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May 6, 2020

Interpreting Diagnostic Tests for SARS-CoV-2

Author Affiliations
  • 1Department of Microbiology, Apollo Hospitals, Chennai, India
  • 2Department of Microbiology, Yokohama City University, Yokohama, Japan
JAMA. 2020;323(22):2249-2251. doi:10.1001/jama.2020.8259

The pandemic of coronavirus disease 2019 (COVID-19) continues to affect much of the world. Knowledge of diagnostic tests for severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is still evolving, and a clear understanding of the nature of the tests and interpretation of their findings is important. This Viewpoint describes how to interpret 2 types of diagnostic tests commonly in use for SARS-CoV-2 infections—reverse transcriptase–polymerase chain reaction (RT-PCR) and IgM and IgG enzyme-linked immunosorbent assay (ELISA)—and how the results may vary over time (Figure).

Figure.  Estimated Variation Over Time in Diagnostic Tests for Detection of SARS-CoV-2 Infection Relative to Symptom Onset
Estimated Variation Over Time in Diagnostic Tests for Detection of SARS-CoV-2 Infection Relative to Symptom Onset

Estimated time intervals and rates of viral detection are based on data from several published reports. Because of variability in values among studies, estimated time intervals should be considered approximations and the probability of detection of SARS-CoV-2 infection is presented qualitatively. SARS-CoV-2 indicates severe acute respiratory syndrome coronavirus 2; PCR, polymerase chain reaction.

aDetection only occurs if patients are followed up proactively from the time of exposure.

bMore likely to register a negative than a positive result by PCR of a nasopharyngeal swab.

Detection of Viral RNA by RT-PCR

Thus far, the most commonly used and reliable test for diagnosis of COVID-19 has been the RT-PCR test performed using nasopharyngeal swabs or other upper respiratory tract specimens, including throat swab or, more recently, saliva. A variety of RNA gene targets are used by different manufacturers, with most tests targeting 1 or more of the envelope (env), nucleocapsid (N), spike (S), RNA-dependent RNA polymerase (RdRp), and ORF1 genes. The sensitivities of the tests to individual genes are comparable according to comparison studies except the RdRp-SARSr (Charité) primer probe, which has a slightly lower sensitivity likely due to a mismatch in the reverse primer.1

In most individuals with symptomatic COVID-19 infection, viral RNA in the nasopharyngeal swab as measured by the cycle threshold (Ct) becomes detectable as early as day 1 of symptoms and peaks within the first week of symptom onset. The Ct is the number of replication cycles required to produce a fluorescent signal, with lower Ct values representing higher viral RNA loads. A Ct value less than 40 is clinically reported as PCR positive. This positivity starts to decline by week 3 and subsequently becomes undetectable. However, the Ct values obtained in severely ill hospitalized patients are lower than the Ct values of mild cases, and PCR positivity may persist beyond 3 weeks after illness onset when most mild cases will yield a negative result.2 However, a “positive” PCR result reflects only the detection of viral RNA and does not necessarily indicate presence of viable virus.3

In some cases, viral RNA has been detected by RT-PCR even beyond week 6 following the first positive test. A few cases have also been reported positive after 2 consecutive negative PCR tests performed 24 hours apart. It is unclear if this is a testing error, reinfection, or reactivation. In a study of 9 patients, attempts to isolate the virus in culture were not successful beyond day 8 of illness onset, which correlates with the decline of infectivity beyond the first week.3 That is in part why the “symptom-based strategy” of the Centers for Disease Control and Prevention (CDC) indicates that health care workers can return to work, if “at least 3 days (72 hours) have passed since recovery defined as resolution of fever without the use of fever-reducing medications and improvement in respiratory symptoms (e.g., cough, shortness of breath); and, at least 10 days have passed since symptoms first appeared.”4

The timeline of PCR positivity is different in specimens other than nasopharyngeal swab. PCR positivity declines more slowly in sputum and may still be positive after nasopharyngeal swabs are negative.3 In one study, PCR positivity in stool was observed in 55 of 96 (57%) infected patients and remained positive in stool beyond nasopharyngeal swab by a median of 4 to 11 days, but was unrelated to clinical severity.2 Persistence of PCR in sputum and stool was found to be similar as assessed by Wölfel et al.3

In a study of 205 patients with confirmed COVID-19 infection, RT-PCR positivity was highest in bronchoalveolar lavage specimens (93%), followed by sputum (72%), nasal swab (63%), and pharyngeal swab (32%).5 False-negative results mainly occurred due to inappropriate timing of sample collection in relation to illness onset and deficiency in sampling technique, especially of nasopharyngeal swabs. Specificity of most of the RT-PCR tests is 100% because the primer design is specific to the genome sequence of SARS-CoV-2. Occasional false-positive results may occur due to technical errors and reagent contamination.

