza B	Swab	81/81	95/95	100 (95.47–100)	100 (96.11–100)	1 (0.98–1.00)	80/81	95/95	98.77 (93.33–99.78)	100 (96.11–100)	0.99 (0.99–1.00)

Abbreviation: CI, confidence interval.

The sequencing results for the discordant samples was positive. However, upon re-testing the samples with the comparative reagent (Allplex™ Respiratory Panel 1) and the AccuPower^® RV1 Real-Time RT-PCR Kit, the Ct values were detected near the cut-off in both kits. Therefore, the samples were low titer samples, and the discrepancy was attributed to the difference in the minimum nucleic acid concentration needed for use in the two tests.

2. Analytical sensitivity and specificity of the AccuPower^® RV1 Kits

The analytical sensitivities of the AccuPower^® RV1 Real-Time RT-PCR Kit were 64.57 copies/reaction (95% CI: 42.66–97.72), 56.23 copies/reaction (95% CI: 37.15–87.10), 38.90 copies/reaction (95% CI: 26.92–56.23), 37.15 copies/reaction (95% CI: 26.92–50.12), and 27.54 copies/reaction (95% CI: 19.05–39.81) for infiuenza A-H1N1 seasonal, H1N1 pdm, H3N2, infiuenza B-Yamagata, and infiuenza B-Victoria, respectively. The analytical sensitivities for the E gene and the SARS-CoV-2-specific gene (RdRp gene and N gene) were 107.15 copies/reaction (95% CI: 81.28–144.54) and 109.65 copies/reaction (95% CI: 79.43–147.91), respectively, with the sputum samples and 42.66 copies/reaction (95% CI: 32.36–54.95) and 26.92 copies/reaction (95% CI: 19.50–36.31), respectively, with the swab samples.

The analytical sensitivities of the AccuPower^® RV1 Multiplex Kits were 47.86 copies/reaction (95% CI: 34.67–66.07), 37.15 copies/reaction (95% CI: 26.30–52.48), 33.11 copies/reaction (95% CI: 22.91–46.77), 33.88 copies/reaction (95% CI: 25.12–45.71), and 26.92 copies/reaction (95% CI: 21.38–33.11) for infiuenza A-H1N1 seasonal, H1N1 pdm, H3N2, infiuenza B-Yamagata, and infiuenza B-Victoria, respectively. The analytical sensitivities for the E gene and the SARS-CoV-2-specific gene (RdRp gene and N gene) were 117.48 copies/reaction (95% CI: 91.20–154.88) and 107.15 copies/reaction (95% CI: 83.18–141.25), respectively, with the sputum samples and 44.67 copies/reaction (95% CI: 33.88–60.26) and 34.67 copies/reaction (95% CI: 26.30–44.67), respectively, with the swab samples.

We tested 40 different viruses and bacterial reference strains using the protocols used for the clinical samples to assess the cross-reactivity results and the detection specificity of the AccuPower^® kits (Table 3). The test results were negative (34 species), except for the specific target (related SARS-CoV-2, Infiuenza A, and Infiuenza B). No non-specific positive reactions were observed.

Table 3 Analytical specificities of the AccuPower^® RV Real-Time RT-PCR Kit and the AccuPower^® RV1 Multiplex Kit

No.	Pathogen	Strain	Result
1	SARS-CoV-2	Seracare (0505-0159)	Positive 1*
2	Human Influenza virus A H1N1 (seasonal)	KBPV VR-33	Positive 2†,
3	Human Influenza virus A H3N2 (Wisconsin/67/05)	Zeptometrix 0810252CFHI	Positive 2
4	Human Influenza virus A H1N1 pdm virus (Michigan/45/15)	Zeptometrix 0810538CFHI	Positive 2
5	Human Influenza virus B (Yamagata/16/88)	Zeptometrix 0810518CFHI	Positive 3‡
6	Human Influenza virus B (Texas/6/11)	Zeptometrix 0810242CFHI	Positive 3
7	Human Coronavirus 229E	KBPV VR-9	Negative
8	Human Coronavirus NL63	Zeptometrix 0810228CF	Negative
9	Human Coronavirus OC43	Zeptometrix 0810024CF	Negative
10	Human Coronavirus OC43	KBPV VR-8	Negative
11	Coronavirus HKU1 (synthetic RNA)	ATCC 3262SD	Negative
12	SARS-Coronavirus (Purified RNA of SARS-Coronavirus strain Frankfurt 1)	EVAg 004N-02005	Negative
13	Parainfluenza virus 1	Zeptometrix 0810014CFHI	Negative
14	Parainfluenza virus 2	Zeptometrix 0810015CFHI	Negative
15	Parainfluenza virus 3	Zeptometrix 0810016CFHI	Negative
16	Parainfluenza virus 4a	Zeptometrix 0810060CFHI	Negative
17	Parainfluenza virus 4b	Zeptometrix 0810060BCFHI	Negative
18	Human Respiratory syncytial virus A	KBPV VR-41	Negative
19	Human Respiratory syncytial virus B	KBPV VR-42	Negative
20	Human Rhinovirus 1 (type A)	KBPV VR-81	Negative
21	Human Rhinovirus 7 (type A)	KBPV VR-82	Negative
22	Human Rhinovirus 8 (type B)	KBPV VR-79	Negative
23	Human Rhinovirus 42 (type B)	KBPV VR-80	Negative
24	Human Metapneumovirus type 3 Type B1	Zeptometrix 0810156CFHI	Negative
25	Human Metapneumovirus type 9 Type A1	Zeptometrix 0810160CFHI	Negative
26	Human Parechovirus 1	Zeptometrix 0810145CF	Negative
27	Human Parechovirus 2	Zeptometrix 0810146CF	Negative
28	Human Parechovirus 3	Zeptometrix 0810147CF	Negative
29	Human Adenovirus type 03 (type B)	KBPV VR-62	Negative
30	Human Adenovirus type 02 (type C)	KBPV VR-58	Negative
31	Human Bocavirus (synthetic DNA)	ATCC 3251SD	Negative
32	Enterovirus 70	KBPV VR-55	Negative
33	Coxsackievirus B5	KBPV VR-17	Negative
34	Echovirus 25	KBPV VR-24	Negative
35	MERS-CoV	Seracare 0505-0002	Negative
36	Chlamydia pneumoniae	ATCC 53592	Negative
37	Haemophilus influenzae	ATCC 31441	Negative
38	Legionella pneumophila	ATCC 33152	Negative
39	Streptococcus pneumoniae	ATCC 49619	Negative
40	Bordetella pertussis	ATCC 12742	Negative

