Detection of SARS-CoV-2 Variants by Multiple Diagnostic Assays Second RESUBMISSION JCV-D-21-00675R2.

INTRODUCTION: The emergence of SARS-CoV-2 Variants of Concern (VOC) and Variants Being Monitored (VBM) have presented additional clinical and public health concerns regarding potential virus transmissibility, disease severity, and immune evasion. It is imperative that diagnostic assays can detect all such variants, and since commercial oligo sequences are commonly not available, empirical testing may be necessary to confirm this. To confirm the sensitivity of the SARS-CoV-2 assays used at the Wadsworth Center for the detection of VOC and VBM, relevant specimens were selected from the specimen archive and tested in the various platforms.
MATERIALS AND METHODS: Patient respiratory specimens submitted from clinal laboratories across the state were selected; three samples per variant were chosen to account for inter assay and variant reproducibility. The four molecular diagnostic platforms for SARS-CoV-2 currently in use at our facility were examined.
RESULTS: A total of 64 specimens were tested, representing 2 VOC, 8 VBM and 4 other variants circulating in New York State. For certain samples, original Ct values provided by sample submitters were much higher, or lower, than those obtained from this study. The investigation of submitter testing platforms, with consideration of the assay's viral targets, confirmed the differences in Ct were not variant specific.
CONCLUSIONS: It was demonstrated that the diagnostic methods investigated in this study detected all the variants tested. Because of the continual evolution of the virus, it is vital to monitor new variants as they emerge for the ability of molecular diagnostic methods to detect them with acceptable sensitivity.

Introduction

The 2019 Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) pandemic spread globally, and the virus has now acquired mutations that impact virulence, transmission, and immune protection. In December of 2020, the SARS-CoV-2 variant B.1.1.7, subsequently designated Alpha, that originally emerged in the UK in September 2020 and quickly became the dominant variant in Europe, was detected in the US. This variant has 23 mutations, including several in the spike gene associated with increased transmissibility [1].

The Center for Disease Control and Prevention (CDC) has classified SARS-CoV-2 variants into three categories: Variant of Interest (VOI), Variant of Concern (VOC), and Variant of High Consequence (VHC), generally characterized as variants associated with reduced efficacy of treatment or vaccine prevention, increased transmissibility and more severe disease [2]. These classifications have changed as the pandemic has progressed and on September 21,2021 a new class of Variants Being Monitored (VBM) was added. [2]

It is vital for patient care and public health surveillance and response, that diagnostic platforms detect all SARS-CoV-2 variants with high levels of sensitivity and specificity. Information on the performance of diagnostic assays with regard to the detection of variants is scarce but two independent single point mutations have been reported to reduce the detection sensitivity for the N2 target of at least one commonly used assay [3]. While constant review of sequence data may assist in identifying mutations that could be problematic for diagnostic detection, sequence information for commercial assays is commonly unavailable for detailed analysis and empirical testing is always reassuring to confirm that assay sensitivity has not been impaired. Therefore, to ensure accurate detection on all testing platforms used at this laboratory, we tested a representative set of specimens containing all available variants of SARS-CoV-2 from the laboratory's archives.

Materials and methods

Patients and specimens

Specimens for this study had been previously submitted for whole genome analysis from SARS-CoV-2 positive patients as part of statewide surveillance. These samples were identified as positive for SARS-CoV-2 with numerous testing methods, by clinical laboratories throughout New York State (NYS), prior to submission to the New York State Department of Health. Whole genome sequencing was performed by the Wadsworth Center Applied Genomic Technology Core and sequences analyzed by the Bioinformatics Core. The sequence database was reviewed and multiple samples per variant identified for testing. The distribution of SARS-CoV-2 variants in NYS over time, from December 2020 to December 2021, is shown in Fig. 1 and demonstrates that the subsequent selection of variants tested was appropriate.

Fig. 1

The figure shows a variant stacked plot indicating the distribution of SARS-CoV-2 variants detected in New York State from December 2020 to December 2021. Sequencing was performed in the Advanced Genomics Technology Core at the Wadsworth Center, on samples submitted from clinical laboratories throughout New York State. Sequence analysis was performed by the Bioinformatics Core.

This work was approved by the New York State Institutional Review Board under study number 02-054.