Detection of Antibodies to SARS-CoV-2

COVID-19 infection can also be detected indirectly by measuring the host immune response to SARS-CoV-2 infection. Serological diagnosis is especially important for patients with mild to moderate illness who may present late, beyond the first 2 weeks of illness onset. Serological diagnosis also is becoming an important tool to understand the extent of COVID-19 in the community and to identify individuals who are immune and potentially “protected” from becoming infected.

The most sensitive and earliest serological marker is total antibodies, levels of which begin to increase from the second week of symptom onset.6 Although IgM and IgG ELISA have been found to be positive even as early as the fourth day after symptom onset, higher levels occur in the second and third week of illness.

For example, IgM and IgG seroconversion occurred in all patients between the third and fourth week of clinical illness onset as measured in 23 patients by To et al7 and 85 patients by Xiang et al.8 Thereafter IgM begins to decline and reaches lower levels by week 5 and almost disappears by week 7, whereas IgG persists beyond 7 weeks.9 In a study of 140 patients, combined sensitivity of PCR and IgM ELISA directed at nucleocapsid (NC) antigen was 98.6% vs 51.9% with a single PCR test. During the first 5.5 days, quantitative PCR had a higher positivity rate than IgM, whereas IgM ELISA had a higher positivity rate after day 5.5 of illness.10

ELISA-based IgM and IgG antibody tests have greater than 95% specificity for diagnosis of COVID-19. Testing of paired serum samples with the initial PCR and the second 2 weeks later can further increase diagnostic accuracy. Typically, the majority of antibodies are produced against the most abundant protein of the virus, which is the NC. Therefore, tests that detect antibodies to NC would be the most sensitive. However, the receptor-binding domain of S (RBD-S) protein is the host attachment protein, and antibodies to RBD-S would be more specific and are expected to be neutralizing. Therefore, using one or both antigens for detecting IgG and IgM would result in high sensitivity.7 Antibodies may, however, have cross-reactivity with SARS-CoV and possibly other coronaviruses.

Rapid point-of-care tests for detection of antibodies have been widely developed and marketed and are of variable quality. Many manufacturers do not reveal the nature of antigens used. These tests are purely qualitative in nature and can only indicate the presence or absence of SARS-CoV-2 antibodies. The presence of neutralizing antibodies can only be confirmed by a plaque reduction neutralization test. However, high titers of IgG antibodies detected by ELISA have been shown to positively correlate with neutralizing antibodies.7 The long-term persistence and duration of protection conferred by the neutralizing antibodies remains unknown.

Conclusions

Using available evidence, a clinically useful timeline of diagnostic markers for detection of COVID-19 has been devised (Figure). Most of the available data are for adult populations who are not immunocompromised. The time course of PCR positivity and seroconversion may vary in children and other groups, including the large population of asymptomatic individuals who go undiagnosed without active surveillance. Many questions remain, particularly how long potential immunity lasts in individuals, both asymptomatic and symptomatic, who are infected with SARS-CoV-2.

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Article Information

Corresponding Author: Sundararaj Stanleyraj Jeremiah, MD, Department of Microbiology and Molecular Biodefense Research, Yokohama City University School of Medicine, 3-9 Fukuura, Kanazawa-ku, Yokohama 236-0004, Japan (rediffjerry@gmail.com).

Published Online: May 6, 2020. doi:10.1001/jama.2020.8259

Conflict of Interest Disclosures: None reported.

