The evaluation of the utility of the GENECUBE HQ SARS-CoV-2 for anterior nasal samples and saliva samples with a new rapid examination protocol.

INTRODUCTION: GENECUBE® is a rapid molecular identification system, and previous studies demonstrated that GENECUBE® HQ SARS-CoV-2 showed excellent analytical performance for the detection of severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) with nasopharyngeal samples. However, other respiratory samples have not been evaluated.
METHODS: This prospective comparison between GENECUBE® HQ SARS-CoV-2 and reference real-time reverse transcriptase polymerase chain reaction (RT-PCR) was performed for the detection of SARS-CoV-2 using anterior nasal samples and saliva samples. Additionally, we evaluated a new rapid examination protocol using GENECUBE® HQ SARS-CoV-2 for the detection of SARS-CoV-2 with saliva samples. For the rapid protocol, in the preparation of saliva samples, purification and extraction processes were adjusted, and the total process time was shortened to approximately 35 minutes.
RESULTS: For 359 anterior nasal samples, the total-, positive-, and negative concordance of the two assays was 99.7% (358/359), 98.1% (51/52), and 100% (307/307), respectively. For saliva samples, the total-, positive-, and negative concordance of the two assays was 99.6% (239/240), 100% (56/56), and 99.5% (183/184), respectively. With the new protocol, total-, positive-, and negative concordance of the two assays was 98.8% (237/240), 100% (56/56), and 98.4% (181/184), respectively. In all discordance cases, SARS-CoV-2 was detected by additional molecular examinations.
CONCLUSION: GENECUBE® HQ SARS-CoV-2 provided high analytical performance for the detection of SARS-CoV-2 in anterior nasal samples and saliva samples.

Introduction

For the diagnosis of coronavirus disease 2019 (COVID-19), accurate and rapid laboratory testing is essential. Molecular examination using real-time reverse transcriptase polymerase chain reaction (RT-PCR) has been considered the gold standard for the identification of SARS-CoV-2 [1], and nasopharyngeal samples have been commonly used for the sample examination, which requires high-level personal protective equipment [2]. For COVID-19 testing, anterior nasal samples and saliva samples have been proposed as alternative samples [3], which can be easily obtained from patients.

GENECUBE® (TOYOBO Co., Ltd., Osaka, Japan) is a Qprobe-PCR-based automated rapid molecular identification system that can detect target genes in a short time and simultaneously analyze up to 12 samples and 4 assays in a single examination [4-9]. The system automatically performs molecular examination directly, including preparation of the reaction mixtures, and amplification and detection of target genes, in 30 minutes.

GENECUBE® HQ SARS-CoV-2 (TOYOBO Co., Ltd.) is the GENECUBE® reagent for detecting the SARS-CoV-2 gene in clinical samples. This reagent was currently approved by the Ministry of Health, Labour and Welfare in Japan in October 2020. We previously evaluated the performance of the assay using 1065 nasopharyngeal samples [10]. Compared with the reference RT-PCR assay, the overall positive- and negative concordance rates were 99.7% (95% confidence interval [CI]: 99.2%–99.9%), 100.0% (95% CI: 93.4%–100.0%) and 99.7% (95% CI: 99.1%–99.9%), respectively. All discordant samples were GENECUBE® HQ SARS-CoV-2-positive and reference RT-PCR-negative, and SARS-CoV-2 was detected by another molecular assay [10]. During the previous evaluation, samples other than nasopharyngeal samples were not used.

In the present study, we evaluated the diagnostic performance of the GENECUBE® HQ SARS-CoV-2 using anterior nasal samples and saliva samples. Additionally, we evaluated a new rapid examination protocol using GENECUBE® HQ SARS-CoV-2 for the detection of SARS-CoV-2.

Materials and methods

The current study was performed at a drive-through PCR center in Tsukuba Medical Center Hospital (TMCH) in Tsukuba, Ibaraki Prefecture, Japan, which intensively performed sample collecting and PCR analysis with nasopharyngeal samples in the Tsukuba district [10, 11]. Patients with and without symptoms were referred from nearby clinics and a local public health center. All of the asymptomatic patients had known contact histories with COVID-19 confirmed/suspected patients.

Anterior nasal samples were prospectively collected from COVID-19-suspected or COVID-19-confirmed patients in addition to nasopharyngeal samples between 11 May 2021 and 5 July 2021, as previously performed [12]. Saliva samples were also prospectively collected in addition to nasopharyngeal samples from COVID-19-confirmed patients between 21 April 2021 and 13 May 2021. All of anterior nasal samples and saliva samples from COVID-19-confirmed patients were obtained on the same day of nasopharyngeal sample collection.

Anterior nasal samples and saliva samples were simultaneously examined using GENECUBE® HQ SARS-CoV-2 (GENECUBE examination) and reference RT-PCR, and the concordance of the two methods was evaluated.

Informed consent was verbally obtained from patients for their participation in the respective part of the current research, and participant consent was documented in the electronic chart of each participant. Written informed consent was not obtained in order to avoid infection transmission through the consent forms. The ethics committee of Tsukuba Medical Center Hospital approved the present study (approval number: 2020–066) with the current protocol, including the method of obtaining informed consent.

This study was performed in line with the principles of the Declaration of Helsinki and adheres to the STARD reporting guidelines.

For negative saliva samples, residual frozen saliva samples collected during SARS-CoV-2 active screening at hospitalization in Tsukuba Medical Center Hospital were used for the current research after anonymization.

Sample collection

For anterior nasal samples, a nasopharyngeal-type flocked swab (Copan Italia SpA, Brescia, Italy) was inserted to a 2 cm depth in one nasal cavity, rotated five times, and held in place for 5 seconds. The swab samples were then diluted in 3 mL of UTM™ (Copan Italia SpA) immediately after sampling, and the UTM™ was then transferred to a microbiology laboratory located next to the drive-through sampling facility of the PCR center.

After arrival, purification, and ribonucleic acid (RNA) extraction were performed with magLEAD (Precision System Science Co., Ltd., Chiba, Japan) with 200 μL of fresh anterior nasal samples. RNA was eluted in 100 μL, which was used for the GENECUBE® examination and the reference RT-PCR examination. All saliva samples were stored at −80°C and were purified with magLEAD after preparation (Fig 1). All of the GENECUBE® examinations and reference RT-PCR examinations were performed simultaneously on the same day.

Fig 1

Workflow of the two extraction methods for the GENECUBE® assay in this study.

The rapid method with magLEAD extraction (b) newly developed in this study takes as little as 10 min for viral RNA extraction, while the standard method (a) takes approximately 25 min. For the rapid protocol, in the preparation of saliva samples, purification and extraction processes were adjusted, and the total process time was shortened. PBS Phosphate-buffered saline. For magLEAD 12gC, the picture was reprinted from [13] under a CC BY license with the permission of Precision System Science Co., Ltd., 2016. For GENECUBE, the picture was reprinted from [14] under a CC BY license with the permission of TOYOBO Co., Ltd., 2021.

GENECUBE® examination with magLEAD and discrepancy analysis

The sample used for the GENECUBE® examination analysis of SARS-CoV-2 was also used for the RT-PCR assays. All assays were performed with the previously described magLEAD conditions [10] (Fig 1). In addition to the standard protocol with magLEAD purification, a rapid protocol created by Hiromichi Suzuki, TOYOBO Co., Ltd. and Precision System Science Co., Ltd. were evaluated for saliva samples and samples for limit of detection (LOD) analysis. For the rapid protocol, in the preparation of saliva samples, purification and extraction processes were adjusted, and the total process time was shortened to approximately 10 minutes. The comparison of the standard protocol and the rapid protocol for each magLEAD purification processes is summarized in Table 1. The rapid protocol is commercially available in Japan as MagDEA Dx SV 200 for GENECUBE®.

