Rapid Diagnostic Testing for SARS-CoV-2 | NEJM – nejm.org

Clinical Course and Diagnostic Testing

Pathophysiology and Timeline of Viremia, Antigenemia, and Immune Response during Acute SARS-CoV-2 Infection.

In some persons, reverse-transcriptase–polymerase-chain-reaction (RT-PCR) tests can remain positive for weeks or months after initial infection with severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), but this positivity rarely indicates replication-competent virus that can result in infection.

The pathophysiology of acute SARS-CoV-2 infection, the clinical course of Covid-19, and the host immunologic response provide a basis for diagnostic testing strategies (Figure 1).10,11 SARS-CoV-2 is predominantly a respiratory airway pathogen, and transmission occurs largely through inhalation of small droplets and aerosols.12 Novel genomic viral variants, including the B.1.617.2 (delta) variant, have higher transmissibility than the original D614G virus, leading to faster dissemination within populations, but they share the same pathophysiology of infection and disease. The WHO recently named the B.1.1.529 (omicron) variant as the sixth “variant of concern,” and available evidence suggests it is more transmissible but less virulent than previous variants.

Symptoms of Covid-19 and Signs or Symptoms of Severe Covid-19.

Symptoms of Covid-19 (Table 1) appear 2 to 14 days after exposure, with an average onset 5 to 6 days after infection.13,14 Most persons with Covid-19 have mild-to-moderate symptoms and recover at home, but some, particularly older or unvaccinated adults and those with underlying medical conditions or immunocompromise, may have serious illness.13 SARS-CoV-2 infection also occurs without causing symptoms or Covid-19, and asymptomatic persons can contribute to viral transmission.15-17 Humoral immunity wanes after initial vaccination,18 but booster immunizations have been shown to reduce the incidence of adverse outcomes.19 Viral load levels and clearance may be similar among vaccinated and unvaccinated adults,20 and adults who have not received a booster immunization have a higher risk of Covid-19–related hospitalization or death than those who have received one.21

Indications and Algorithms for Rapid Diagnostic Tests (RDTs) for SARS-CoV-2.

The Centers for Disease Control and Prevention defines a close contact as a person who was less than 6 feet away for 15 minutes or more over a 24-hour period.13,23 Potential high-risk transmission settings include an airplane, a concert or sporting event, and a crowded or poorly ventilated indoor area.13,22,23 Covid-19 denotes coronavirus disease 2019.

Three common indications for diagnostic SARS-CoV-2 testing, as recommended by the WHO22 and the Centers for Disease Control and Prevention (CDC),23 range from high to low pretest probability of infection (Figure 2). First, anyone with Covid-19 symptoms, regardless of vaccination status, should undergo testing for SARS-CoV-2. Second, asymptomatic persons, regardless of vaccination status, who are close contacts of someone with known or probable SARS-CoV-2 infection should undergo diagnostic testing. Persons who are unvaccinated or who have not received a vaccine booster within the previous 6 months have a higher pretest probability of infection than those who are fully vaccinated, whereas others have a low or moderate pretest probability of infection. Third, testing should be considered in asymptomatic persons who have been in a setting where the risk of transmission is high, such as in an airplane or at a sporting event. Use of an RDT may also be considered in persons who plan to be in a group setting, even though they may have a low pretest probability of infection; this testing should occur as close to the time of the gathering as possible.

Diagnostic testing for acute SARS-CoV-2 infection can be performed with either molecular NAATs or antigen-based assays, and both are available as RDTs.22,23 Molecular NAATs detect the presence of viral gene targets, including the N, S, and E genes and the open reading frame 1ab (ORF 1ab). Reverse-transcriptase–polymerase-chain-reaction (RT-PCR) assays are the most widely used diagnostic SARS-CoV-2 NAATs worldwide.24 Antigen-based tests, also called immunoassays, detect domains of the surface proteins, including the nucleocapsid, spike, and receptor-binding domains, that are specific to SARS-CoV-2. Although both techniques are highly specific, NAATs are generally more sensitive than antigen-based tests because they amplify target genomic sequences. Tests to detect host IgG or IgM antibodies to SARS-CoV-2 should not be used to diagnose acute infection.

The clinical performance of diagnostic SARS-CoV-2 testing extends beyond pathogen targets such as viral proteins and RNA and includes clinical characteristics (e.g., the patient’s viral load and the time since exposure or symptom onset), operational testing attributes (e.g., the specimen type, swab technique, transport conditions, and laboratory technique), and analytic test properties (e.g., sample preparation and signal amplification).7,25 Although NAATs are highly sensitive and accurate, they can remain positive for weeks to months after infection.26,27 Viral culture studies suggest that SARS-CoV-2 may be capable of replicating only for 10 to 14 days after symptom onset, so NAATs may detect remnant viral RNA well past the time period of recovering replication-competent virus.26,27 Conversely, antigen-based assays remain positive for 5 to 12 days after symptom onset and perform better in persons with a high viral load,28 which correlates with disease severity and death.29 Thus, antigen-based tests may correlate better with replication-competent SARS-CoV-2 than molecular tests and may provide information about potential transmissibility.30

RDTs for Acute SARS-CoV-2 Infection

The Food and Drug Administration (FDA) and WHO have each conducted an expedited review process to accelerate the temporary approval of diagnostic SARS-CoV-2 tests.31,32 As of December 2021, the FDA had granted emergency use authorization (EUA) status to 28 RDTs for the diagnosis of acute SARS-CoV-2 infection, and more FDA-authorized tests are expected.31 In the European Union, more than 140 different companies have had an antigen-based RDT registered on the “common list” for approved use.33 The molecular and antigen-based RDTs with EUA status have various pathogen targets, detection methods, swabbing sites to obtain specimens, indications for use, and performance characteristics (see Table S1 in the Supplementary Appendix, available with the full text of this article at NEJM.org).

