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| ====3.1.6 Variant testing==== | | ====3.1.7 Multiplex testing==== |
| As the pandemic has progressed, you may have heard talk of a "delta" variant of SARS-CoV-2, which is reportedly more contagious and virulent than the initial strain that kicked off the pandemic.<ref name="CDCDelta21">{{cite web |url=https://www.cdc.gov/coronavirus/2019-ncov/variants/delta-variant.html |title=Delta Variant: What We Know About the Science |author=Centers for Disease Control and Prevention |publisher=Centers for Disease Control and Prevention |date=26 August 2021 |accessdate=18 September 2021}}</ref> One or more variants of a virus are expected as time progresses, and some of those variants can cause significantly more problems than the source virus. As such, analytical testing of the virus over time is vital to public health. | | As the pandemic has progressed and test manufacturers have become more experienced with SARS-CoV-2 test development, multiplex testing has become an option. The multiplex assay—an immunoassay test able to measure multiple analytes in a single test—is certainly not new in itself. In 1989, R. Ekins developed the ambient analyte theory, which stated that miniaturizing an immunoassay can lead to an improved limit of detection (LOD). That work influenced the future development of microarray multiplex technology principles.<ref name="TigheELISA15">{{Cite journal |last=Tighe |first=Patrick J. |last2=Ryder |first2=Richard R. |last3=Todd |first3=Ian |last4=Fairclough |first4=Lucy C. |date=2015-04 |title=ELISA in the multiplex era: Potentials and pitfalls |url=https://onlinelibrary.wiley.com/doi/10.1002/prca.201400130 |journal=PROTEOMICS – Clinical Applications |language=en |volume=9 |issue=3-4 |pages=406–422 |doi=10.1002/prca.201400130 |issn=1862-8346 |pmc=PMC6680274 |pmid=25644123}}</ref> As of September 2021, eighteen ''in vitro'' molecular diagnostic tests are shown as receiving EUAs by the FDA, four of them even authorized for CLIA waived testing. |
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| The purpose of variant testing can be described in two ways, one for public health reasons and another for clinical care reasons. On the public health side, analysis of SARS-CoV-2 variants provides an unbiased, population-level view "of the specific viral strains in circulation and monitors changes in the viral genome over time."<ref name="BuchanSARS21">{{cite web |url=https://www.amp.org/AMP/assets/File/clinical-practice/COVID/AMP_RC_VariantTestingforSARSCOV2_4_28_21.pdf |format=PDF |title=SARS-CoV-2 Variant Testing |work=Rapid Communication |author=Buchan, B.W.; Wolk, D.M.; Yao, J.D. |publisher=Association for Molecular Pathology |date=28 April 2021 |accessdate=18 September 2021}}</ref> With enough public health laboratories conducting this type of analysis—typically whole-genome sequencing (WGS) using [[next-generation sequencing]] (NGS) techniques—a clearer picture of how an outbreak spreads is gained, as well as what variants are taking hold and further threatening human populations (even those that are vaccinated). This information is typically shared through the public health system for surveillance and reporting purposes, though the affected patients themselves may never see the data.<ref name="BuchanSARS21" />
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| On the clinical care side, analysis of SARS-CoV-2 variants provides further insights into improving COVID-19 patient outcomes. Buchan ''et al.'' identify three potential insights that clinicians may gain, noting that variant testing allows the clinician<ref name="BuchanSARS21" />:
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| * to distinguish between an existing, persistent infection caused by one viral strain vs. re-infection by a different viral strain;
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| * to determine whether a patient not responding to a treatment is affected by a specific viral spike protein (S) gene mutation that is "potentially resistant or less susceptible to
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| neutralizing antibodies or monoclonal antibodies"; and
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| * to detect in the serum or plasma of a patient post-vaccination "viral S gene substitutions in specific variants that are potentially resistant or less susceptible" to the antibodies the vaccine generates.
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| If, for example, a patient is diagnosed with a variant that is tied to heightened disease severity, the clinician can opt for additional treatments early on to counteract the variant's effects on the patient. This testing is done in a hospital or reference lab by WGS or by targeting a portion of the genome (e.g., a spike protein) or a specific mutation (using RT-PCR). However, according to Buchan ''et al.'', the contributions a mutation makes to a "variant's attributes is not entirely understood, and there is no definitive evidence that directly links a given mutation to poor outcomes, significantly reduced efficacy of SARS-CoV-2 therapies, or vaccine coverage."<ref name="BuchanSARS21" />
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| That said, and leaving the public health element to the side, if you are a laboratory conducting clinical analyses of SARS-CoV-2 specimens, the likelihood of including viral sequencing and sequence analysis for variant testing may be low for your facility. Such testing is a multi-step process requiring a non-trivial set of resources, often available to large commercial diagnostic laboratories.<ref name="BuchanSARS21" /><ref name="WilliamsEnhanc21">{{cite web |url=https://health.mo.gov/emergencies/ert/alertsadvisories/pdf/update21921.pdf |format=PDF |title=Enhancing Public Health Surveillance for Variant SARSCoV-2 Viruses in Missouri |author=Williams, R.W. |publisher=Missouri Department of Health & Senior Services |date=19 February 2021 |accessdate=18 September 2021}}</ref> The CDC represents one possible workflow for genomic sequencing as such<ref name="CDCRole21">{{cite web |url=https://www.cdc.gov/coronavirus/2019-ncov/variants/cdc-role-surveillance.html |title=CDC’s Role in Tracking Variants |author=Centers for Disease Control and Prevention |publisher=Centers for Disease Control and Prevention |date=08 September 2021 |accessdate=18 September 2021}}</ref>:
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| # A specimen containing the SARS-CoV-2 virus is received by the lab and promptly entered into the [[laboratory information system]] (LIS).
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| # The RNA of the SARS-CoV-2 virus is extracted from the sample and then converted to complimentary DNA. It is then enriched and loaded into the appropriate NGS instrument.
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| # The instrument runs the sequencing and raw data is collected, with the lab maintaining quality control steps. The raw data is turned into actionable sequence data.
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| # The sequence data is verified for suitability, with a resequencing occurring if found to be inadequate. Otherwise, the data is then analyzed and interpreted.
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| If your diagnostic lab has or is planning on adding sequencing tools to supplement clinical diagnostics, it may make sense to consider adding variant testing to your available options. But in reality, this sort of testing may largely be left to large institutions, such as the University of Rochester Vaccine Treatment Evaluation Unit or the Yale School of Public Health.<ref name="DupuyCOVID21">{{cite web |url=https://apnews.com/article/fact-checking-549965482111 |title=COVID-19 variants tested through genome sequencing |author=Dupuy, B. |work=Reuters Fact-Checking |date=28 July 2021 |accessdate=18 September 2021}}</ref>
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