Table 1

The comparison of the standard and rapid protocols for each magLEAD purification process.

Process	Rapid protocol MagDEA Dx SV 200 for GENECUBE®	Standard protocol MagDEA Dx SV 200
Lysis process	1.5 min	4.0 min
Binding to Magnetic Beads process	1.0 min	2.0 min
Washing process	0.5 min × 1	1.5 min × 2
Elution process	1.0 min	5.0 min
Other process: Motor operation, sample and buffer preparation, liquid dispensing, B/F separation, eluate collection, etc.
Total time	Approximately 10 min	Approximately 25 min
For the Rapid protocol MagDEA Dx SV 200 for GENECUBE®, operation speeds, including the interval of each process, were made as fast as possible.

If discordance was recognized between GENECUBE® and the reference RT-PCR, an additional evaluation was performed with Xpert® Xpress SARS-CoV-2 and GeneXpert® (Cepheid Inc., Sunnyvale, CA, USA) [15] analyses for anterior nasal samples according to the manufacturer’s instructions documented in the package insert, and with an RT-PCR with LightMix® Modular SARS-CoV (COVID19) E-gene (Roche Diagnostics KK) [16] for saliva samples along with re-evaluation with the NIID RT-PCR method.

Reference real-time RT-PCR method

Reference RT-PCR examinations were performed with purified samples using a method developed by the National Institute of Infectious Diseases (NIID), Japan, for SARS-CoV-2 [17, 18], which was used Briefly, 5 μL of the extracted RNA was used for one-step quantitative RT-PCR with the THUNDERBIRD® Probe One-step qRT-PCR kit (TOYOBO Co., Ltd.) and the LightCycler® 96 Real-time PCR System (Roche Diagnostics KK, Basel, Switzerland). A duplicate analysis for N2 genes was performed for the evaluation of SARS-CoV-2. EDX SARS-CoV-2 Standard (Bio-Rad Laboratories, Inc., Hercules, CA, USA) and sterile purified water (Merck & Co., Inc., Kenilworth, NJ, USA) were used as positive and negative controls, respectively. The calibration curves were generated with 5, 50 and 500 copies/reaction of EDX SARS-CoV-2 Standard.

Estimation of the limit of detection (LOD) for GENECUBE® HQ SARS-CoV-2 with nasopharyngeal samples and saliva samples

To estimate the LOD for GENECUBE® HQ SARS-CoV-2, we made four different concentrations of samples (2500 copies/mL, 1000 copies/mL, 500 copies/mL, 250 copies/mL) with SARS-CoV-2 reference material (AccuPlex™ SARS-CoV-2 Reference Material Kit, SeraCare; SeraCare Life Sciences, Inc., Milford, MA, USA) and matrix (UTM™; three pooled nasopharyngeal samples and two pooled saliva samples). In total, six samples were made at each concentration. The GENECUBE® examination was performed four times, and the reference RT-PCR was performed twice for each sample.

Statistical analyses

The positive-, negative-, and total concordance rates of the GENECUBE® examinations compared with the reference RT-PCR were calculated using the Clopper and Pearson methods with 95% confidence intervals. All calculations were conducted using the R 3.3.1 software program (The R Foundation, Vienna, Austria).

Results

Evaluation of LOD for the reference RT-PCR and GENECUBE® with SARS-CoV-2 reference material and pooled negative samples

The details of the results of the LOD evaluation for the three SARS-CoV-2 detection methods with SARS-CoV-2 reference material and pooled negative samples are listed in Table 2, summarized in Table 3 and S1 Fig.

Table 2

Detailed results of the estimated LOD test for three SARS-CoV-2 detection methods.

Ratio of reference material and sample	Copies/mL	Sample	GENECUBE® (Standard method with magLEAD)	GENECUBE® (Rapid method with magLEAD)	Real-time RT-PCR (N2 NIID method)
			N of detection/N of test (detection rate)			Ct value (Copies/test)
reference material: sample = 1:1	2500	Total	24/24 (100)	24/24 (100)	12/12 (100)	-
		UTM	4/4 (100)	4/4 (100)	2/2 (100)	31.9 (48)/32.4 (34)
		Pooled nasopharyngeal sample 1	4/4 (100)	4/4 (100)	2/2 (100)	32.2 (39)/31.9 (50)
		Pooled nasopharyngeal sample 2	4/4 (100)	4/4 (100)	2/2 (100)	32.7 (28)/32.3 (36)
		Pooled nasopharyngeal sample 3	4/4 (100)	4/4 (100)	2/2 (100)	32.2 (40)/32.4 (35)
		Pooled saliva sample 1	4/4 (100)	4/4 (100)	2/2 (100)	32.3 (38)/32.5 (33)
		Pooled saliva sample 2	4/4 (100)	4/4 (100)	2/2 (100)	32.3 (37)/32.7 (28)
reference material: sample = 1:4	1000	Total	24/24 (100)	24/24 (100)	10/12 (83)	-
		UTM	4/4 (100)	4/4 (100)	2/2 (100)	34.1 (11)/33.5 (16)
		Pooled nasopharyngeal sample 1	4/4 (100)	4/4 (100)	2/2 (100)	33.7 (15)/33.7 (15)
		Pooled nasopharyngeal sample 2	4/4 (100)	4/4 (100)	2/2 (100)	33.5 (16)/33.6 (15)
		Pooled nasopharyngeal sample 3	4/4 (100)	4/4 (100)	2/2 (100)	34.1 (11)/33.1 (21)
		Pooled saliva sample 1	4/4 (100)	4/4 (100)	1/2 (50)	33.3 (19)/ND
		Pooled saliva sample 2	4/4 (100)	4/4 (100)	1/2 (50)	33.8 (14)/ND
reference material: sample = 1:9	500	Total	23/24 (96)	24/24 (100)	4/12 (33)	-
		UTM	4/4 (100)	4/4 (100)	2/2 (100)	34.5 (8)/35.3 (5)
		Pooled nasopharyngeal sample 1	4/4 (100)	4/4 (100)	0/2 (0)	ND/ND
		Pooled nasopharyngeal sample 2	3/4 (75)	4/4 (100)	2/2 (100)	34.3 (10)/34.2 (10)
		Pooled nasopharyngeal sample 3	4/4 (100)	4/4 (100)	0/2 (0)	ND/ND
		Pooled saliva sample 1	4/4 (100)	4/4 (100)	0/2 (0)	ND/ND
		Pooled saliva sample 2	4/4 (100)	4/4 (100)	0/2 (0)	ND/ND
reference material: sample = 1:19	250	Total	19/24 (79)	18/24 (75)	3/12 (25)	-
		UTM	4/4 (100)	4/4 (100)	2/2 (100)	36.2 (3)/34.8 (7)
		Pooled nasopharyngeal sample 1	4/4 (100)	4/4 (100)	0/2 (0)	ND/ND
		Pooled nasopharyngeal sample 2	4/4 (100)	2/4 (50)	1/2 (50)	35.1 (5)/ND
		Pooled nasopharyngeal sample 3	2/4 (50)	3/4 (75)	0/2 (0)	ND/ND
		Pooled saliva sample 1	3/4 (75)	2/4 (50)	0/2 (0)	ND/ND
		Pooled saliva sample 2	2/4 (50)	3/4 (75)	0/2 (0)	ND/ND

The AccuplexTM SARS-CoV-2 reference material (5000 copies/mL) was diluted with UTM or pooled samples and subjected to magLEAD extraction with the standard or rapid method. Each extract was then assayed four times by GENECUBE and twice by NIID RT-PCR.

