Ying Liu, M.D., Pathology Resident
Anna Romanelli, Ph.D., Medical Director of Clinical Microbiology Laboratory

As communities across the U.S. have struggled to cope with the effects of the COVID-19 pandemic, many have focused on the lack of widespread testing as a major roadblock to reopening the country and go back to some sort of normalcy. However, as progress has been made on this front, concern has shifted to testing accuracy, predominantly with antibody testing, which is designed to identify prior infection. The goal of this blog article is to review the challenges and implications associated with false negative COVID-19 testing.

Challenges with COVID-19 Diagnostic Testing:

In general, antibody tests identify prior infection, while molecular diagnostic testing identifies current active infection. There are two main issues that are worth understanding for COVID-19 diagnostic testing. The first issue relates to who gets tested. At the start of the pandemic in the U.S, availability of testing kits was limited, which placed restrictions on who got tested. During this time, to get a coronavirus diagnostic test, a person was asked to meet certain criteria, such as having symptoms of COVID-19, the disease caused by the coronavirus, or having had close contact with a person confirmed to be infected. In turn, asymptomatic people who are thought to be the major “spreaders” of COVID-19 were not being tested due to the paucity of testing kits. Fast forward to three months later, testing is being performed on a wider scale to include both symptomatic and asymptomatic individuals. Implementing this broader strategy of testing demonstrated a much higher incidence of COVID-19 than previously reported. In order to accurately interpret this uptick in COVID-19 positive cases, we need to understand how well the current diagnostic tests rule out COVID-19 infection. This leads us to our second issue which is the testing itself.

There is already growing concern that the diagnostic test for COVID-19 is not reliable. Current testing has the potential to come back positive in some people who are not infected with SARS-CoV-2 and negative in people who are in fact infected. A false positive result could mistakenly label a person infected, which has implications causing unnecessary isolation mandates and needless contact tracing. A false negative result, on the other hand, is significantly more consequential, because an infected person who may be asymptomatic will not be required to quarantine and has the potential to infect others. When people have false negative results, they tend to go back to their normal routine or “life before COVID-19”. Social distancing is not being practiced, masks are “optional” in their daily lives and they put themselves in high-risk environments. With imperfect tests, a negative result means only that a person is less likely to be infected. Important testing parameters to keep in mind for a negative test are pre-test probability and the test's sensitivity and specificity. The greater the sensitivity, the less likely it will miss real cases. The greater the specificity, the more likely uninfected individuals will be correctly deemed negative. The current SARS-CoV-2 diagnostic PCR assay performed at UC Davis has a sensitivity of greater than 97.5% and specificity of 100%. It can detect several orders of magnitude above the limit of detection using a typical nasopharyngeal swab specimen.

SARS-CoV-2 is an enveloped positive sense RNA virus and current molecular tests are designed to target one or more common regions, include the envelope (E), nucleocapsid (N), spike protein (S), and the open reading frame (ORF) region. Assays used by the Center for Disease Control and Prevention (CDC) and others target multiple regions within a gene (1,2). In a meta-analysis review, the authors performed a systematic review and critical appraisal of literature, they screened all the publications from major database, and selected five published studies involving 957 patients in China (either confirmed cases or cases under suspicion of Covid-19). The false negative rates of RT-PCR SARS-CoV-2 assays were calculated using a multilevel mixed-effect logistic regression model and resulted in a range from 2% to 29%, which also indicated up to 29% of patients could have an initial RT-PCR false-negative result (3). However, because of the heterogeneity of sensitivity among the five studies, the certainty of the evidence was low. Even though the evidence is limited, the concern of false negative RT-PCR results is not negligible.

It is also important to be aware that detection of viral RNA does not necessarily indicate the virus is transmissible. The duration of virus shedding dropped after 20 days post-onset of symptoms. The genetic material of dead viral particles can remain within the epithelial cells and can be detected. This was supported by a recent study, viral shedding from sputum has been shown to extend beyond symptom duration (4,5). Although we have learned a lot about this virus in a short period of time, these are still fundamental questions we have to answer in order to make intelligent decisions on how to approach testing (6).

Another challenge is the need for establishing reference standards for measuring the sensitivity of SARS-CoV-2 test in asymptomatic patients as it is crucial to identify people who are infected, so they are isolated to limit their interactions with people who are at higher risk. Unfortunately, there is no data available to measure test sensitivity in asymptomatic patients. It was believed that viral loads in asymptomatic patient may be different than patients with symptoms. However, that is also debatable, as one other study analyzing a small number of patients also reported viral load of asymptomatic patients was as high as that of symptomatic patients (7).

