Test Id : COVNG

This test should only be requested on known SARS-CoV-2 RNA-positive upper or lower respiratory tract specimens, with polymerase chain reaction target cycle threshold value of to 30.0 or less or transcription-mediated amplification generated relative light units of 1200 or more.

This test should not be used to detect the presence or absence of SARS-CoV-2 in an individual, with or without symptoms or signs of COVID-19. For these cases, order COVOO / Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) RNA Detection, Varies.

Ship specimens refrigerated (if <72 hours from collection to arrive at Mayo Clinic Laboratories [MCL]) or frozen (if 72 hours or more from collection to arrive at MCL)

The following question must be answered at the time of test ordering:

Does the patient have a positive SARS-CoV-2, COVID19 polymerase chain reaction test result within the last 5 days? Answer "Yes" or "No".

Note: Test orders for submitted specimens with a "No" answer to this question will be canceled.

Call 800-533-1710 to have this test added to a previously collected specimen that tested positive for SARS-CoV-2, COVID19 with COVOO, COVID, or COFLU. A new specimen would not be needed if there is sufficient specimen volume remaining.

Specimen Type: Nasopharyngeal (NP), oropharyngeal (OP ie, throat), nasal mid-turbinate, or nares/nasal swab

Supplies: Swab, Sterile Polyester(T507)

Collection Container/Tube:

Preferred: Sterile polyester swab

Acceptable: Dacron-tipped swab with plastic shaft

Submission Container/Tube: Universal transport media, viral transport media, or equivalent (eg, Copan UTM-RT, BD VTM, MicroTest M4, M4-RT, M5). Media should not contain guanidine thiocyanate (GTC).

For more information on acceptable transport media, see www.fda.gov/medical-devices/emergency-situations-medical-devices/faqs-diagnostic-testing-sars-cov-2

Specimen Volume: Entire specimen with a minimum of 1.5 mL (maximum 3 mL) of transport media

Collection Instructions:

1. Collect specimen by swabbing back and forth over nasal or pharyngeal mucosa surface to maximize recovery of cells.

2. NP and OP swab specimens may be combined at collection into a single vial of transport media but only 1 swab is required for analysis.

3. Swab must be placed into transport medium. Swab shaft should be broken or cut so that there is no obstruction to the sample or pressure on the media container cap.

4. Do not send in glass tubes, vacutainer tubes, or tubes with push caps.

5. Do not overfill with more than 3 mL total volume of media.

Specimen Type: Nasopharyngeal aspirate or nasal washings

Container/Tube: Sterile container

Specimen Volume: Minimum of 1.5 mL

Additional Information: Do not aliquot into viral transport media, glass tubes, vacutainer tubes, or tubes with push caps.

Specimen Type: Nasopharyngeal aspirate or nasal washings, bronchoalveolar lavage (BAL) fluid, bronchial washings, endotracheal aspirate, sputum

Container/Tube: Sterile container

Specimen Volume: Minimum of 1.5 mL

Additional Information: Do not aliquot into viral transport media, glass tubes, vacutainer tubes, or tubes with push caps.

See Specimen Required

Distinguishing between persistent infection with the same viral strain and reinfection with a new viral strain in an individual with recurrent positive molecular test results for SARS-CoV-2

Detection and identification of vaccine-escape SARS-CoV-2 variants with spike (S) gene variant of interest

Detection and identification of SARS-CoV-2 variants containing S gene variants of interest that reduce the efficacy of vaccine-induced antibodies, convalescent plasma, and/or monoclonal antibody therapy for COVID-19

Detection and identification of SARS-CoV-2 variants containing RdRp codon mutations of interest that reduce the efficacy of nucleoside analogs used in the therapy of COVID-19

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is a novel, positive-sense, single-stranded RNA virus that causes COVID-19, and it contains an approximately 30,000 base pair long RNA genome that is prone to spontaneous genetic mutation. Worldwide scientific reports indicated emergence of specific variants of SARS-CoV-2 that are associated with increased transmissibility of this virus among susceptible humans, increased severity of disease, and/or reduced neutralization by vaccine-induced antibodies, therapeutic monoclonal antibodies, and convalescent plasma since December 2020.

A group of viruses within the same genus sharing the same distinctive set of mutations in the viral genome is called a variant. If enough mutations accumulate in a lineage, the viruses may evolve clear-cut differences in how they function. These lineages come to be known as strains. The United States SARS-CoV-2 inter-agency group, comprising the Department of Health and Human Services, Biomedical Advances Research and Development Authority, Centers for Disease Control and Prevention, Food and Drug Administration, National Institutes of Health, and Department of Defense, has developed a variant classification scheme that defines 3 classes of SARS-CoV-2 variants: variant of interest, variant of concern, and variant of high consequence (www.cdc.gov/coronavirus/2019-ncov/variants/variant-surveillance.html). The relative proportions of these variants present among the reported COVID-19 infections at the US national and state levels are available at www.cdc.gov/coronavirus/2019-ncov/cases-updates/variant-proportions.html.

A SARS-CoV-2 variant of interest contains specific genetic mutations that are associated with predicted increase in transmissibility or disease severity, possible impact on serologic and/or molecular diagnostic assays, and changes to the ACE2 receptor binding domain that may result in reduced neutralization by antibodies generated from previous infection or vaccination and reduced efficacy of monoclonal antibody therapy. Current SARS-CoV-2 variants of interest in the US are the B.1.525, B.1.526, and P.2 lineages, all of which share the D614G codon mutation in the S gene of the virus, and this mutation is associated with increased transmission of this virus.