Ct cycle threshold, LOD limit of detection, ND not detected, NIID National Institute of Infectious Diseases, RT-PCR reverse transcription polymerase chain reaction

Table 3

Summary of the estimated LOD test results for three SARS-CoV-2 detection methods.

Sample (Copies/mL)	GENECUBE® (Standard method with magLEAD)	GENECUBE® (Rapid method with magLEAD)	Real-time RT-PCR (N2 NIID method)
	N of detection/N of test (detection rate)	N of detection/N of test (detection rate)	N of detection/N of test (detection rate)
2500	24/24 (100)	24/24 (100)	12/12 (100)
1000	24/24 (100)	24/24 (100)	10/12 (83)
500	23/24 (96)	24/24 (100)	4/12 (33)
250	19/24 (79)	18/24 (75)	3/12 (25)

LOD limit of detection, N number, NIID National Institute of Infectious Diseases, RT-PCR reverse transcription polymerase chain reaction

The reference NIID real-time RT-PCR method showed positive results for all UTM-based samples (range: 250–5000 copies/mL), while the detection rate was 100% down to 1000 copies/mL for pooled nasopharyngeal samples and down to 2500 copies/mL for pooled saliva-based samples. None of the 500 copies/mL of pooled saliva-based samples and 250 copies/mL of pooled saliva-based samples were detected by the NIID real-time RT-PCR method.

The standard protocols with magLEAD and GENECUBE® showed positive results for all UTM-based samples. The detection rate was 100% down to 1000 copies/mL for pooled nasopharyngeal-based samples and down to 500 copies/mL for pooled saliva-based samples. The detection rate of 500 copies/mL pooled nasopharyngeal-based samples was 91.7% (11/12).

For the rapid protocol with magLEAD and GENECUBE®, the method showed positive results for all UTM-based samples. The detection rate was 100% down to 500 copies/mL for pooled nasopharyngeal-based samples and pooled saliva-based samples.

Comparison of the reference RT-PCR and GENECUBE® for the detection of SARS-CoV-2 with anterior nasal samples

In this study, we prospectively evaluated 359 fresh anterior nasal samples, including 59 samples with positive SARS-CoV-2 results for simultaneously collected nasopharyngeal samples (cycle threshold (Ct) < 20, n = 40; Ct ≥ 20 to < 30, n = 16; Ct ≥ 30, n = 3) (S1 Table). Of the 359 anterior nasal samples, 298 (83.0%) were obtained from asymptomatic patients.

The comparison of the reference RT-PCR and GENECUBE® (standard protocol) for the detection of SARS-CoV-2 with anterior nasal samples is summarized in Tables 4–6. For anterior nasal samples prospectively obtained from suspected COVID-19 patients (Table 4), the total, positive and negative concordance of the 2 assays were 100% (320/320), 100% (18/18) and 100% (302/302), respectively. With the addition of the enriched positive patients, the total, positive and negative concordance of the 2 assays were 99.7% (358/359), 98.1% (51/52) and 100% (307/307), respectively. When GENECUBE® (standard protocol) with anterior nasal samples were compared with the reference RT-PCR with nasopharyngeal samples, the total, positive and negative concordance were 97.8% (351/359), 86.4% (51/59) and 100% (300/300), respectively.

Table 4

Concordance rate of the GENECUBE® HQ SARS-CoV-2 with real-time RT-PCR for anterior nasal samples obtained from suspected COVID-19 patients.

		Real-time RT-PCR (N2 NIID method) (Anterior nasal samples)
		Positive	Negative
Standard method with magLEAD extraction for GENECUBE® (Anterior nasal sample)	Positive	18	0
	Negative	0	302
Positive concordance rate (%)		100 (81.5–100)
Negative concordance rate (%)		100 (98.8–100)
Total concordance rate (%)		100 (98.9–100)

NIID National Institute of Infectious Diseases, RT-PCR reverse transcription polymerase chain reaction

Data in parentheses are 95% confidence intervals.

Table 6

Concordance rate between the GENECUBE® HQ SARS-CoV-2 with anterior nasal samples and real-time RT-PCR with nasopharyngeal samples, both of which were simultaneously obtained from suspected or confirmed COVID-19 patients.

		Real-time RT-PCR (N2 NIID method) (Nasopharyngeal samples)
		Positive	Negative
Standard method with magLEAD extraction for GENECUBE® (Anterior nasal samples)	Positive	51	0
	Negative	8	300
Positive concordance rate (%)		86.4 (75.0–94.0)
Negative concordance rate (%)		100 (98.8–100)
Total concordance rate (%)		97.8 (95.7–99.0)

NIID National Institute of Infectious Diseases, RT-PCR reverse transcription polymerase chain reaction

Data in parentheses are 95% confidence intervals.

Comparison of the reference RT-PCR and GENECUBE® for the detection of SARS-CoV-2 with saliva samples

For the comparison between the reference RT-PCR and GENECUBE® for the detection of SARS-CoV-2, 60 frozen samples (symptomatic patients, 32; asymptomatic patients, 28) obtained from confirmed COVID-19 patients by nasopharyngeal samples and 180 frozen negative saliva samples were examined.

The evaluation of the standard protocol with magLEAD and GENECUBE® is described in Table 7, and the evaluation of the rapid protocol with magLEAD and GENECUBE® is described in Table 8. The result of one sample was invalid initially by both GENECUBE® examinations, and the sample required four-fold dilution with lysis buffer for the GENECUBE® examinations.

Table 7

Concordance rate of the standard method with magLEAD extraction for GENECUBE® with real-time RT-PCR for saliva samples*.

		Real-time RT-PCR (N2 NIID method)
		Positive	Negative
Rapid method with magLEAD extraction for GENECUBE®	Positive	56	1**
	Negative	0	183
Positive concordance rate (%)		100 (93.6–100)
Negative concordance rate (%)		99.5 (97.0–100)
Total concordance rate (%)		99.6 (97.7–100)

NIID National Institute of Infectious Diseases, RT-PCR reverse transcription polymerase chain reaction.

Data in parentheses are 95% confidence intervals.

* 60 frozen samples obtained from confirmed COVID-19 patients by nasopharyngeal samples and 180 negative saliva samples were used.

**The discordant samples were tested by real-time RT-PCR with Roche LightMix Modular SARS and Wuhan CoV E-gene and all were positive (S3 Table).

Table 8

Concordance rate of the rapid method with magLEAD extraction for GENECUBE® with real-time RT-PCR for saliva samples*.

		Real-time RT-PCR (N2 NIID method)
		Positive	Negative
Rapid method with magLEAD extraction for GENECUBE®	Positive	56	3**
	Negative	0	181
Positive concordance rate (%)		100 (93.6–100)
Negative concordance rate (%)		98.4 (95.3–99.7)
Total concordance rate (%)		98.8 (96.4–99.7)

NIID National Institute of Infectious Diseases, RT-PCR reverse transcription polymerase chain reaction

Data in parentheses are 95% confidence intervals.