References
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Nalla  AK, Casto  AM, Huang  MW,  et al.  Comparative performance of SARS-CoV-2 detection assays using seven different primer/probe sets and one assay kit.   J Clin Microbiol. 2020;JCM.00557-20. Published online April 8, 2020. doi:10.1128/JCM.00557-20PubMedGoogle Scholar
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Zheng  S, Fan  J, Yu  F,  et al.  Viral load dynamics and disease severity in patients infected with SARS-CoV-2 in Zhejiang province, China, January-March 2020: retrospective cohort study.   BMJ. 2020;369:m1443. Published online April 21, 2020. doi:10.1136/bmj.m1443PubMedGoogle ScholarCrossref
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Wölfel  R, Corman  VM, Guggemos  W,  et al.  Virological assessment of hospitalized patients with COVID-2019.   Nature. 2020. Published online April 1, 2020. doi:10.1038/s41586-020-2196-xPubMedGoogle Scholar
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CDC. Return-to-work criteria for healthcare workers. Updated April 30, 2020. Accessed May 3, 2020. https://www.cdc.gov/coronavirus/2019-ncov/hcp/return-to-work.html
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Wang  W, Xu  Y, Gao  R,  et al.  Detection of SARS-CoV-2 in different types of clinical specimens.   JAMA. 2020. Published online March 11, 2020. doi:10.1001/jama.2020.3786PubMedGoogle Scholar
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Lou  B, Li  T, Zheng  S,  et al Serology characteristics of SARS-CoV-2 infection since the exposure and post symptoms onset. medRxiv. Preprint posted March 27, 2020. doi:10.1101/2020.03.23.20041707
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    6 Comments for this article
    EXPAND ALL
    Interpreting a Variety of Diagnostic Tests for SARS-CoV-2 and COVID-19
    Michael McAleer, PhD (Econometrics), Queen's | Asia University, Taiwan
    The detailed and informative paper on interpreting diagnostic tests for the SARS-CoV-2 virus that causes the COVID-19 disease is based on two types of diagnostics in common use, namely :

    (1) Reverse transcriptase–polymerase chain reaction (RT-PCR); 

    (2) IgM and IgG enzyme-linked immunosorbent assay (ELISA).

    It is striking that the results of the diagnostic tests can and do vary over time, which lends itself to the possibility of rigorous time series analysis, if appropriate data were to be made available. 

    The null hypothesis of any diagnostic test of the virus and the associated disease, as given in (1), is that
    a negative test is presumed, namely:

    Null Hypothesis (H0): No confirmation of the virus.

    The critical issue in using any diagnostic test, or combination of a variety of tests, is how to achieve a correct diagnosis of a positive outcome.  

    For a detailed discussion of diagnostic testing of COVID-19, see (2), which suggests a number of diagnostics of the viral infection, none of which seems to have been used in the invaluable Viewpoint.

    It is well known that the purpose of any diagnostic test is to achieve high power, which involves the rejection of a false null hypothesis, leading to a true positive diagnosis.

    In cases where several diagnostic tests are available, with none being paramount, the use of a variety of tests will lead to a more accurate diagnosis by increasing the number of tests.

    Based on available data for adult populations without being immunocompromised, the estimated variation over time in the outcomes of the diagnostic tests is considerable, in terms of amplitude, shape, and the length of time since the onset of the disease symptoms.

    As the estimated time intervals and rates of detection are based on separate reports, each of which has different assumptions and distinct methodologies, the data are approximations, such that the probability of detection of the disease, based on maximizing the power of the various test procedures, is presented qualitatively rather than quantitatively.

    The collection and availability of time series data from different studies, according to the age of patients, existing comorbidities, types of preexisting conditions, including the various stages such as for cancer patients, their current treatments, and genetic markers, would lead to an invaulable unbalanced panel data set.

    Such a rich set of data could be used to predict the probability of a more accurate detection of infections through a range of diagnostic tests.

    References

    1. McAleer, M. 2020. Is one diagnostic test for COVID-19 enough? Journal of Risk and Financial Management, 13(4:77), 1-3.