Antibody test:

Antibody testing can identify prior infection by using a surface protein or an array of virus peptides to capture antibodies specific to the virus in patients' blood. It has been shown in a recent study analyzing 134 samples, using colloidal gold-based immunochromatographic strip method targeting the viral antibody, the sensitivity of the assay is up to 93%, 8-14 days after onset of symptoms (8). There are limitations regarding the utilization of antibody-based testing. A better understanding of the importance of the “testing window” or timeline of antibody testing and what the test result means are important to overcome the limitations. The timeline of development of new antibody can be delayed because validation of a new test using the antibodies from the blood of infected people always takes time. Clinically, immunoglobulins (Ig) such as IgM, IgA, or IgG are used for immunoassays. IgG is more specific than IgA and IgM, and the levels of IgG may correlate with immunity, but it typically appears 7 days post infection. IgM may appear as early as 3 days post infection, but IgM and IgA are less specific. It is believed by day 14 post infection, although virus-targeting antibody is detectable, this lag period indicates this test cannot be used for early detection of infection within the first week or so. The fact that this test is most accurate 2 weeks after onset of symptoms makes it unrealistic to be used for the diagnosis of this highly infectious disease (9,10). We have to be aware that if testing is performed during this “window period” (too soon after exposure where antibody production is non-detectable), false negative serology results may occur. Ongoing research is trying to determine if virus-targeting antibodies provide some immunity to SARS-CoV-2, how long the antibodies last and whether past infection will protect from future infection.

A recent study screened 9,530 specimens using Diazyme SARS-CoV-2 IgG serology assay, and all positive results (N=164) were reanalyzed using the neutralization assay, the Roche total immunoglobin assay, and the Abbott IgG assay. The relationship between the levels of neutralizing antibodies and the magnitude of positive SARS-CoV-2 serology was correlated. The authors demonstrated the three widely available clinical serology assays positively correlated with SARS-CoV-2 neutralization activity, and confirmed that using a two-platform approach for COVID-19 serology positive individuals greatly improved positive predictive value for neutralization (11). Of note, the UC Davis serology assay actually has similar platforms as those used in the current study.

Conclusion:

In summary, performing a single COVID-19 test could lead to false negative results and misdiagnosis. Clinicians should not trust unexpected negative results in a patient with typical symptoms and known exposure (assuming false negative) if pretest probability is high. Therefore, a combined diagnostic workflow integrated with both molecular detection (detects current active infection) and antibody testing (prior infection/immunity) should be implemented to provide a high quality and cost-effective diagnostic solution. This approach should also include this diagnostic workflow at different time points throughout the course of disease.

References:

  1. Wu F, Zhao S, Yu B, et al. A new coronavirus associated with human respiratory disease in China. Nature. 2020;579:265-269.
  2. UC Davis Labroatary Best Practise Blog (updated on July 15, 2020) https://blog.ucdmc.ucdavis.edu/labbestpractice/index.php/2020/06/16/review-of-covid-19-testing-methods/ Accessed on Aug 15, 2020.
  3. Arevalo-Rodriguez I, Buitrago-Garcia D, Simancas-Racines D, et al. False-negative results of initial RT-PCR assays for COVID-19: a systematic review. medRxiv. April 21, 2020.
  4. Rawlins EL, Hogan BL. Ciliated epithelial cell lifespan in the mouse trachea and lung. Am J Physiol Lung Cell Mol Physiol. 2008;295(1):L231-L234.
  5. Wölfel R, Corman VM, Guggemos W, et al. Virological assessment of hospitalized patients with COVID-2019. 2020;581(7809):465-469.
  6. Woloshin S, Patel N, Kesselheim A. False negative tests for SARS-CoV-2 infection-challenges and implications. New Engl. J. Med. 2020;383:e38.
  7. Lee S, Kim T, Lee E, et al. Clinical course and molecular viral shedding among asymptomatic and symptomatic patients with SARS-CoV-2 infection in a community treatment center in the republic of Korea. JAMA Intern Med.Published online August 06, 2020.
  8. Pan Y, Li X, Yang G, et al. Serological immunochromatographic approach in diagnosis with SARS-CoV-2 infected COVID-19 patients. medRxiv. Jan 1, 2020.
  9. Hu E. COVID-19 Testing: Challenges, Limitations and Suggestions for improvement. Preprints. 2020,2020040155.
  10. Zhang W, Du RH, Li B, et al. Molecular and serological investigation of 2019-nCoV infected patients: implication of multiple shedding routes, Emerging Microbes & Infections. 2020;9(1):386-389.
  11. Suhandynata RT, Hoffman MA, Huang D, et al. Commercial Serology Assays Predict Neutralization Activity Against SARS-CoV-2. July 10, 2020.