* 60 frozen samples obtained from confirmed COVID-19 patients by nasopharyngeal samples and 180 negative saliva samples were used.

**The discordant samples were tested by real-time RT-PCR with Roche LightMix Modular SARS and Wuhan CoV E-gene and all were positive (S3 Table).

For the evaluation of the standard protocol with magLEAD and GENECUBE® (Table 7), the total-, positive-, and negative concordance of the two assays was 99.6% (239/240), 100% (56/56), and 99.5% (183/184), respectively.

For the evaluation of the rapid protocol with magLEAD and GENECUBE® (Table 8), the total-, positive-, and negative concordance of the two assays was 98.8% (237/240), 100% (56/56), and 98.4% (181/184), respectively.

Discussion

During the analytical evaluation with 359 anterior nasal samples and 240 saliva samples, the GENECUBE® evaluation with GENECUBE® HQ SARS-CoV-2 showed high concordance rates compared with the reference RT-PCR. The estimated LoDs study indicated that the GENECUBE® evaluation conducted with GENECUBE® HQ SARS-CoV-2 maintained a high analytical performance for nasopharyngeal samples and saliva samples, with detection successful for as little as 1000 copies/mL for both types of samples.

In the current study concerning the GENECUBE examination, the rapid method with magLEAD extraction showed equivalent analytical performance to the standard method, both in the estimated LOD study and the comparative study with reference via real-time RT-PCR. Of note, two saliva samples obtained from COVID-19-confirmed patients were positive with the rapid protocol and negative with the standard protocol. This slight difference might be due to differences in the centrifugation condition (Fig 1) and number of washes performed (Table 1), which can result in the loss of virus and RNA; however, the difference was not proven in the estimated LOD study, and the superiority with respect to the analytical performance cannot be concluded based on the present findings.

Saliva has been considered a good alternative for the detection of SARS-CoV-2 in COVID-19 patients [3] and has been widely used in COVID-19 practice. Among rapid molecular identification systems, GeneXpert® showed good analytical performance for the detection of SARS-CoV-2 with saliva samples [19]; however, the application of saliva to rapid molecular identification systems remains a challenge owing to saliva’s viscosity and RT-PCR inhibition. In our current study, we used 60 saliva samples with positive nasopharyngeal sample results for SARS-CoV-2 (S2 Table), and the rapid protocol detected SARS-CoV-2 in most samples (98.3%; 59/60). The rapid protocol can detect SARS-CoV-2 with high performance in approximately 35 minutes with saliva samples, and the protocol is expected to have clinical utility, especially for the rapid accurate identification of SARS-CoV-2-infected patients with collecting nasopharyngeal samples is considered difficult. The current study used frozen saliva samples for validation. While the analytical performance for the detection of SARS-CoV-2 has been reported to be equal between fresh and frozen saliva samples [20], the freeze-thaw method has been known as one of the RNA/DNA extraction methods [21] which might influence the sensitivity [22]. Therefore, a further evaluation should be performed with fresh samples.

Anterior nasal samples are also a good alternative method for COVID-19 sampling [3]. The method has been reported as less painful and induced fewer coughs or sneezes compared with nasopharyngeal sampling [12]. The application of self-collected anterior nasal sampling has also been reported [23]. In the current study, the analytical performance of the GENECUBE® examination was almost identical to the reference RT-PCR. However, there were seven negative results using anterior nasal samples, which were obtained from patients with positive nasopharyngeal samples (S1 Table). The detection rate was 88.1% (52/59), which was inferior to that with saliva samples; however, saliva samples were not simultaneously collected with anterior nasal samples in the present study, so we cannot compare the sensitivity. In addition, we obtained anterior nasal samples from a single nasal cavity; however, the viral loads obtained with nasal sampling differ between nares [24], so sampling from both nasal cavities is preferable [25].

There are several limitations in this study that should be mentioned. First, reference real-time RT-PCR was used for the comparison, and a discrepant analysis was used for the validation, which can cause bias unless a composite reference standard is used as a reference [26-32]. While the current study evaluated the concordance with the reference real-time RT-PCR findings, the sensitivity and specificity of the GENECUBE® examination were not accurately confirmed. Second, the current research was performed at a PCR center in Japan. The influence of LODs of the GENECUBE® evaluation for genetic variants of SARS-CoV-2 was not evaluated in this study. Third, the current evaluated rapid protocol showed excellent performance for the detection of SARS-CoV-2; however, the sample size was insufficient to conclude that the protocol can be used in clinical practice; additional evaluation in studies with large samples is required. Fourth, the current GENECUBE® examination can analyze only 12 samples at a single run, and the amplification curve is not displayed. The system must be improved for better examination. Fourth, for the evaluation of the GENECUBE® examination using anterior nasal samples, the proportion of low viral load samples (Ct ≥ 30) was small, which could have improved thus concordance rates of the current study.

In conclusion, the GENECUBE® examination with GENECUBE® HQ SARS-CoV-2 provided high analytical performance for the detection of SARS-CoV-2 in anterior nasal samples and saliva samples.

Detection rate of three SARS-CoV-2 detection methods for each concentration of samples.

The same set of samples was extracted with the standard or rapid method with magLEAD and analyzed with GENECUBE® and real-time RT-PCR (N2 NIID method) for each sample concentration. The vertical axis shows the detection rate (%). The horizontal axis shows the comparison of each three SARS-CoV-2 detection methods, and each of the bar graph types shows the sample concentration.

(TIF)

Click here for additional data file.

Results of SARS-Cov-2 detection for anterior nasal samples.

(DOCX)

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Results of SARS-Cov-2 detection for saliva samples.

(DOCX)

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Detailed data for the three cases with discordant findings among the three SARS-CoV-2 detection methods for saliva samples.

(DOCX)

Click here for additional data file.

21 Sep 2021

PONE-D-21-27242Evaluation of GENECUBE® HQ SARS-CoV-2 for anterior nasal samples and saliva samples with a new rapid examination protocolPLOS ONE

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Comments to the Author

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Reviewer #1: Partly

Reviewer #2: Partly

Reviewer #3: Yes

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2. Has the statistical analysis been performed appropriately and rigorously?

Reviewer #1: No

Reviewer #2: Yes

Reviewer #3: Yes

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Reviewer #1: No

Reviewer #2: Yes

Reviewer #3: Yes

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Reviewer #2: Yes

Reviewer #3: Yes

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5. Review Comments to the Author

Please use the space provided to explain your answers to the questions above. You may also include additional comments for the author, including concerns about dual publication, research ethics, or publication ethics. (Please upload your review as an attachment if it exceeds 20,000 characters)

Reviewer #1: As a whole this is a good and convincing paper, but I have some concerns – both minor and major.

Journal policy concern: Authors state that there will be some restrictions on data sharing, without specifying what those are. They later state that all relevant data are in the manuscript. This seems internally contradictory.

Minor concern: Line 69: Authors state that “This reagent was approved in October, 2021.” Who was the approving body?

