Editors’ Note: This article was accepted on April 28, 2020, and was accurate at that time. Because information about SARS-CoV-2 and COVID-19 is evolving rapidly, please verify these recommendations and information.

When SARS-CoV-2 appeared, we had no tests. The disease spread unchecked and unmeasured.

Today, we have several tests, each with its own unique roles and limitations. How should these be used, and what strengths and weaknesses affect their use?

Nucleic Acid-Based Tests

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These tests have been the work horses in diagnosing acute cases of COVID-19. The two primary analytic methods are reverse transcriptase polymerase chain reaction (RT-PCR) and loop-mediated isothermal amplification (LAMP). Regardless of the technique, the basic principle is the same: RNA from viral particles are bound to complementary DNA sequences, which are then copied. Repeated cycles of copying produce exponential amplification that, in sufficient quantity, reaches a defined threshold for a positive test. Absent specific SARS-CoV-2 RNA, minimal amplification occurs, and the test never reaches the threshold for positivity.

These tests are designed for those actively infected and shedding virus. Their analytic sensitivity and specificity are considered excellent, with limitations related to mismatches between the DNA primers and small alterations in the SARS-CoV-2 RNA genome. The DNA primers used may match fragments of other RNA found in samples but typically not to the extent where cross-reactivity impairs amplification of the target.

The sensitivities for these tests are limited by two main factors. The first issue is the process through which the specimen is obtained. The most widely recognized version of this test is the nasopharyngeal swab. An inadequate sampling technique will diminish the quality of the specimen, lowering sensitivity.

Second, viral load and shedding also decrease with time, contributing to diminishing sensitivity.¹ Overall clinical sensitivity in practice appears similar to our expectations for common influenza tests, in a range approximating 70 to 80 percent.¹ As many of us have  already experienced in our clinical workflow, a single negative nasopharyngeal swab does not adequately exclude COVID-19 infection when the remaining clinical picture is supportive. A positive test is, however, virtually unimpeachable.

Antibody Tests

Serological tests are designed to detect the presence of antibodies to SARS-CoV-2. The antibodies of interest include the acute-phase immunoglobulin (IgM), the late-phase (IgG), and occasionally IgA.¹ These tests have been promoted widely as a critical part of plans to reopen America and are in frequent use in population prevalence studies.

Antibody tests are more complicated than DNA-based tests, however. Two main types, enzyme-linked immunosorbent assay (ELISA) and lateral-flow immunoassay (LFIA), are in use. These tests are more difficult to develop because the developers of these assays must synthesize their own novel viral fragments. This involves an analysis of the actual protein coat of the virus, typically focusing on the unique features of the spike protein and cell-entry apparatus. When serum or plasma containing antibodies to SARS-CoV-2 are mixed with the assay antigens, the test reporter systems provide a positive result.

The most pressing issue with these antibody tests is accuracy.

The ELISA tests require significant time and reagent cost but offer the advantage of quantitative antibody titers. These tests are valuable for accurately identifying high levels of circulating antibody for those being considered as possible donors for convalescent plasma donation (although the efficacy of this strategy remains unknown). The prolonged turnaround time and biohazard safety requirements for ELISA reduce its practicality for widespread testing.

In far greater use are LFIA-based devices, which are the widely seen cartridge-based tests. These tests do not typically report a quantitative measurement but provide positive and control color-change lines using a technique similar to home pregnancy tests. The major advantages of these tests are speed and cost. However, they lack the quantitative precision of ELISA.

The most salient issue with these antibody tests is accuracy. The sensitivity limitations of antibody tests are readily apparent because even acute-phase IgM responses are not immediate, usually taking a few days to develop. Therefore, a single antibody test should not be the sole mechanism for diagnosing acute infections. Consideration ought to be given to the time of symptom onset to determine the likelihood of a false negative, as well as either a DNA-based test or a plan to repeat the antibody test in a few days, if negative.

The other accuracy issues stem from antigen synthesis. The challenge for antigen synthesis involves creating a match for a piece of the virus that is both unique to SARS-CoV-2 while also stable enough not to result in mismatches as the virus naturally mutates. The most stable components of SARS-CoV-2 are also the ones conserved across multiple other coronaviruses, resulting in cross-reactivity and false positives. Several common-cold coronaviruses (eg, HKU1, NL63, OC43, 229E, etc.) are known to react with the antibodies in several developed serological tests. A false-positive test may endanger an individual by suggesting potential immunity from SARS-CoV-2 where none exists.

Antibody tests are also being deployed to evaluate the spread of COVID-19 in some communities. This renders false positives especially important. In communities in which little infection is thought to have occurred, the number of false positives can exceed the number of true positives, even in a test with high specificity. These false positives lead to overestimation of the number of potentially immune persons  and may misinform public policy decisions.

Conclusion

Each of the several SARS-CoV-2 tests can play a role in detecting individual cases and evaluating the spread of COVID-19. Results must be carefully interpreted in the context of how common (and therefore likely) the disease is. Otherwise, both false-negative and false-positive results from these tests may ultimately place patients, their families, and health care professionals at elevated risk.

The opinions expressed herein are solely those of Dr. Radecki and do not necessarily reflect those of his employer or academic affiliates

References

1. He X, Lau EHY, Wu P, et al. Temporal dynamics in viral shedding and transmissibility of COVID-19. Nat Med. 2020;26(5):672-675.
2. Wang W, Xu Y, Gao R, et al. Detection of SARS-CoV-2 in different types of clinical specimens. JAMA. 2020;323(18):1843-1844.
3. IDSA COVID-19 antibody testing primer. Infectious Diseases Society of America website. Accessed May 11, 2020.