Microbiology & parasitology
Microbiology - viruses
Diagnostic testing of SARS-CoV-2 (COVID-19)


Topic Completed: 8 May 2020

Minor changes: 7 August 2020

Copyright: 2020, PathologyOutlines.com, Inc.

PubMed Search: SARS-CoV-2 [title] diagnostic testing

See Also: Lung pathology topic

Samantha C. Wu, B.A.
Patricia Tsang, M.D., M.B.A.
Page views in 2020 to date: 1,149
Cite this page: Wu SC, Tsang P. COVID-19 (SARS-CoV-2) testing. PathologyOutlines.com website. https://www.pathologyoutlines.com/topic/microbiologyvirusesdiagtestSARSCOV2.html. Accessed August 8th, 2020.
Definition / general
Clinical features
  • Symptoms mostly mild to moderate but can be severe or fatal, especially among the elderly and those with underlying illnesses
  • Typical signs and symptoms: fever, cough and dyspnea
  • Other symptoms include chills, muscle pain, sore throat and loss of taste or smell
  • Incubation period: 2 - 14 days (Cureus 2020;12:e7560)
  • Children of all ages are at risk for COVID-19 infection but complications generally appear to be less common than in adults (CDC: Coronavirus Disease 2019 - Information for Pediatric Healthcare Providers [Accessed 19 May 2020])
    • Symptoms may be similar to those of common viral respiratory infections, requiring appropriate suspicion for COVID-19 as well as consideration for other infectious etiologies
    • A serious multisystem inflammatory syndrome in children (MIS-C) has been reported (up to 21 years of age)
Essential features of laboratory testing
Testing priorities
  • Guidelines for patient testing established by the U.S. Centers for Disease Control (CDC)
    • Previously considered “nonpriority”, some asymptomatic individuals are now considered “priority” for testing due to the possibility of asymptomatic infection and viral shedding that can lead to disease transmission
    • Guidance updated on May 3, 2020 to test certain asymptomatic individuals as a part of public health surveillance or a mitigation strategy in long term care facilities and other settings (CDC: Evaluating and Testing Persons for Coronavirus Disease 2019 (COVID-19) [Accessed 19 May 2020])
    • Broader testing guidelines reflect the need for more aggressive infection control measures as well as the increased availability of commercial assays, reagents and supplies
  • High priority:
    • Hospitalized patients with symptoms
    • Healthcare facility workers, workers in congregate living settings and first responders with symptoms
    • Residents in long term care facilities or other congregate living settings, including prisons and shelters, with symptoms
  • Priority:
    • Persons with symptoms of potential COVID-19 infection including fever, cough, shortness of breath, chills, muscle pain, new loss of taste or smell, vomiting or diarrhea or sore throat
    • Persons without symptoms who are prioritized by health departments or clinicians, for any reason, including but not limited to public health monitoring, sentinel surveillance or screening of other asymptomatic individuals according to state and local plans
  • Special considerations for healthcare personnel:
    • Testing may be considered if there has been exposure to a person with suspected (not yet confirmed) COVID-19
    • Even mild signs and symptoms (e.g., sore throat) of COVID-19 should be evaluated in healthcare personnel who may be potentially exposed, in order to protect vulnerable patients they may come in contact with
  • Special considerations for children and young adults with MIS-C
    • May represent a post-viral syndrome or cytokine storm caused by body’s immune response
    • Incubation period may be up to 4 weeks (longer than that seen in adults with respiratory symptoms)
    • Serology testing more likely positive than RT-PCR for SARS-CoV-2 in one small cohort of 10 Italian children (Lancet 2020 May 13 [Epub ahead of print])