Major concern: Authors note that if discrepancies were found between GeneCube analysis and the RTPCR reference method, a third method was employed to resolve the discrepancy. This procedure, known as “discrepant analysis” is biased. [1-7]. Bias results from the fact that testing of non-discrepant samples (based on two assays) may, in principle, result in a discrepancy when the “resolver” test is applied; however, there is no chance to detect this discrepancy because the resolver is never applied to the non-discrepant tests. This may give the appearance that both of the initial test methods are better than they actually are. What the authors did was not as bad as it could have been since they apparently did not use the discrepant analysis to create or adjust sensitivity numbers. However, use of a composite reference standard for assessing the performance of methods does not suffer from this bias [8,9], and gives a more accurate portrayal of the relative sensitivity of both methods. While the impact of this inappropriate technique on the conclusions is negligible, its appearance in the literature promotes its further use. I think the paper would be significantly improved (and shortened somewhat) if none of the discrepant analysis is included.

Minor concern: Sampling from a single nares is to be avoided, as there is sufficient data on respiratory infections to show that sampling from both nares improves sensitivity. I include one reference, but there are many others I haven’t taken the time to look up [10]. I think the authors should note in their discussion the fact that sampling both nares is preferable.

Minor concern: The number of samples assessed in the LOD study is too small to draw strong conclusions about the LOD (see CLSI document EP7 and https://www.ncbi.nlm.nih.gov/pmc/articles/PMC2556583/). Pooling of sample types (UTM, nasal swab, saliva) to achieve N is not appropriate, since these samples may have differing sources of assay interference. I think it is fair to say the authors have approximated the LOD, however.

1. Miller WC. Bias in discrepant analysis: When two wrongs don’t make a right. Journal of Clinical Epidemiology. 1998;51: 219–231. doi:10.1016/S0895-4356(97)00264-3

2. Hadgu A. Discrepant analysis: A biased and an unscientific method for estimating test sensitivity and specificity. Journal of Clinical Epidemiology. 1999;52: 1231–1237. doi:10.1016/S0895-4356(99)00101-8

3. Hadgu A. The discrepancy in discrepant analysis. Lancet. 1996;348: 592–593. doi:10.1016/S0140-6736(96)05122-7

4. McAdam AJ. Discrepant analysis and bias: A micro-comic strip. Journal of Clinical Microbiology. American Society for Microbiology; 2017. pp. 2878–2879. doi:10.1128/JCM.00969-17

5. Hadgu A, McAdam AJ. Discrepant analysis is an inappropriate and unscientific method (multiple letters) [4]. Journal of Clinical Microbiology. 2000. pp. 4301–4302.

6. Miller WC. Can we do better than discrepant analysis for new diagnostic test evaluation? Clinical infectious diseases : an official publication of the Infectious Diseases Society of America. 1998. pp. 1186–1193. doi:10.1086/514996

7. Green TA, Black CM, Johnson RE. Evaluation of bias in diagnostic-test sensitivity and specificity estimates computed by discrepant analysis. Journal of Clinical Microbiology. 1998;36: 375–381. doi:10.1128/jcm.36.2.375-381.1998

8. Baughman AL, Bisgard KM, Cortese MM, Thompson WW, Sanden GN, Strebel PM. Utility of composite reference standards and latent class analysis in evaluating the clinical accuracy of diagnostic tests for pertussis. Clinical and Vaccine Immunology. 2008;15: 106–114. doi:10.1128/CVI.00223-07

9. Tang S, Hemyari P, Canchola JA, Duncan J. Dual composite reference standards (dCRS) in molecular diagnostic research: A new approach to reduce bias in the presence of imperfect reference. Journal of Biopharmaceutical Statistics. 2018;28: 951–965. doi:10.1080/10543406.2018.1428613

10. van Wesenbeeck L, Meeuws H, D’Haese D, Ispas G, Houspie L, van Ranst M, et al. Sampling variability between two mid-turbinate swabs of the same patient has implications for influenza viral load monitoring. Virology Journal. 2014;11. doi:10.1186/s12985-014-0233-9

Reviewer #2: The manuscript submitted by Naito et al entitled “Evaluation of GENECUBE HQ SARS-CoV-2 for anterior nasal samples and saliva samples with a new rapid examination protocol” details a study that evaluates a molecular assay for detection of SARS-CoV-2. The assay was previously evaluated for NP specimens, but this assay evaluated the use of both NS, saliva, and saliva with a rapid processing protocol. In total 359 NS were evaluated and 240 saliva samples. Overall, the results demonstrated that the assay was accurate for all specimen types tested; However, there are some discrepancies in numbers that need to be clarified to ensure the proper comparisons were made. There are also a few additional clarifications needed prior to publication listed below:

Major Comments

For the enriched positive patients that had confirmed COVID-19 results, how was this detected. Did patients come back in after the results came through or was it a rapid test. Also, by cherry picking these patients you mess with the pre-test probability and would likely improve the assay performance, especially as the LoD appears to be better for GENECUBE vs RT-PCR assay. I would suggest breaking out the results as Total, prospective, call back subjects to be clear and see effects on true prospective testing.

The definition of the rapid method is lacking in specifics. What steps were modified in the maglead extraction process that reduced the method by 20 minutes. As this is written it would not be possible to replicate the study or adopt the new method for a clinical lab to validate and use for faster TAT.

Ln223-227: This is a bit unclear. There are 3 FP results based on rapid. 1 was the FP found in 4a, but the other 2 are new. Why were these 2 specimens tested 8 times on the lightmix test? One was picked up 50% of the time and the other was 12.5%.

Ln245-250: Why is table 4a and 4b not using the NP PCR result as the reference method. This analysis was done is ST2, which should be the results used for the evaluation and not comparing saliva tested on the two platforms (Is the reference PCR validated for saliva?). If you use the presentation of data in table 4a and 4b this would suggest that saliva with GENECUBE is more sensitive, but if you compare it to the PCR from NP (gold standard) then it seems like it would be slightly less sensitive of a sample type. I would delete 4a and 4b and replace with data comparing to NP. This is the same for NS comparison data as ln 257 indicates that PPA was actually 88.1% compared to the 98.1% you present in table 3.

Minor Comments

Ln 73: Was the pos and negative concordance both 99.7% in the NP study as you only give 2 values for the overall, pos-, neg concordance.

Were discordant specimens tested on the Xpert run as package insert (i.e. testing directly from VTM).

Table 2: The current layout is a bit confusing at first with the spacing for “Standard method with magLead…”

LoD study: The standard method is to perform 20 replicates at each concentration in pooled negatives and the LoD is then defined as the lowest concentration that was 19/20. What was the rationale for using multiple matrices and at the end having 24 tests for GENECUBE and 12 for the reference method.

Ln197: Was the copies/mL determined based on the CT value and the LoD of the specimen? If so what was the CT value for the reference test and the Xpert assay.

Why were the saliva samples frozen initially? Could this have skewed results as there is some data that a freeze thaw can help sensitivity of saliva samples.

Reviewer #3: Thank you for inviting me to peer review this manuscript. The authors have studied the GENECUBE ® HQ SARS-CoV-2 using anterior nasal samples and saliva samples by using a new protocol. This study aims to evaluate the detection of SARS-CoV-2 with saliva samples for the first time and using a rapid protocol. Here are some comments which could be considers to improve the manuscript:

1. Line 69 “This reagent was approved in October, 2021.”. The date needs to be corrected.

2. Line 86, the situation of the infected cases is not explained. How did the authors select them and in which phase of disease they were? Did they have symptoms or they were asymptomatic cases?