*Detects only SARS-CoV-2; ^†Detects only Influenza A; ^‡Detects only Influenza B.

Abbreviations (strain): KBPV, Korea Bank for Pathogenic Viruses; EVAg, European Virus Archive-Global.

Data on SARS-CoV-2 and infiuenza virus co-infection are limited. Infiuenza infection was classified as one of the characteristics of SARS-CoV-2 positive patients, and a small number of positive cases were studied. However, many studies have reported a correlation between the likelihood of co-infection and the severity or mortality rate due to co-infection [11-13]. Therefore, co-infection of SARS-CoV-2 and infiuenza viruses can pose serious risks. We can prepare for potential pandemics due to such co-infections by routinely testing for respiratory viruses other than SARS-CoV-2.

The AccuPower^® RV1 Real-Time RT-PCR Kit contains dried primary materials required for PCR in one tube optimized for the ExiStation™ series. ExiStation™ can rapidly extract and analyze nucleic acid using an exclusive software. This minimizes the hands-on time after sample processing and any human error that may occur during the preparation of the reaction mixture. The test was completed within 2 hours and 30 minutes. Additionally, the AccuPower^® RV1 Multiplex Kit is compatible with the ABI7500 instrument (Applied Biosystems, Foster City, CA), CFX96 instrument (Bio-Rad, Hercules, CA), and the Exicycler™.

We evaluated the cross-reactivity with 40 respiratory pathogens, including SARS-CoV-2, infiuenza A, and infiuenza B, and confirmed that there was no cross-reactivity to pathogens with similar symptoms.

We evaluated the limit of detection (LoD) in the sputum and nasopharyngeal/oropharyngeal swabs. The LoD in sputum was approximately 100 copies/test, and that in the swabs was approximately 40 copies/test in both kits. The rate of positivity is higher in sputum samples than that in the swabs and is less affected by the onset of symptoms or the time of collection [14]. However, sputum samples are usually viscous and contain many PCR and extraction inhibitors; therefore, we recommend performing a pre-treatment process for nucleic acid extraction for real-time PCR. Homogenization of sputum with proteinase K, dithiothreitol, or N-acetyl-L-cysteine (NALC) can increase the sensitivity of the test, and hence, the rate of positivity [15, 16].

We confirmed the clinical sensitivity and specificity by comparing the confirmation results of the AccuPower^® RV1 Real-Time RT-PCR Kit and the AccuPower^® RV1 Multiplex Kit using residual clinical samples collected from three institutions. The sensitivity and specificity were 100%, which is consistent with the confirmation results for both positive and negative samples of SARS-CoV-2. One infiuenza A sample and some infiuenza B samples displayed a discrepant result in the AccuPower^® RV1 Multiplex Kit. Sensitivity and specificity were 98.77%. The kappa values for all targets were ≥0.99 and were considered clinically valid when compared with the results of the confirmation tests.

A limitation of our study is that we did not include asymptomatic, pre-symptomatic, and co-infection cases, as only residual samples were used. Further studies, including these cases, need to be conducted.

In conclusion, the AccuPower^® RV1 Kits produced results comparable to the Allplex™ and Standard M Kit. The AccuPower^® RV1 Kits showed a higher concordance with the results of the comparative methods. Thus, the AccuPower^® RV1 Kits are a valuable tool to detect infiuenza A, B, SARS-CoV-2, and their co-infections in clinical laboratories.

This research was supported by a grant from the Korea Health Technology R&D Project through the Korea Health Industry Development Institute (KHIDI), funded by the Ministry of Health & Welfare, Republic of Korea (grant number: HI20C2038). Some bioresources from the National Biobank of Korea and the Korea Disease Control and Prevention Agency (grant number: 2020-045) were used.

None declared.