  • Special considerations for neonates (CDC: Evaluation and Management Considerations for Neonates At Risk for COVID-19 [Accessed 28 May 2020])
    • Testing recommended for all neonates born to mothers with confirmed or suspected COVID-19, whether or not there are any signs and symptoms in the neonate or the mother
    • RNA testing by RT-PCR on nasopharyngeal, oropharyngeal or nasal swab samples
Evolution of diagnostic testing
Technology platforms
  • Current commercial in vitro diagnostic assays typically feature:
    • Qualitative rRT-PCR for amplification of viral RNA in respiratory samples
    • Dual or triple amplification targets may include genetic sequences from nucleocapsid (N) gene, envelope (E) gene, spike (S) protein or ORF1ab
    • First commercial molecular assay to receive emergency use authorization: Roche cobas (March 12, 2020)
  • Rapid point of care assay by Abbott Diagnostics granted emergency use authorization on March 27, 2020 based on isothermal nucleic acid amplification
  • Serology assay, typically either a rapid diagnostic test (lateral flow assay) or enzyme linked immunoassay (ELISA) to detect IgM and IgG antibody immune response in the blood
FDA's list of serological assays that should not be distributed
  • U.S. FDA recommends health care providers to "be aware that not all marketed serological tests have been evaluated by the FDA"
  • 53 EUA serological assays have been removed from the FDA website notification list and should no longer be used for COVID-19 testing (FDA: What Tests Should No Longer Be Distributed for COVID-19? [Accessed 29 June 2020])
  • An independent study conducted by a federally funded national laboratory has found that 15 of 20 commercial SARS-CoV-2 antibody assays should not be marketed (FDA: Independent Evaluations of COVID-19 Serological Tests [Accessed 29 June 2020])
    • Study assays were each compared against 110 frozen serum samples with known IgM and IgG status (30 positive and 80 negative)
    • Performance characteristics were evaluated, including sensitivity, specificity, positive predictive value and negative predictive value (assumed prevalence of 5%)
    • EUA authorization was granted for 5 serological assays based on this study:
      • Biohit Healthcare (Hefei) Co., Ltd. SARS-CoV-2 IgM/IgG Antibody Test Kit
      • Euroimmun SARS-COV-2 ELISA (IgG)
      • Hangzhou Biotest Biotech, Co., Ltd. Covid-19 IgG/IgM Rapid Test Cassette
      • Hangzhou Laihe Biotech Co., Ltd. Novel Coronavirus (2019-nCoV) IgM/IgG Antibody Combo Test Kit (Colloidal Gold)
      • Healgen COVID-19 IgG/IgM Rapid Test Cassette
    • 15 serological test kit manufacturers received “should not be distributed” status or market withdrawal notice based on unacceptable assay performance:
      • Abacus Pharma International; Accudiagnostics; Atlas-Link (Beijing); Aurora Biomed Inc.; Biomedomics; ChemBio; Chemtron Biotech, Inc.; GP Getein Biotech, Inc.; Phamatech; SD BIOSENSOR, Inc.; Shanghai Fosun Long March Medical Science Co., Ltd.; TESTSEALABS; Tianjin Beroni Biotechnology Co., Ltd.; W.H.P.M, Inc.; Zhongshan Bio-Tech Co LTD
ICD, CPT and Medicare rate
  • The first ICD-10-CM diagnostic code listed below can be used for positive COVID-19 laboratory test results, while the last three below for negative or unknown test results:
    • U07.1: COVID-19 with positive or presumptive -positive test results
    • Z11.59: asymptomatic, no known exposure, results unknown or negative
    • Z03.818: possible exposure to COVID-19, infection ruled out
    • Z20.828: contact with COVID-19, suspected exposure
  • The following CPT billing codes and their corresponding Medicare reimbursement rates can be used for SARS-CoV-2 laboratory testing (CMS: Medicare Administrative Contractor (MAC) COVID-19 Test Pricing [Accessed 7 August 2020], ACOG: Coding for COVID-19 Testing [Accessed 7 August 2020]):