3. It seems that the authors have collected the anterior nasal samples and saliva samples from different cases. It was better to take both sample types from each studied case to be able to compare them and investigate the accuracy of the samples to detect the infection. (It seems that such work is mentioned very briefly in the Discussion)

4. There is no explanation for positive and negative controls.

5. New Method and Standard Method are not explained in the Materials and Methods. In Line 133, “For the rapid protocol, in the preparation of saliva samples, purification and extraction processes were adjusted, and the total process time was shortened to approximately 10 minutes.” How this happened and what is the main difference making the novel method this short?

6. The obtained resulted are not discussed deeply. As an example, Line 181 “The detection rate was 100% down to 1000 copies/mL for pooled …” by considering these results, is this method useful for early detection or it is just useful for the chronic cases? How could a Dr. decide this strategy is a good choice for a specific case (like asymptomatic cases, accurate or chronic infections)?

7. Line 186. Compared with the standard test, how could the Rapid Strategy decrease the LoD of the nasopharyngeal samples from 1000 copies/mL to 500 copies/mL? (not discussed again).

8. Line 245 “In our current study, we used 60 saliva samples with positive nasopharyngeal sample results for SARS-CoV-2 (Supplementary Table 2), and the rapid protocol detected SARS-CoV-2 in most samples (98.3%; 59/60)”. It is the first time that the authors are refer to performing some tests on the nasopharyngeal and saliva samples taken from one case. It is better to be explained (materials and methods) and reported (results) in the previous sections before the Discussion.

9. The authors could also add graph(s) to report the results in a more useful way.

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Reviewer #1: No

Reviewer #2: No

Reviewer #3: Yes: Tina Shaffaf

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19 Oct 2021

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Response: Informed consent was verbally obtained from patients for their participation in the respective parts of the current research, and participant consent was documented in the electronic chart of each participant. Written informed consent was not obtained in order to avoid infection transmission through the consent forms. The ethics committee approved the present study with the current protocol, including the method of obtaining informed consent. We have now mentioned this in the Methods section.

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Response: We removed any funding-related text from our manuscript and included our amended statements within our cover letter. We have provided our amended Funding Statement below. Please change the online submission form on our behalf.

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Response: We have now uploaded the completed content permission form as an “Other” file along with the submission. In addition, we added the following text to the caption of the copyrighted figure:

“For magLEAD, the picture was reprinted from [13] under a CC BY license with the permission of Precision System Science Co., Ltd., 2016” and “For GENECUBE, てthe picture was reprinted from [14] under a CC BY license with the permission of TOYOBO Co., Ltd., 2021”.

[13] Precision System Science Co., Ltd. magLEAD 6gC & magLEAD 12gC High-Quality, Low Cost, Automated Nucleic Acid Extraction System. 2021 [Cited 5 August 2021]. Available from https://www.pss.co.jp/product/magtration/lead6-12gc.html

[14] TOYOBO Co., Ltd. Fully Automated Gene Analyzer GENECUBE® (model C). 2021 [Cited 5 August 2021]. Available from https://www.toyobo.co.jp/products/bio/gene/genecube_c/index.html.

5. Review Comments to the Author

Reviewer #1: As a whole this is a good and convincing paper, but I have some concerns – both minor and major.

Response: We appreciate your review and suggestions for our manuscript. We have now revised the manuscript based on the suggestions, with changes shown in red.

Suggestion: Journal policy concern: Authors state that there will be some restrictions on data sharing, without specifying what those are. They later state that all relevant data are in the manuscript. This seems internally contradictory.

Response: We have now added all of our data as supplementary tables 1 and 2 and modified our statement.

Suggestion: Minor concern: Line 69: Authors state that “This reagent was approved in October, 2021.” Who was the approving body?

Response: The reagent was approved by the Ministry of Health, Labour and Welfare in Japan in October 2020. We changed the year of approval from 2021 to 2020, which was an error in our description.

Major concern: Authors note that if discrepancies were found between GeneCube analysis and the RTPCR reference method, a third method was employed to resolve the discrepancy. This procedure, known as “discrepant analysis” is biased. [1-7]. Bias results from the fact that testing of non-discrepant samples (based on two assays) may, in principle, result in a discrepancy when the “resolver” test is applied: however, there is no chance to detect this discrepancy because the resolver is never applied to the non-discrepant tests. This may give the appearance that both of the initial test methods are better than they actually are. What the authors did was not as bad as it could have been since they apparently did not use the discrepant analysis to create or adjust sensitivity numbers. However, use of a composite reference standard for assessing the performance of methods does not suffer from this bias [8,9], and gives a more accurate portrayal of the relative sensitivity of both methods. While the impact of this inappropriate technique on the conclusions is negligible, its appearance in the literature promotes its further use. I think the paper would be significantly improved (and shortened somewhat) if none of the discrepant analysis is included.

Response: We completely agree with your comment and admit that this is a limitation of our current study: Reference real-time RT-PCR was used for the comparison, and a discrepant analysis was used for the validation, which can cause bias unless a composite reference standard is used as a reference. While the current study evaluated the concordance with the reference real-time RT-PCR findings, the sensitivity and specificity of the GENECUBE® examination were not accurately confirmed. Regarding the discrepant analysis, the description was deleted from the Results section of the manuscript, and the table concerning the results of the discrepancy analysis of saliva was moved to the supplementary materials.

Minor concern: Sampling from a single nares is to be avoided, as there is sufficient data on respiratory infections to show that sampling from both nares improves sensitivity. I include one reference, but there are many others I haven’t taken the time to look up [10]. I think the authors should note in their discussion the fact that sampling both nares is preferable.

Response: We have now mentioned this in the Discussion section as follows: “[…] however, the viral loads obtained with nasal sampling differ between nares, so sampling from both nasal cavities is preferable.”

Minor concern: The number of samples assessed in the LOD study is too small to draw strong conclusions about the LOD (see CLSI document EP7 and https://www.ncbi.nlm.nih.gov/pmc/articles/PMC2556583/). Pooling of sample types (UTM, nasal swab, saliva) to achieve N is not appropriate, since these samples may have differing sources of assay interference. I think it is fair to say the authors have approximated the LOD, however.

Response: The current evaluation was insufficient to determine the LODs. We have now revised the text concerning the evaluation and withdrew our strong conclusion concerning LODs, which mentioned the superiority of the GENECUBE examination.

1. Miller WC. Bias in discrepant analysis: When two wrongs don’t make a right. Journal of Clinical Epidemiology. 1998:51: 219–231. doi:10.1016/S0895-4356(97)00264-3

2. Hadgu A. Discrepant analysis: A biased and an unscientific method for estimating test sensitivity and specificity. Journal of Clinical Epidemiology. 1999:52: 1231–1237. doi:10.1016/S0895-4356(99)00101-8

3. Hadgu A. The discrepancy in discrepant analysis. Lancet. 1996:348: 592–593. doi:10.1016/S0140-6736(96)05122-7

4. McAdam AJ. Discrepant analysis and bias: A micro-comic strip. Journal of Clinical Microbiology. American Society for Microbiology: 2017. pp. 2878–2879. doi:10.1128/JCM.00969-17

5. Hadgu A, McAdam AJ. Discrepant analysis is an inappropriate and unscientific method (multiple letters) [4]. Journal of Clinical Microbiology. 2000. pp. 4301–4302.