    2. Sharfstein, Joshua M., Becker. S, and Michelle M. Mello. 2020. Diagnostic testing for the novel coronavirus. JAMA. Published online March 9, 2020. doi:10.1001/jama.2020.3864.
    CONFLICT OF INTEREST: None Reported
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    Role of IgA in the Anti-SARS-CoV-2 Antibody Response
    Ger Rijkers, PhD | Laboratory for Medical Microbiology and Immunology, St. Elisabeth Hospital, Tilburg, The Netherlands
    In their Viewpoint on Interpreting Diagnostic Tests for SARS-CoV-2 Sethuraman and colleagues may have left out a major player of the humoral immune response against respiratory viruses, and that is IgA. By now there are a number of publications which demonstrate a clear-cut IgA anti-SARS-CoV-2 response (1-3). In our own series of over 35 patients, close analysis (3 serum samples per week) showed a robust IgA response with a kinetic pattern similar to that of the IgG response: on average 10 days after onset of symptoms, 50% of patients were positive for IgA and IgG antibodies and by week 3 plateau levels for IgA and IgG were reached. In the Figure of this paper the legend thus could be adapted by labeling of the green dashed curve as representing IgG and IgA antibodies.

    References

    1. Padoan A, Sciacovelli L, Basso D, et al. IgA-Ab response to spike glycoprotein of SARS-CoV-2 in patients with COVID-19: A longitudinal study. Clin Chim Acta. 2020; 507:164-166. doi: 10.1016/j.cca.2020.04.026.

    2. Béné MC, de Carvalho M, Eveillard M, Lebri Y. Good IgA bad IgG in SARS-CoV-2 infection? Clin Infect Dis. 2020. pii: ciaa426. doi: 10.1093/cid/ciaa426.

    3. Okba NMA, Müller MA, Li W, Wang C, et al. Severe Acute Respiratory Syndrome Coronavirus 2- Specific Antibody Responses in Coronavirus Disease 2019 Patients. Emerg Infect Dis. 2020;26(7).
    CONFLICT OF INTEREST: None Reported
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    Looking Forward to Updating This With Results of Newly Emerging Antigen Test
    William Prendergast, MD | Retired Ophthalmologist, former Clin. Asst. Prof. @ OHSU
    Excellent summary of testing available up to now. Brings together a lot of useful information. I sincerely hope that we can look forward to the authors updating this survey with information about antigen testing, which, I am reading in today's newspaper, was just given emergency approval by the FDA.
    CONFLICT OF INTEREST: None Reported
    Prognostic Value of Antibody-Relevance
    Liz Jenny-Avital, MD | Jacobi Medical Center
    I would love to see an analysis of Ab (even neutralizing Ab) among those who survive and those who die. Neutralizing Ab may not be protective, at least not in patients already sick enough to be hospitalized. Such an analysis might better inform our current belief in the value of convalescent plasma (CP). I believe that checking Ab in sick patients admitted to hospital might temper the enthusiasm for use of CP.

    That said, I think Ab may be useful for diagnosing some later COVID-associated morbidity (eg clot) when nasopharyngeal PCR is negative.
    CONFLICT OF INTEREST: None Reported
    Synchronous Seroconversion of IgG and IgM
    Vedat Aslan, MD | Antalya Education and Research Hospital
    If antigen on the surface T cell receptor matches the B cells' MHC receptor, it triggers proliferation and antibody production.

    Some B cells that are approved by T cells turn to short-life plasma cells that produce only IgM antibodies. This takes a short time. IgM antibodies are less specific and have low affinity. They can not pass through normal tissues.

    Some of these B cells go to germinal centers of lymphoid tissues. After somatic hypermutation and class switch, they turn to long-lived plasma cells and memory cells that produce IgA, IgG or IgE. This takes a longer
    time.

    Synchronous seroconversion of IgG and IgM means SARS-CoV-2 is not a new virus for the patient because the IgG response needs about two weeks in response to new antigens.

    There are two possibilities:

    1: SARS or another coronaviruses' surface antigens are the same as SARS-CoV-2 and so the antibody response is the same. Most Chinese people are immune to most of the coronaviruses. They can carry the virus but do not develop serious pneumonia more than European and American people.

    2: The antibody test used for diagnosis has cross reactivity to other coronaviruses.
    CONFLICT OF INTEREST: None Reported
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    Humoral Response in Asymptomatic Patients
    Bader Alwadaany, MSN | Kuwait PAAET, college of Nursing
    As it is still empirically baffling to pinpoint the factors influencing display of COVID 19 symptoms it would be informative to examine seroconversion for IgG, A and M and their titre levels in asymptomatic patients.
    CONFLICT OF INTEREST: None Reported
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