At least three unique original patient specimens per variant were retrieved from freezer archives and tested across the four diagnostic molecular platforms in use at the Wadsworth Center. For the two VOCs, Delta and Omicron, at least six patient specimens were tested to ensure a more in-depth examination of the variants in this category. Testing of each sample was completed in one day to avoid data discrepancies which may be caused by multiple freeze-thaw cycles.

SARS-CoV-2 test platforms

The test platforms used at the Wadsworth Center are listed below, and the target genes for the assays are shown in Tables 1 ,2 , and 3 . For all platforms, testing was performed as specified in the corresponding package insert or instruction for use documents. Submitting laboratories used a wide variety of tests and therefore target genes for their assays are not individually listed.

Table 1

Results of testing at Submitting Laboratories and Wadsworth Center for Variants of Concern

VARIANT CLASSIFICATION		Submitters’ Ct values		Wadsworth Ct values
VOC	Sample ID			NeuMoDx		Cepheid	GenMark	CDC LDT
		Target 1	Target 2	N gene	Nsp2 gene	E	N1	N1	N2
B.1.617.2 Delta	PS1	Not provided		20.65	22.11	24.20	Detected	22.84	34.22
	SP2	20.20	20.60	21.95	22.54	Depleteda	Detected	23.55	27.07
	PS3	13.40	N/A	21.88	22.63	27.60	Detected	28.77	30.23
	PS4	Not provided		16.08	16.59	16.30	Detected	18.32	18.75
	PS5	Not provided		17.03	17.65	17.20	Detected	19.35	19.35
	PS6	20.40	N/A	18.47	19.32	19.10	Detected	20.33	20.65
	PS7	13.68	N/A	22.99	24.08	25.20	Detected	29.33	29.66
	PS8	16.76	N/A	21.15	21.75	21.40	Detected	24.24	22.57
	PS9	14.27	N/A	19.77	20.01	18.50	Detected	17.12	17.45
	PS10	17.33	N/A	20.29	21.48	21.90	Detected	23.48	23.54
	PS11	23.40	N/A	22.19	22.78	22.60	Detected	23.62	25.18
AY.4 lineage Delta	PS1	14.60	N/A	21.57	22.51	22.90	Detected	27.10	27.29
	PS2	21.50	N/A	28.75	29.53	31.20	Detected	33.92	33.92
	PS3	Not provided		17.04	17.44	17.10	Detected	19.13	19.37
	PS4	12.00	N/A	20.59	21.58	21.40	Detected	23.63	22.75
	PS5	21.03	N/A	20.76	21.07	21.00	Detected	22.24	22.64
	PS6	22.72	N/A	23.72	23.72	22.80	Detected	24.73	25.24
	PS7	17.84	N/A	25.94	27.32	28.30	Detected	31.95	32.15
B.1.1.529 Omicron	PS1	20.70	N/A	16.22	16.78	19.80	Detected	20.02	19.03
	PS2	13.50	N/A	19.49	19.19	23.30	Detected	22.71	22.28
	PS3	34.00	N/A	Unresolvedb		35.90	Detected	36.58	34.47
	PS4	19.50	N/A	19.81	20.00	23.20	Detected	23.23	22.53
	PS5	19.90	N/A	17.54	17.83	21.10	Detected	20.65	19.91
	PS6	18.60	N/A	15.94	16.61	19.30	Detected	19.36	18.68

“Depleted” indicates that all the specimen had been used for testing and there was no residual sample left for any further tests.

An “Unresolved” result is generated by the NeuMoDx for this assay when the amplification starts too late in the cycle to be called positive. This usually occurs when the sample viral load is very low.

Table 2

Results of testing at Submitting Laboratories and Wadsworth Center for Variants Being Monitored

