Clinical laboratory testing routinely provides actionable results, which help direct patient care in the inpatient and outpatient settings. Since December 2019, a novel coronavirus (SARS-CoV-2) has been causing disease (COVID-19 [coronavirus disease 2019]) in patients, beginning in China and now extending worldwide. In this context of a novel viral pandemic, clinical laboratories have developed multiple novel assays for SARS-CoV-2 diagnosis and for managing patients afflicted with this illness. These include molecular and serologic-based tests, some with point-of-care testing capabilities. Herein, we present an overview of the types of testing available for managing patients with COVID-19, as well as for screening of potential plasma donors who have recovered from COVID-19.
Beginning in December 2019, an outbreak of “pneumonia of unknown cause” was detected in Wuhan City, Hubei Province, China. Ultimately, the 2019 novel coronavirus, or SARS-CoV-2, was identified as the causative agent and subsequently isolated and sequenced. Since that time, SARS-CoV-2 has spread worldwide, causing a severe illness known as COVID-19 (coronavirus disease 2019), which led the World Health Organization (WHO) to declare it a pandemic on March 11, 2020. Since the beginning of the outbreak, clinical laboratories have been developing various assays to aid in detecting SARS-CoV-2 and clinically managing patients with COVID-19.
The 3 categories of tests used to detect current or past viral infection are molecular, serologic, and antigen-detection assays (Table 1). In this context, a molecular assay is used to determine whether a patient is actively infected with the pathogen of interest. Reverse transcription polymerase chain reaction (RT-PCR) is a common laboratory technique used to detect respiratory viral pathogens, such as influenza and respiratory syncytial virus (RSV). Currently, this is the main type of test being utilized to determine whether patients are infected with SARS-CoV-2.
RT-PCR is a sensitive technique for RNA detection, whereby RNA is reverse transcribed into complementary DNA (cDNA) and cDNA targets specific for the pathogen of interest are amplified. If SARS-CoV-2 RNA is present in a patient specimen, typically collected as a nasopharyngeal (NP) or anterior nasal swab, it will be detected by this assay. Depending on the platform, these assays can be completed in less than 1 hour to several hours, once the specimen arrives in the laboratory and is loaded onto the platform.
The caveats to interpreting results from this assay type are that doing so does not inform us whether a patient previously had the infection; rather, this type of assay only detects patients actively shedding virus (current infection or carriage state) or those who have residual viral RNA present. Therefore, these assays are most useful in acute settings to detect patients with COVID-19, where the results can inform appropriate isolation protocols and ensure that appropriate personal protective equipment (PPE) protocols are utilized when treating these patients. As of the date of publication of this article, of the 102 commercial laboratories and/or test kit manufacturers approved for emergency use by the United States Food and Drug Administration (FDA) for SARS-CoV-2 testing, 81 of them were molecular assays (Table 2). We note that many of these commercial assays require a laboratory to have vendor-specific instrumentation and equipment to utilize these test kits. The FDA also has authorized 37 molecular-based laboratory developed tests (LDTs) that can be used in the single laboratory that developed the test.
Given that true NP specimens are often difficult to obtain, the FDA has stated that oropharyngeal (OP), nasal midturbinate, and anterior nares swabs are acceptable when using an NP swab is not possible. Also, the FDA recently granted an Emergency-Use Authorization (EUA) to Rutgers Clinical Genomics Laboratory-Rutgers University for an RT-PCR LDT for qualitative detection of SARS-CoV-2 in saliva specimens as well as OP, NP, anterior nasal, and midturbinate nasal swabs. Saliva testing presents potential benefits of eliminating the need for swabs and decreasing the risk posed to the health-care workers collecting these specimens. However, this specimen type might require additional dilution or pretreatment due to its viscosity. Also, viral RNA might be more difficult to detect in this specimen type, although the results of a previous study found that saliva and NP specimens were comparable for detection of respiratory viruses by RT-PCR.
The possibility of false negative results with molecular assays should also be considered. One report documents a patient with multiple RT-PCR NP/OP specimens that tested negative; this patient ultimately had SARS-CoV-2 detected in a bronchoalveolar lavage (BAL) fluid specimen. For lower respiratory tract specimens, currently, the FDA recommends testing BAL fluid only under certain clinical circumstances such as invasive mechanical ventilation. Sputum should be tested from patients who develop a productive cough, although the FDA does not recommend induction of sputum for SARS-CoV-2 testing.
The other main type of assay is serological. These assays determine the exposure history and/or immune status of a patient. They detect the presence of antibodies against SARS-CoV-2 antigens in serum, plasma, or whole blood specimens. After initial viral infection, there is a delay before the production of antibodies by the immune system (Figure 1). During this time, known as the window period, a patient who is infected with SARS-CoV-2 but has no detectable antibodies, would have negative test results on a serologic assay. Typically, when the immune system mounts a response against a virus, short-lived immunoglobulin (Ig)M antibodies are initially produced, followed by a more durable IgG antibody response. However, there are limited data thus far in the literature regarding the longevity of anti–SARS-CoV-2 antibodies.
SARS-CoV-2 estimated seroconversion rates.
The graph in Figure 1 demonstrates estimated viral RNA, IgM, and IgG detection levels for SARS-CoV-2 based on the limited published literature to date. The estimated median seroconversion time is 7 to 12 days; most patients with COVID-19 have detectable antibodies approximately 15 days after the onset of symptoms. Due to the subjectivity in determining symptom onset, these dates can be highly variable. Also, viral RNA has been shown to peak in the first week of illness and then gradually decline.