A SARS-CoV-2 variant of concern contains specific genetic mutations that are associated with increase in transmissibility, severe disease (increased hospitalization or death), failures of serologic and/or molecular diagnostic assays, and significant reduction in neutralization by antibodies generated from previous infection or vaccination, and reduced efficacy of monoclonal antibody therapy or vaccines. Current SARS-CoV-2 variants of interest in the US are the B.1.1.7, B.1.351, B.1.427, B.1.429, and P.1 lineages.

A SARS-CoV-2 variant of high consequence has clear evidence of significantly reduced effectiveness of current preventive measures, therapeutic agents, and medical interventions, when compared to previously circulating variants. At present, there are no such variants in US or globally.

Not applicable

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2)-specific S gene mutations are detected with this assay at a minimum 50% frequency for detecting codon substitutions in the viral sequence.

An "Inconclusive" result indicates that testing failed due to poor sequence quality resulting from the presence of inhibitory substances and/or low amount of virus (ie, polymerase chain reaction [PCR] target cycle threshold [Ct] value of >30.0) present in the submitted specimen. A new specimen should be collected and submitted for retesting if clinically necessary.

The table below indicates the clinical implications of known SARS-CoV-2 variants of interest and variants of concern:

a. Based on in vitro (experimental) data only

b. Reduced efficacy of bamlanivimab, etesevimab, and casirivimab

c. All current Pango lineage designations that start with AY and BA are sublineages of the Delta and Omicron variants, respectively

d. Reduced efficacy of bamlanivimab

e. Reduced efficacy of bamlanivimab and casirivimab

Current antiviral drugs, such as remdesivir, that are used to treat COVID-19 are nucleoside analogs that inhibit viral RNA-dependent polymerase (RdRp) of coronaviruses (including SARS-CoV-2) and prevent viral replication. Genotypic mutations occurring alone or in combination in the RdRp-encoding region of the SARS-CoV-2 nsp12 gene have been reported to be associated with resistance to these antiviral drugs. The RdRp codon mutations associated with such antiviral resistance are: F480L, D484Y, and V557L.

The table below indicates the clinical implications of known codon-substitutions in the SARS-CoV-2 spike protein (S) encoding region:

*Based on in vitro (experimental) data only.

**This dual codon mutation also causes failure of certain molecular detection assays.

The ability to amplify the SARS-CoV-2 target sequences and detect S gene mutations with this assay is limited in specimens with viral polymerase chain reaction target cycle threshold values of greater than 30.0 (ca. <50,000 copies/mL).

Viral variants present at less than 50% of the total viral population in a given clinical specimen will not be detected with this assay, as the nucleic acid substitution detection threshold is set at 50%.

1. Lauring AS, Hodcroft EB: Genetic variants of SARS-CoV-2-what do they mean? JAMA. 2021 Feb 9;325(6):529-531. doi: 10.1001/jama.2020.27124

2. Walensky RP, Walke HT, Fauci AS: SARS-CoV-2 variants of concern in the United States-challenges and opportunities. JAMA. 2021 Mar 16;325(11):1037-1038. doi: 10.1001/jama.2021.2294

3. Centers for Disease Control and Prevention: SARS-CoV-2 variant classifications and definitions. Available at www.cdc.gov/coronavirus/2019-ncov/cases-updates/variant-surveillance/variant-info.html

4. World Health Organization: Weekly epidemiological update on COVID-19 - 8 June 2021. Available at www.who.int/publications/m/item/weekly-epidemiological-update-on-covid-19---8-june-2021

5. Scripps Research: SARS-CoV-2 (hCoV-19) Mutation Situation Reports. Available at https://outbreak.info/situation-reports

6. European Centre for Disease Prevention and Control. SARS-CoV-2 variants of concern. ECDC; June 24, 2021. Accessed June 28, 2021. Available at www.ecdc.europa.eu/en/covid-19/variants-concern

This test utilizes the commercially available Ion AmpliSeq SARS-CoV-2 Research Panel, a next-generation sequencing assay based on a sequencing by synthesis method. The assay amplifies 237 sequences ranging from 125 to 275 base pairs in length covering 99% of the SARS-CoV-2 genome. Clinical respiratory tract specimens undergo manual total nucleic acid extraction and purification using MagMAX Viral / Pathogen Nucleic Acid Isolation Kit. The eluate undergoes automated reverse-transcription-polymerase chain reaction of viral sequences, DNA library preparation (including enzymatic shearing, adapter ligation, purification, normalization), DNA template preparation, and sequencing on the automated Genexus integrated sequencer. No-template control and a positive SARS-CoV-2 control are included in each assay run for quality control purposes. Viral sequence data are assembled using the Iterative Refinement Meta-Assembler (IRMA) application (50% base substitution frequency threshold) to generate unamended plurality consensus sequences of SARS-CoV-2 for analysis with the latest versions of the web-based application tools: https://pangolin.cog-uk.io/ for SARS-CoV-2 lineage assignment; https://clades.nextstrain.org/ for viral clade assignment and phylogenetic analysis; https://covdb.stanford.edu/sierra/sars2/by-sequences/ for codon mutation calling, in comparison to wild-type reference sequence of Wuhan-Hu-1, lineage B, clade 19A.(Package insert: Ion AmpliSeq SARS-CoV-2 Research Panel. Life Technologies Corporation; rev. B.0, publ. no. MAN0019278, 10/08/2020)

Varies

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This test was developed and its performance characteristics determined by Mayo Clinic in a manner consistent with CLIA requirements. It has not been cleared or approved by the US Food and Drug Administration.

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