6. Miller WC. Can we do better than discrepant analysis for new diagnostic test evaluation? Clinical infectious diseases : an official publication of the Infectious Diseases Society of America. 1998. pp. 1186–1193. doi:10.1086/514996

7. Green TA, Black CM, Johnson RE. Evaluation of bias in diagnostic-test sensitivity and specificity estimates computed by discrepant analysis. Journal of Clinical Microbiology. 1998:36: 375–381. doi:10.1128/jcm.36.2.375-381.1998

8. Baughman AL, Bisgard KM, Cortese MM, Thompson WW, Sanden GN, Strebel PM. Utility of composite reference standards and latent class analysis in evaluating the clinical accuracy of diagnostic tests for pertussis. Clinical and Vaccine Immunology. 2008:15: 106–114. doi:10.1128/CVI.00223-07

9. Tang S, Hemyari P, Canchola JA, Duncan J. Dual composite reference standards (dCRS) in molecular diagnostic research: A new approach to reduce bias in the presence of imperfect reference. Journal of Biopharmaceutical Statistics. 2018:28: 951–965. doi:10.1080/10543406.2018.1428613

10. van Wesenbeeck L, Meeuws H, D’Haese D, Ispas G, Houspie L, van Ranst M, et al. Sampling variability between two mid-turbinate swabs of the same patient has implications for influenza viral load monitoring. Virology Journal. 2014:11. doi:10.1186/s12985-014-0233-9

Reviewer #2: The manuscript submitted by Naito et al entitled “Evaluation of GENECUBE HQ SARS-CoV-2 for anterior nasal samples and saliva samples with a new rapid examination protocol” details a study that evaluates a molecular assay for detection of SARS-CoV-2. The assay was previously evaluated for NP specimens, but this assay evaluated the use of both NS, saliva, and saliva with a rapid processing protocol. In total 359 NS were evaluated and 240 saliva samples. Overall, the results demonstrated that the assay was accurate for all specimen types tested: However, there are some discrepancies in numbers that need to be clarified to ensure the proper comparisons were made. There are also a few additional clarifications needed prior to publication listed below:

Response: We appreciate your review and suggestions for our manuscript. We have now revised the manuscript based on the suggestions, with changes shown in red.

Major Comments

Suggestion: For the enriched positive patients that had confirmed COVID-19 results, how was this detected. Did patients come back in after the results came through or was it a rapid test. Also, by cherry picking these patients you mess with the pre-test probability and would likely improve the assay performance, especially as the LoD appears to be better for GENECUBE vs RT-PCR assay. I would suggest breaking out the results as Total, prospective, call back subjects to be clear and see effects on true prospective testing.

Response: We have now included additional data in Table 4a, which excluded anterior nasal samples obtained from confirmed COVID-19 patients, in the Results section.

Suggestion: The definition of the rapid method is lacking in specifics. What steps were modified in the magLEAD extraction process that reduced the method by 20 minutes. As this is written it would not be possible to replicate the study or adopt the new method for a clinical lab to validate and use for faster TAT.

Response: We added Table 1 to show the results of the comparison of the standard and rapid protocols for each magLEAD purification processes in the Methods section. The new extraction method is now commercially available in Japan as MagDEA Dx SV 200 for GENECUBE® 12gC, so researchers can validate the current findings. We have now mentioned this in the revised manuscript.

Suggestion: Ln223-227: This is a bit unclear. There are 3 FP results based on rapid. 1 was the FP found in 4a, but the other 2 are new. Why were these 2 specimens tested 8 times on the lightmix test? One was picked up 50% of the time and the other was 12.5%.

Response: All three discordant saliva samples were obtained from COVID-19 patients confirmed with simultaneously obtained nasopharyngeal samples (supplementary table 2). For #11, #39, additional RT-PCR (E-gene) was performed with two additional purification methods, as RT-PCR (E-gene) was negative for the initial purified sample. Multiple tests (eight times) were performed because the viral loads of SARS-CoV-2 in the saliva samples were considered to be very low. These data are considered to be supplementary, so we moved the discrepancy analysis to a supplementary table.

Suggestion: Ln245-250: Why is table 4a and 4b not using the NP PCR result as the reference method. This analysis was done is ST2, which should be the results used for the evaluation and not comparing saliva tested on the two platforms (Is the reference PCR validated for saliva?). If you use the presentation of data in table 4a and 4b this would suggest that saliva with GENECUBE is more sensitive, but if you compare it to the PCR from NP (gold standard) then it seems like it would be slightly less sensitive of a sample type. I would delete 4a and 4b and replace with data comparing to NP. This is the same for NS comparison data as ln 257 indicates that PPA was actually 88.1% compared to the 98.1% you present in table 3.

Response: The reference method has been validated for saliva, and we added a reference to the Method section. For anterior nasal samples, all samples were simultaneously obtained with nasopharyngeal samples, and we added a comparison between GENECUBE® HQ SARS-CoV-2 with anterior nasal samples and real-time RT-PCR with nasopharyngeal samples to the Results section. For saliva samples, while positive saliva samples were simultaneously obtained with nasopharyngeal samples, negative saliva samples were not simultaneously obtained with nasopharyngeal samples, so we cannot make the table suggested. We added the limitation in discussion section.

Minor Comments

Suggestion: Ln 73: Was the pos and negative concordance both 99.7% in the NP study as you only give 2 values for the overall, pos-, neg concordance.

Response: This was our mistake. We have now added the data concerning total concordance in red.

Suggestion: Were discordant specimens tested on the Xpert run as package insert (i.e. testing directly from VTM).

Response: We tested the discordant specimens directly from UTM according to the manufacturer’s instructions included on the package insert. We have now added the description to the Methods section in red.

Suggestion: Table 2: The current layout is a bit confusing at first with the spacing for “Standard method with magLead…”

Response: We changed the layout to improve the appearance.

Suggestion: LoD study: The standard method is to perform 20 replicates at each concentration in pooled negatives and the LoD is then defined as the lowest concentration that was 19/20. What was the rationale for using multiple matrices and at the end having 24 tests for GENECUBE and 12 for the reference method.

Response: As proposed, it was inappropriate to draw conclusions about LoDs with the current LoD study, although the current protocol is useful for investigating the influence of the respiratory sample matrix on the analytical performance of GENECUBE examinations and is also useful for estimating LoDs. We have now revised the expression to “estimated LoDs”, and the assessment of the results was added to the Discussion section in red.

Suggestion: Ln197: Was the copies/mL determined based on the CT value and the LoD of the specimen? If so what was the CT value for the reference test and the Xpert assay.

Response: For quantitative RT-PCR, the Ct value of reference RT-PCR with 5, 50, and 500 copies/reaction of positive control was used. We have now added the quantitative method to the Methods section in red.

Suggestion: Why were the saliva samples frozen initially? Could this have skewed results as there is some data that a freeze thaw can help sensitivity of saliva samples.

Response: The current study was performed with two GENECUBE examinations (standard protocol and rapid protocol) and reference RT-PCR. Due to a lack of human resources, we were unable to perform GENECUBE examinations and reference RT-PCR with fresh saliva samples. We have now mentioned this as a limitation in the Discussion section. While the analytical performance for the detection of SARS-CoV-2 has been reported to be equal between fresh and frozen saliva samples [11], the freeze-thaw method has been known as one of the RNA/DNA extraction methods [12] which might influence the sensitivity [13]. We have now mentioned this in the Discussion section.