    CPT code Medicare rate Description
    86328 $45.23 Immunoassay for SARS-CoV-2 (COVID-19) antibody(ies), qualitative or semiquantitative, single step method (e.g. reagent strip used in the point-of-care setting)
    86769 $42.13 Immunoassay for SARS-CoV-2 (COVID-19) antibody(ies); qualitative or semiquantitative; multiple step method
    87635 $51.31 Infectious agent detection by nucleic acid (DNA or RNA); SARS-CoV-2 (COVID-19), amplified probe technique
    87426 NA Antigen detection by immunoassay, (e.g., enzyme immunoassay [EIA], enzyme linked immunosorbent assay [ELISA], immunochemiluminometric assay [IMCA]) qualitative or semiquantitative, multiple step method; SARS-CoV or SARS-CoV-2
    U0001 $35.91 2019 novel coronavirus real time RT-PCR diagnostic test panel at a CDC lab
    U0002 $51.31 2019-nCoV coronavirus, SARS-CoV-2/2019-nCoV (COVID-19) using any technique, multiple types of subtypes at a non-CDC lab
    U0003 $100 Infectious agent detection by nucleic acid (DNA or RNA); SARS-CoV-2 (COVID-19), amplified probe technique, making use of high throughput technologies as described by CMS-2020-01-R
    U0004 $100 2019-nCoV Coronavirus, SARS-CoV-2/2019-nCoV (COVID-19), any technique, multiple types or subtypes (includes all targets), non-CDC, making use of high throughput technologies as described by CMS-2020-01-R
Diagrams / tables
  • Below is a partial list of commercially available SARS-CoV-2 assays in the U.S. and a comparison of the key features based on manufacturers’ claims gleaned from package inserts and news releases (FDA: Emergency Use Authorizations - In Vitro Diagnostics EUAs [Accessed 24 April 2020])
  • Independent studies are needed to verify the comparative performances of these assays; due to the rapid rollout of these emergency use authorization assays, peer reviewed publications will take time to catch up
  • The analytical sensitivities (limits of detection) vary widely among assays