Serological tests can be unique to one class of immunoglobulins; can detect IgM and IgG antibodies simultaneously; or can be total antibody tests, which detect IgA antibodies as well. Depending on the exact protocol and platform, such assays can typically be completed in 1 to 2 hours once a specimen arrives in the laboratory and is loaded onto the relevant platform. We note, however, that at the time of publication of this article, there are a few commercial assays available on large, automated analyzers, from diagnostic manufacturers including Roche Diagnostics, Abbott Diagnostics, and Ortho Clinical Diagnostics (with offerings more recently available from Beckman Coulter, Inc and Siemens AG). However, to our knowledge, there are no objective, peer-reviewed data on their performance characteristics.
Many available commercial serological assays use a lateral flow assay format; for many of these assays, there are unsubstantiated, or even false, claims about test performance. The estimated median seroconversion time is 7 to 12 days, with virtually all patients with COVID-19 having detectable antibodies approximately 15 days after onset of symptoms.[8–10] However, the results of a recent acute antibody response study demonstrated simultaneous or sequential IgM and IgG seroconversion, with a slight decrease in IgM antibody titers 3 weeks after symptom onset. Therefore, these assays will be most helpful in determining the exposure status of an individual and in assessing the immune response of that person to SARS-CoV-2.
Because SARS-CoV-2 is a new virus, it is not clear whether an immune response confers immunity and how long the immune response will last—that is, whether it is durable and sustained (years) or if it is short-lived (1–2 months). These assays can be particularly helpful for individuals who may have had symptoms consistent with COVID-19 but who were never tested with an RT-PCR test (due to the severe limitations in testing capabilities in many areas) and now have recovered from their illness. Currently, the Centers for Disease Control and Prevention (CDC) does not recommend using serology testing to determine eligibility to return to the in-person workforce. Individuals who were symptomatic can stop self-isolation as long as their symptoms have improved with 3 days of no fever and at least 10 days have elapsed since the onset of their symptoms.
Finally, these tests can also be used in serosurveillance, in which data are obtained to calculate the prevalence of anti–SARS-CoV-2 antibodies in the community. Such information can help epidemiologists better understand the true burden of disease and to model continued viral-transmission dynamics based on the percentage of the population that is immune vs susceptible. Given that approximately 80% of COVID-19 cases are mild to moderate in severity[10,13] and that molecular testing has been restricted to the most severely ill patients, the true number of cases has likely not been revealed by molecular-based assays. Thus, serological testing can provide a more accurate enumeration of the number of past infections. As with all laboratory testing, however, the results of these assays will only be accurate and maximally useful based on their performance characteristics, including sensitivity and specificity.
COVID-19 testing is also important for identifying potential patients who have recovered from COVID-19 and have detectable antibodies against SARS-CoV-2 (convalescent donors) for clinical trials. Currently, studies are underway in which convalescent donors can donate plasma, which can then be transfused into critically ill patients with COVID-19. A review of the published literature to date indicates that treatment with plasma from convalescent donors demonstrates beneficial effects, although further evaluation with clinical trials remains imperative. Potential donors require a serologic assay to detect the presence of anti–SARS-CoV-2 antibodies in their plasma. For this type of plasma to be beneficial, the antibodies present should have neutralizing activity (ie, the antibodies bind to and neutralize infection by active SARS-CoV-2 virus). Such testing is not currently performed in clinical laboratories but rather in research laboratories. Ideally, convalescent plasma donors would be noninfectious (symptom-free for >14 days) and have high titers of virus-neutralizing antibodies (as determined by serologic testing).
Point-of-care (POC) testing is beginning to be available for SARS-CoV-2. POC testing refers to a broad category of diagnostic tests that can be performed where patient care occurs. Functionally, these tests have a rapid turnaround time (TAT) and can potentially be performed by various nonlaboratory clinical personnel. These assays can be molecular or serologic. One molecular POC test by Abbott Diagnostics uses isothermal nucleic acid amplification (a technique similar to polymerase chain reaction [PCR]) to detect SARS-CoV-2 in approximately 15 minutes. The results of recent studies[15–17] have demonstrated low sensitivity for the Abbott ID Now assay with specimens collected in transport media. Thus, the EUA for this test was modified for testing only from direct/dry swabs.
The Cellex serologic POC qSARS-CoV-2 IgG/IgM Rapid Test (Cellex, Inc.) utilizes a lateral flow immunoassay, which qualitatively detects IgM and/or IgG antibodies from whole-blood specimens. A blood specimen flows by capillary action along the cassette and, if anti–SARS-CoV-2 IgM or IgG antibodies are present, they will bind to recombinant SARS-CoV-2 antigens present on the test strip. The presence of these antibody-antigen complexes are then detected by a colorimetric change, which is revealed when the complexes are captured by anti–human IgG or anti–human IgM antibodies. The results are available in approximately 15 to 20 minutes. Of note, currently POC serology tests must be performed in conjunction with a CLIA (Clinical Laboratory Improvement Amendments)–licensed laboratory and cannot be performed in locations with only a CLIA waiver, such as a physician office.
Finally, there is now an antigen detection assay available from Quidel Corporation (Sofia 2 SARS Antigen FIA) that uses a lateral flow immunofluorescent sandwich assay technique for detection of the nucleocapsid protein antigen of SARS-CoV and SARS-CoV-2. In theory, viral proteins can be detected using one of a number of antigen capture methods (eg, antibodies, aptamers). Such tests are used routinely for other viral assays (eg, human immunodeficiency virus [HIV] p24 antigen as part of 4th– and 5th-generation HIV tests), and also for hepatitis B surface antigen. Like molecular assays, antigen detection tests can be used to detect active SARS-CoV-2 infection.
In summary, molecular and serological tests provide meaningful data for treating patients with COVID-19. However, each category has different clinical utility, different characteristics, and different limitations. Clinical laboratories continue to develop new assays and implement increased testing capabilities to meet the high demands for patient testing during this pandemic.