11. Fukumoto T, Iwasaki S, Fujisawa S, Hayasaka K, Sato K, Oguri S, et al. Efficacy of a novel SARS-CoV-2 detection kit without RNA extraction and purification. Int J Infect Dis. 2020:98:16-17: https://doi.org/10.1016/j.ijid.2020.06.074

12. Paz S, Mauer C, Ritchie A, Robishaw JD, Caputi M. A simplified SARS-CoV-2 detection protocol for research laboratories. PLoS One. 2020:15: e0244271: https://doi.org/10.1371/journal.pone.0244271

13. Ott IM, Strine MS, Watkins AE, Boot M, Kalinich CC, Harden CA, et al. Simply saliva: stability of SARS-CoV-2 detection negates the need for expensive collection devices. madRxiv. 2020: https://doi.org/10.1101/2020.08.03.20165233

Reviewer #3: Thank you for inviting me to peer review this manuscript. The authors have studied the GENECUBE ® HQ SARS-CoV-2 using anterior nasal samples and saliva samples by using a new protocol. This study aims to evaluate the detection of SARS-CoV-2 with saliva samples for the first time and using a rapid protocol. Here are some comments which could be considers to improve the manuscript:

Response: We appreciate your review and suggestions for our manuscript. We have now revised the manuscript based on the suggestions, with changes shown in red.

Suggestion: Line 69 “This reagent was approved in October, 2021.”. The date needs to be corrected.

Response: This was our mistake. We have now changed the year of approval from 2021 to 2020.

Suggestion: Line 86, the situation of the infected cases is not explained. How did the authors select them and in which phase of disease they were? Did they have symptoms or they were asymptomatic cases?

Response: We have now clarified the situation of the patients in the Methods section and the number of patients with symptoms in the Results section.

Suggestion: It seems that the authors have collected the anterior nasal samples and saliva samples from different cases. It was better to take both sample types from each studied case to be able to compare them and investigate the accuracy of the samples to detect the infection. (It seems that such work is mentioned very briefly in the Discussion)

Response: As you suggested, saliva samples were not simultaneously collected with anterior nasal samples in this study; we therefore cannot compare the sensitivity. We have now mentioned this as a limitation in the Discussion section.

Suggestion: There is no explanation for positive and negative controls.

Response: EDX SARS-CoV-2 Standard (Bio-Rad Laboratories, Inc., Hercules, CA, USA) and sterile purified water (Merck & Co., Inc., Kenilworth, NJ, USA) were used as positive and negative controls, respectively. We have now mentioned this in the Methods section.

Suggestion: New Method and Standard Method are not explained in the Materials and Methods. In Line 133, “For the rapid protocol, in the preparation of saliva samples, purification and extraction processes were adjusted, and the total process time was shortened to approximately 10 minutes.” How this happened and what is the main difference making the novel method this short?

Response: We added Table 1 to show the results of the comparison of the standard and rapid protocols for each magLEAD purification processes in the Methods section.

Suggestion: The obtained resulted are not discussed deeply. As an example, Line 181 “The detection rate was 100% down to 1000 copies/mL for pooled …” by considering these results, is this method useful for early detection or it is just useful for the chronic cases? How could a Dr. decide this strategy is a good choice for a specific case (like asymptomatic cases, accurate or chronic infections)?

Response: We have now described our assessment of the rapid method and the current limitations associated with the study of anterior nasal samples and saliva samples in the Discussion section. The rapid protocol with a GENECUBE examination for saliva is considered useful for the rapid accurate identification of SARS-CoV-2-infected patients when collecting nasopharyngeal samples is difficult.

Suggestion: Line 186. Compared with the standard test, how could the Rapid Strategy decrease the LoD of the nasopharyngeal samples from 1000 copies/mL to 500 copies/mL? (not discussed again).

Response: The total detection rate in the estimated LOD study was considered equal between the two protocols, although the detection rate was slightly higher than that with the standard method in a comparative study of saliva samples. This slight difference might be due to differences in the centrifugation condition (Fig. 1) and number of washes performed (Table 1), which can result in the loss of virus and RNA; however, the difference was not proven in the estimated LOD study, and the superiority with respect to the analytical performance cannot be concluded based on the present findings. We have now mentioned this in the Discussion.

Suggestion: Line 245 “In our current study, we used 60 saliva samples with positive nasopharyngeal sample results for SARS-CoV-2 (Supplementary Table 2), and the rapid protocol detected SARS-CoV-2 in most samples (98.3%: 59/60)”. It is the first time that the authors are refer to performing some tests on the nasopharyngeal and saliva samples taken from one case. It is better to be explained (materials and methods) and reported (results) in the previous sections before the Discussion.

Response: We have now described the simultaneous sampling collection in the Methods section and the number in the Results section.

Suggestion: The authors could also add graph(s) to report the results in a more useful way.

Response: We have now added a supplementary figure (S1) to support the viewing of table 2 in the supporting information file.

Submitted filename: Response to reviewers.docx

Click here for additional data file.

29 Nov 2021

PONE-D-21-27242R1The evaluation of the utility of the GENECUBE HQ SARS-CoV-2 for anterior nasal samples and saliva samples with a new rapid examination protocolPLOS ONE

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Reviewer #1: All comments have been addressed

Reviewer #2: All comments have been addressed

Reviewer #3: All comments have been addressed

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Reviewer #2: Yes

Reviewer #3: Yes

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Reviewer #1: (No Response)

Reviewer #2: Yes

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Reviewer #2: Thank you for the changes to the manuscript, which improves the clarity of study. One minor suggestion I would have is to add a limitation about the lack of low viral load samples tested in the evaluation. In ln 202 only 3 samples were CT >30. This is a small proportion of your positives and that could have improved concordance as too few were near the limit of detection.

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7 Dec 2021

Suggestion: Thank you for the changes to the manuscript, which improves the clarity of study. One minor suggestion I would have is to add a limitation about the lack of low viral load samples tested in the evaluation. line 202, only 3 samples were CT >30. This is a small proportion of your positives and that could have improved concordance as too few were near the limit of detection.

Response: Thank you very much for your suggestion. As you pointed out, the proportion of low viral load samples (Ct ≥ 30) was small for the evaluation of the GENECUBE® examination using anterior nasal samples, which could have improved thus concordance rates of the current study. We therefore added this point as one limitation associated with our study in the limitation section of the discussion.

Submitted filename: Response to reviewers.docx

Click here for additional data file.

17 Dec 2021

The evaluation of the utility of the GENECUBE HQ SARS-CoV-2 for anterior nasal samples and saliva samples with a new rapid examination protocol

PONE-D-21-27242R2

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22 Dec 2021

PONE-D-21-27242R2

The evaluation of the utility of the GENECUBE HQ SARS-CoV-2 for anterior nasal samples and saliva samples with a new rapid examination protocol

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Table 5

Concordance rate of the GENECUBE® HQ SARS-CoV-2 with real-time RT-PCR for anterior nasal samples obtained from suspected or confirmed COVID-19 patients.

		Real-time RT-PCR (N2 NIID method) (Anterior nasal samples)
		Positive	Negative
Standard method with magLEAD extraction for GENECUBE® (Anterior nasal sample)	Positive	51	0
	Negative	1*	307
Positive concordance rate (%)		98.1 (89.7–100)
Negative concordance rate (%)		100 (98.8–100)
Total concordance rate (%)		99.7 (98.5–100)

NIID National Institute of Infectious Diseases, RT-PCR reverse transcription polymerase chain reaction

Data in parentheses are 95% confidence intervals.

*The discordant sample was tested by Xpert® Xpress SARS-CoV-2 and GeneXpert® and was positive.