In order for an RDT to receive temporary approval by the FDA, WHO, and European Union regulatory agencies, it must have at least 80% sensitivity (positive percent agreement) and 98% specificity (negative percent agreement), as compared with a reference standard of laboratory-based RT-PCR testing, although the WHO has allowed for specificity of 97% or greater.22,31,32,34 Approval by the FDA is also based on a prospective cohort study involving at least 30 persons with SARS-CoV-2 infection and 30 persons without SARS-CoV-2 infection.31 An EUA from the European Union is based on performance data that may be obtained either through a prospective clinical study or through retrospective in vitro laboratory testing.33,34 The regulatory agencies require monitoring and reporting of test performance with respect to viral variants, although these requirements have not been well enforced; they do not require independent verification of clinical validation data provided by each test manufacturer.31,32,34

For several molecular RDTs that are intended for use in low-complexity settings, the FDA has issued EUA status with a Clinical Laboratory Improvement Amendments (CLIA) certificate of waiver (which can be obtained by community health centers, nursing homes, schools, churches, and other gathering places for collecting specimens and performing testing). Some of these RDTs are also approved for home-based use.31 These molecular RDTs, which use RT-PCR, loop-mediated isothermal amplification, or nicking enzyme-assisted amplification to detect the viral RNA of SARS-CoV-2, provide results in 13 to 55 minutes. All molecular RDTs are approved for use in symptomatic persons, and a few also have approval for the screening of asymptomatic persons.

Similarly, many antigen-based RDTs have received FDA EUA status for use in settings that have received a CLIA waiver or for home-based use.31 These antigen-based RDTs are immunoassays that use SARS-CoV-2–specific antibodies to bind viral proteins (mostly nucleocapsid) and generate either a visual or fluorescence signal. Most are lateral-flow assays on a nitrocellulose membrane, whereas others involve the use of thin microfluidic test strips, magnetic beads, or an immunofluorescence readout to enhance protein capture and detection.28 All antigen-based RDTs are approved for use in symptomatic persons and provide results in 10 to 30 minutes. Several have EUA status for screening of asymptomatic persons; most of these tests are intended to be used twice over a period of 3 days, although a small number with high sensitivity for detecting asymptomatic infection are approved for use without serial testing.31,35

Although direct-comparison studies are limited and often retrospective, antigen-based RDTs have a lower sensitivity than molecular RDTs, as compared with a reference standard of laboratory-based RT-PCR tests, particularly among persons who have a low viral load or no replication-competent virus.36-38 However, antigen-based RDTs can detect infection early in the disease course (within 5 to 7 days after symptom onset) when viral loads are high (i.e., a low RT-PCR cycle threshold); these high viral loads account for most transmissions.39-41 Studies have shown varying degrees of clinical accuracy (sensitivity, 36 to 82%; specificity, approximately 98 to 100%) when various antigen-based RDTs are used for screening asymptomatic persons.35,42,43

Although home-based RDTs broaden the use of testing, they have been shown to be more accurate when performed by trained health care providers than by untrained persons.44,45 Persons who perform tests at home should carefully follow test kit instructions.

Interpretation of Results of Testing and Screening

The appropriate interpretation of RDTs for SARS-CoV-2 testing and screening depends on the clinical indication and the pretest probability of infection (Figure 2). Among persons with a moderate-to-high pretest probability, which includes symptomatic persons and asymptomatic persons who have had close contact with a person with Covid-19, a positive RDT indicates a confirmed SARS-CoV-2 infection. However, RDTs may have false negative results, and repeat testing should be considered in cases of high clinical suspicion or worsening symptoms and in the serial screening of asymptomatic persons. A second negative RDT 2 days after the initial test or a negative laboratory-based NAAT would help to rule out SARS-CoV-2 infection. All symptomatic persons and asymptomatic persons who have not been fully vaccinated and who have had exposure to an infected contact should quarantine while awaiting test results. Although the standard CDC definition of “full vaccination” has been 2 weeks after the second dose in a two-dose vaccination series, many experts (including this author) propose that the definition should include a booster vaccination in persons who are eligible to receive one.

In persons with a low pretest probability of infection (e.g., asymptomatic persons without a known SARS-CoV-2 exposure), a single negative RDT provides reassurance that SARS-CoV-2 infection is unlikely. However, given imperfect specificity, a positive RDT may indicate a false positive result. If there is low clinical suspicion or a low prevalence of Covid-19 in the population, then repeat testing should be performed. A second positive RDT or positive laboratory-based NAAT would confirm SARS-CoV-2 infection. All asymptomatic persons (vaccinated or unvaccinated) with potential or known exposure should monitor for symptoms for 14 days.

In persons with exposure to SARS-CoV-2, testing is generally not useful in the first 48 hours after exposure, since the virus will not have achieved a sufficient viral load.13 The most appropriate window for testing is generally considered to be 5 to 7 days after exposure, which is the average peak of symptoms and viral load.13 Therefore, for a single-test strategy, asymptomatic, exposed persons could use an RDT 5 to 7 days after exposure. For a two-test strategy, which is the FDA-approved indication for most RDTs for asymptomatic screening, a second RDT should be performed 2 days after a negative test. All symptomatic persons should be tested at the onset of symptoms and, if test results are negative, repeat testing should be considered if clinical suspicion remains high or symptoms worsen.13 In persons with low pretest probability of infection who have a positive RDT, a confirmatory test should be performed promptly.

Routine serial screening strategies with frequent testing have been proposed and implemented to quickly detect SARS-CoV-2 and reduce transmission.46-50 However, when the population prevalence of SARS-CoV-2 is low, the probability of a false positive RDT increases.51,52

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