Table 1. Performance Characteristics of Some EUA SARS-CoV-2 Assays
Manufacturer In Vitro Diagnostic EUAs Technology Claimed LoD Sensitivity Specificity
Abbott Abbott RealTime SARS-CoV-2 Assay rRT-PCR 100 copies/mL 100% at 1-2x LoD 100%
Abbott ID NOW COVID-19 Test Isothermal nucleic acid amplification 125 copies/mL 100% at 2-5x LoD 100%
Becton Dickonson BD SARS-CoV-2 Reagents rRT-PCR 40 copies/mL 95% at 1-2x LoD, 100% at 3-5x LoD 100%
Becton Dickonson BioGX SARS-CoV-2 Reagents rRT-PCR 40 copies/mL 95% at 1-2x LoD, 100% at 3-5x LoD 100%
BGI Genomics Real-Time Fluorescent RT-PCR Kit for Detecting SARS-2019-nCoV rRT-PCR Throat: 150 copies/mL; BALF: 100 copies/mL Throat: 95% at 1x LoD; BALF: 100% at 1x LoD 100%
*Bio-Rad Platelia SARS-CoV-2 Total Ab assay ELISA (IgM, IgA, IgG) 100% serum (n=27), 83% plasma (n=24) 99.6%
BioFire BioFire COVID-19 Test rRT-PCR 330 copies/mL 100% at 1x LoD 100%
*Cellex qSARS-CoV-2 IgG/IgM Rapid Test Lateral flow immunoassay (IgM, IgG) 94% (n=128, no symptoms to severe) 95.6%
Cepheid Xpert Xpress SARS-CoV-2 rRT-PCR 250 copies/mL 100% at 2x LoD 100%
Co-Diagnostics Logix Smart Coronavirus Disease 2019 (COVID-19) Kit rRT-PCR 4290 copies/mL of sputum 100% at 1x LoD 100%
DiaCarta QuantiVirus SARS-CoV-2 Test Kit rRT-PCR ABI QuantStudio 5: 200 copies/mL; ABI 7500 Fast Dx: 100 copies/mL ABI QuantStudio: 95% at 1x LoD; ABI 7500 Fast Dx: 100% at 1x LoD 100%
DiaSorin Simplexa COVID-19 Direct Assay rRT-PCR 500 copies/mL 100% at 1x LoD 100%
GenMark ePlex SARS-CoV-2 Test RT-PCR, electrochemical detection 1 x 105 copies/mL 94.4% at 1x LoD 100%
Gnomegen Gnomegen COVID-19 RT-Digital PCR Detection Kit rRT-PCR, digital, nanofluidic chip 60 copies/mL 100% at 1-2x LoD 100%
Hologic Panther Fusion SARS-CoV-2 Assay rRT-PCR 0.01 TCID50/mL 100% at 1-5x LoD 100%
InBios Smart Detect SARS-CoV-2 rRT-PCR Kit rRT-PCR 7500 Fast Dx: 1100 copies/mL; CFX96 Touch: 860 copies/mL 100% at 1x LoD 100%
Ipsum COV-19 IDx Assay rRT-PCR 8.5 x 103 copies/mL 100% at 1x LoD 100%
Luminex NxTAG CoV Extended Panel Assay rRT-PCR 5.0 x 103 copies/mL 95% at 2x LoD 100%
Luminex ARIES SARS-CoV-2 Assay rRT-PCR 7.5 x 104 copies/mL 100% at 1x LoD 100%
Mesa Biotech Accula SARS-CoV-2 rRT-PCR, lateral flow 200 copies/60uL reaction (pooled nasal & throat) 100% at 2-50x LoD 100%
NeuMoDx NeuMoDx SARS-CoV-2 Assay rRT-PCR 150 copies/mL 100% at 1.5x LoD 100%
*Ortho Vitros Anti-SARS-CoV-2 Total Reagent Pack and Total Calibrator Immunometric luminescent reaction (IgG) 92.3% at 1-5 days, 88.9% at 6-15 days, 75% at 16-22 days post-PCR 100%
PerkinElmer PerkinElmer New Coronavirus Nucleic Acid Detection Kit rRT-PCR ORF1ab: 8.3 copies/mL; N: 27.2 copies/mL ORF1ab: 100% at 1.5x LoD; N: 100% at 1.5x LoD 100%
Primerdesign Primerdesign Ltd COVID-19 genesig Real-Time PCR assay rRT-PCR 330 copies/mL 94.7% at 1-2x LoD, 100% at 3-5x LoD 100%
QIAGEN QIAstat-Dx Respiratory SARS-CoV-2 Panel (22 bacterial & viruses) rRT-PCR 500 copies/mL 100% at 1-2x LoD 100%
Quidel Lyra SARS-CoV-2 Assay rRT-PCR 800 copies/mL 100% at 1x LoD 100%
Roche cobas SARS-CoV-2 Assay rRT-PCR 0.007 TCID50/mL 100% at 1.5x LoD 100%
*Roche Elecsys Anti-SARS-CoV-2 Sandwich immunoassay (antibodies) 65.5% at ≤ 6 days, 88.1% at 7-13 days, 100% ≥ 14 days post-PCR 99.8%
Thermo Fisher TaqPath COVID-19 Combo Kit rRT-PCR 10 copies/140uL 100% at 1x LoD 100%
* Serology assay
Abbreviations: LoD = limit of detection; rRT-PCR = real time reverse transcriptase polymerase chain reaction