VARIANT CLASSIFICATION		Submitters’ Ct values		Wadsworth Ct Values
VBM	Sample ID			NeuMoDx		Cepheid	GenMark	CDC LDT
		Target 1	Target 2	N gene	Nsp2 gene	E	N1	N1	N2
B.1.1.7 Alpha	PS1	20.40	20.80	18.33	19.38	20.00	Detected	19.25	21.75
	PS2	26.40	27.00	26.66	27.24	27.20	Detected	26.48	27.03
	PS3	16.10	16.90	18.99	19.29	20.40	Detected	19.89	21.69
B.1.351 Beta	PS1	17.00	17.50	26.58	28.35	29.00	Detected	26.27	26.46
	PS2	17.90	18.10	14.01	14.99	16.10	Detected	16.50	16.26
	PS3	22.50	N/A	28.06	28.65	32.90	Detected	33.52	34.94
P.1 Gamma	PS1	19.10	23.20	16.57	17.60	17.20	Detected	16.97	16.96
	PS2	17.70	N/A	17.36	18.30	17.20	Detected	18.23	18.39
	PS3	19.20	N/A	19.42	20.11	20.40	Detected	21.24	21.36
B.1.427 Epsilon	PS1	18.60	17.50	18.13	18.84	19.50	Detected	19.75	19.64
	PS2	21.00	19.70	15.24	16.02	16.30	Detected	17.28	16.95
	PS3	Not provided		24.43	24.94	25.20	Detected	25.51	25.65
B.1.429 Epsilon	PS1	23.06	23.23	19.55	20.55	20.40	Detected	20.96	20.92
	PS2	17.61	17.33	13.32	15.04	16.90	Detected	15.65	15.71
	PS3	25.95	26.20	24.08	24.94	25.10	Detected	24.34	25.31
B.1.525 Eta	PS1	17.70	17.61	25.42	26.68	28.80	Detected	24.89	25.93
	PS2	20.94	21.04	17.94	19.58	19.90	Detected	18.73	18.77
	PS3	Not provided		16.10	16.90	16.60	Detected	17.62	17.32
B.1.526 Iota	PS1	16.60	16.50	13.30	14.45	15.10	Detected	15.21	15.52
	PS2	16.40	N/A	23.19	25.66	28.10	Detected	28.03	28.50
	PS3	19.20	N/A	24.55	25.44	33.20	Detected	24.77	25.77
B.1.621 Mu	PS1	16.10	N/A	15.58	16.87	17.00	Detected	20.08	18.20
	PS2	25.17	N/A	29.13	29.75	30.70	Detected	33.45	31.85
	PS3	18.90	N/A	19.01	19.63	18.90	Detected	21.60	20.30
P.2 Zeta	PS1	26.98	27.41	21.75	22.86	Depleteda	Detected	22.08	21.51
	PS2	16.06	16.93	19.05	20.15	19.20	Detected	20.76	20.09
	PS3	22.10	19.80	19.74	20.69	21.40	Detected	21.30	20.70

“Depleted” indicates that all the specimen had been used for testing and there was no residual sample left for any further tests.

Table 3

Results of testing at Submitting Laboratories and Wadsworth Center with Other Variants Commonly Circulating in NYS During 2021.

VARIANT CLASSIFICATION		Submitters’ Ct values		Wadsworth Ct Values
Other Lineages	Sample ID			NeuMoDx		Cepheid	GenMark	CDC LDT
		Target 1	Target 2	N gene	Nsp2 gene	E	N1	N1	N2
B.1.526.1	PS1	23.43	22.89	26.62	28.16	42.20; Undetected	Undetected; Detected	27.35	28.80
	PS2	25.18	26.31	25.84	26.45	26.10	Detected	25.37	25.27
	PS3	26.52	26.76	17.66	18.30	20.20	Detected	19.08	19.20
B.1.526.2	PS1	Not provided		17.19	18.79	19.00	Detected	19.09	18.39
	PS2	25.15	24.51	29.47	30.04	34.20	Detected	30.37	32.79
	PS3	26.54	27.70	20.08	21.62	27.40	Detected	26.76	26.82
B.1.243	PS1	16.23	16.68	18.58	18.79	18.50	Detected	19.07	18.74
	PS2	6.70*	N/A	17.80	18.37	17.40	Detected	18.49	18.41
	PS3	27.50	28.20	29.56	30.00	29.80	Detected	30.95	30.49
B.1.1.519	PS1	17.84	20.50	17.48	18.83	19.50	Detected	17.63	19.36
	PS2	21.18	23.72	22.94	24.48	24.50	Detected	23.31	25.20
	PS3	26.23	27.08	23.78	24.78	25.70	Detected	24.52	26.09

Cn value obtained by the ABBOTT Real Time Sars-CoV-2 Assay in combination with the Real Time M2000 system at submitter site.

NeuMoDx molecular SARS-CoV-2 assay (Germantown, MD) – in vitro real time RT-PCR using NeuMoDx 288 instrument for qualitative detection of SARS-CoV-2. (https://www.fda.gov/media/136565/download)

Cepheid Xpert (Sunnyvale, CA) –Automated diagnostic instrument Multiplex Xpert Xpress SARS-CoV-2/Flu/RSV assay, using the Cepheid GeneXpert Infinity instrument. (https://www.fda.gov/media/142437/download)

GenMark ePlex Respiratory Pathogen Panel 2 (Carlsbad, CA) – multiplex in vitro diagnostic test using ePlex instrument for the simultaneous qualitative detection and identification of multiple respiratory viral and bacterial nucleic acid including SARS-CoV-2. (https://www.fda.gov/media/142905/download)

CDC 2019-Novel Coronavirus (2019-nCoV) Real-Time RT-PCR Diagnostic Panel - with MagNA Pure 96 (Roche, Indianapolis-Marion County, Indiana) extraction and real-time RT-PCR performed on 7500DX. (https://www.fda.gov/media/134922/download)

Results and discussion

Results from all testing platforms, including original data provided by the submitters, are summarized in Tables 1, 2, and 3. Except for the GenMark ePlex, all other diagnostic platforms generate Ct values. For most samples, Wadsworth testing produced similar Ct results across the platforms. However, due to the variable testing methods used by original submitters, initial testing data did not always correlate with those generated during this study.

Results obtained during testing of specimen PS1 of variant B.1.526.1 on GenMark and Cepheid Platforms suggest the viral load could be very close to the limit of detection. However, the submitter's results for this specimen are comparable to Wadsworth's results with the NeuMoDx and CDC methods. Whether there was a matrix-related mechanical issue with this specimen passing through the cartridge assays is unknown. The fact that both GenMark and Cepheid are sample to answer platforms could be an explanation for this irregularity. Whatever the cause, it is not a universal occurrence for this variant since other specimens containing the variant did not show this effect.

In Table 3, variant B.1.243, specimen PS 2; the submitter's result was generated from the ABBOTT Real Time Sars-Cov-2 Assay in combination with the Real Time M2000 system. Testing on this instrument records CN values after the first 10 cycles, and therefore the CN count is always much lower than the Ct values produced by other real-time assays [4]. Notably, the Ct values produced by the NeuMoDx, Cepheid-Xpert and CDC methods for this specimen are comparable to each other. Most importantly, all the variants tested were detected on all platforms.

Alignments of Omicron sequences from various countries, with the primer and probe sequences in the CDC 2019-Novel Coronavirus (2019-nCoV) Real-Time RT-PCR Diagnostic Panel, show a mismatch at the 5’ end of the N1 probe at position 28,274 for 93% of the sequences (see supplemental figure). However, all six Omicron samples tested in this study contained this mutation and all were detected, even the sample with a low viral load (PS3). An analysis of the N gene mutations in Omicron variants BA.2 and BA.3 did not indicate any potential for adverse impact by any of them on the performance of the four assays in this study (data not shown). However, at this time we did not have any samples of those viruses available for empirical testing.

Currently, all variants previously in the VOI classification have been re-classified as VBM. Also, no SARS-CoV-2 variants have emerged that have been assigned VHC. Since such a variant has the potential for markedly reduced effectiveness of medical countermeasures and prevention measures, it is our hope that such a variant does not emerge. Nevertheless, with the ongoing evolution of SARS-CoV-2 and emergence of new variants, it is important to continuously monitor all circulating viruses to optimize clinical treatment and public health mitigation measures and intervention protocols.

Potential challenges to SARS-CoV-2 variant detection include changes in the genome, at a site corresponding to a target region of a diagnostic assay, such that it disrupts the detecting reaction. Continual review of circulating SARS-CoV-2 virus sequences is imperative. Thus, assays can be adjusted as necessary for ongoing sensitive detection of emerging variants.

In this study, multiple samples of numerous SARS-CoV-2 variants circulating in NYS were tested against the currently used detection assays at the Wadsworth Center. The few detection irregularities that occurred in this study were not consistent across the variants tested and did not appear to be inherent to any of the variants but localized to something unusual in an individual specimen. From the collective data, all variants tested were successfully detected across all molecular platforms used at this center with a high level of sensitivity. We therefore conclude that all the SARS-CoV-2 variants tested, which are representative of the major recently circulating types in NYS, can be detected by these platforms.

Funding

The sequencing work was partially funded by the New York Community Trust.

Declaration of Competing Interest

The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.