Table 2. Categories of SARS-CoV-2 EUA Assays and Examples
Category In Vitro Diagnostic EUAs Instruments Throughput Cycle Time
Point of Care Setting
Abbott ID NOW COVID-19 Test ID NOW Nonbatch 5 min (+), 13 min (-)
Cepheid Xpert Xpress SARS-CoV-2 GeneXpert Xpress System Nonbatch modular 45 min per cartridge
Mesa Biotech Accula SARS-CoV-2 Accula/Silaris Dock Nonbatch cassette 30 min
Respiratory Pathogen Panel
Qiagen QIAstat-Dx Respiratory SARS-CoV-2 Panel (22 bacterial & viruses) QIAstat Dx Analyzer System Nonbatch modular About 1 hour per cartridge
Serology
Bio-Rad Platelia SARS-CoV-2 Total Ab None 96 wells per microplate (manual) 1st incubation: 60 min 2nd incubation: 30 min
Cellex qSARS-CoV-2 IgG/IgM Rapid Test None Nonbatch cassette 15-20 min per cassette
Ortho VITROS Immunodiagnostic Anti-SARS-CoV-2 Total Reagent and Calibrator VITROS Eci/EDiQ/3600 or VITROS 5600/XT 7600 Automated 37 min incubation plus 48 min testing
Roche Elecsys Anti-SARS-CoV-2 cobas e411, e601, e602, e801 Automated 18 min
Modular Cartridge / Cassette / Pouch
BioFire COVID-19 Test FilmArray 2.0 or Torch Nonbatch modular 50 min per pouch
Cepheid Xpert Xpress SARS-CoV-2 GeneXpert Dx / Infinity Nonbatch modular 45 min per cartridge
GenMark ePlex SARS-CoV-2 Test ePlex Nonbatch modular cartridge
Luminex ARIES SARS-CoV-2 Assay ARIES System Nonbatch cassette 2 hours per cassette
Qiagen QIAstat-Dx Respiratory SARS-CoV-2 Panel (22 bacterial & viruses) QIAstat Dx Analyzer System Nonbatch modular About 1 hour per cartridge
High Throughput Capability
Abbott RealTime SARS-CoV-2 Assay m2000 RealTime 96 wells per batch
BGI Real-Time Fluorescent RT-PCR Kit for Detecting SARS-2019-nCoV ABI 7500 Real Time PCR 96 wells per batch
DiaCarta QuantiVirus SARS-CoV-2 Test Kit ABI QuantStudio 5 / 7500 Fast Dx Real-Time PCR Batch
DiaSorin Simplexa COVID-19 Direct Assay LIAISON MDX Batch
Hologic Panther Fusion SARS-CoV-2 Assay Panther Fusion 1150 samples per 24 hours < 3 hours
InBios Smart Detect SARS-CoV-2 rRT-PCR Kit ABI 7500 Fast Dx Real-Time PCR / CFX96 Touch Real-Time PCR 96 wells per batch
Luminex NxTAG CoV Extended Panel Assay Luminex MAGPIX & bioMerieux easyMAG / EMAG 96 wells per batch < 2.5 hours thermal cycler run time
NeuMoDx SARS-CoV-2 Assay NeuMoDx 96 and 288 Molecular Systems Random access, modular
Ortho VITROS Immunodiagnostic Anti-SARS-CoV-2 Total Reagent and Calibrator VITROS Eci/EDiQ/3600 or VITROS 5600/XT 7600 100 wells per reagent pack 37 min incubation plus 48 min testing
PerkinElmer New Coronavirus Nucleic Acid Detection Kit ABI 7500 Real-Time PCR 96 wells per batch
Primerdesign Ltd COVID-19 genesig Real-Time PCR Assay ABI 7500 / Bio-Rad CFX Connect / Roche LightCycler 4800 II Batch
Quidel Lyra SARS-CoV-2 Assay ABI 7500 Fast Dx/Standard, Roche LightCycler 480, Qiagen Rotor-Gene Q, BioRad CFX96 Touch, Thermofisher Quantstudio 7 Pro Batch
Roche cobas SARS-CoV-2 Assay cobas 6800/8800 384/960 per 8 hours 3.5 hours
Thermal Fisher TaqPath COVID-19 Combo Kit Applied Biosystems 7500 Fast Dx 96 wells per batch 4 hours (incl. sample prep)
Board review style question #1
Which of the following is true regarding serology (nonmolecular) testing for SARS-CoV-2?

  1. Dual or triple genetic sequences are targeted to increase the performance of the assay
  2. It is typically based on reverse transcription followed by nucleic acid amplification
  3. Elevated IgA antibody to COVID-19 indicates suitability for convalescent plasma donation
  4. Suitable respiratory specimens include the nasopharyngeal swab and bronchial lavage
  5. The lack of an antibody response does not rule out acute infection
Board review answer #1
E. The lack of an antibody response does not rule out acute infection

Comment Here

Reference: Diagnostic testing of SARS-CoV-2
Board review style question #2
Which of the following groups of individuals is considered a top priority for COVID-19 testing according to the CDC?

  1. Children with earache
  2. Pre-employment screening for the retail industry
  3. Symptomatic healthcare workers and hospitalized patients
  4. Asymptomatic transportation personnel with contact exposure to a person with COVID-19
Board review answer #2
C. Symptomatic healthcare workers and hospitalized patients are a top priority for COVID-19 testing

Comment Here

Reference: Diagnostic testing of SARS-CoV-2
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