In May 2020, six weeks after the first COVID-19 patient in Philadelphia arrived at the Hospital of the University of Pennsylvania, the Penn Physician Blog noted that testing would be of paramount concern in the weeks and months ahead to ensure the safety of individuals returning to the workplace and public spaces.
This has proved to be the case, and in the ensuing months, Penn Medicine has become a leading resource for public-access testing. At the same time, concerns about testing availability, type, safety, efficacy and use have entered the public compass, and with them, a wealth of statistics, data and clinical guidance. This article seeks to supplement the previous report with information that has subsequently come to light.
COVID-19 Testing Types
RT-PCR; Molecular; Viral Testing
The most widely used assay for COVID-19, molecular, or real-time reverse transcriptase polymerase chain reaction (RT-PCR) testing, is used to detect acute infection with SARS-CoV-2 in the upper and lower respiratory tract. RT-PCR is relatively simple to administer and highly sensitive.
According to the CDC, RT-PCR tests are recommended to diagnose acute infection of both symptomatic and asymptomatic individuals, to guide contact tracing, treatment options, and isolation requirements.
PCR is a technique that can be used to quickly and selectively amplify trace amounts of DNA. Because SARS-CoV-2 lacks native DNA, laboratories use reverse transcriptase, a polymerase enzyme found in retroviruses (e.g., HIV, HepB) to transcribe single-stranded RNA into complementary DNA, which can then be amplified (a process during which millions of copies of the DNA are produced) to permit even infinitesimal levels of genetic materials to yield a result.
A positive test result for COVID-19 indicates that RNA from SARS-CoV-2 was detected, and the patient is presumptively infected with the virus. While the individual is presumed to be contagious, a limitation of all tests used to detect nucleic acid in respiratory pathogens is their inability to differentiate between infective and non-infective viruses. A negative result does not exclude the possibility of COVID-19.
Time to Result
By comparison to traditional methods of DNA cloning and amplification, the process of analyzing RT-PCR tests is swift, leading to their somewhat confusing designation as “real-time” tests. The results of laboratory-based RT-PCR tests are typically available within several hours to days. Point-of-care test results may be available within an hour.
Administering the Tests: Alternative Options
The gold-standard for RT-PCR testing involves specimens from swabs inserted through the nose (nasopharyngeal, or NP) to a depth of about 5 inches; another approach inserts a swab orally to reach the throat. Because these tests present a number of practical issues, including the need for specific settings and mandatory PPE, researchers and test providers have shown an interest in alternative approaches for sampling. The caveat for these approaches is that they maintain the efficacy and sensitivity seen with standard tests.
To date, the leading candidates for alternative RT-PCR include saliva sampling and nasal (as opposed to nasopharyngeal) swabbing. According to a recent meta-analysis, these approaches have not yielded levels of efficacy equivalent to NP swabbing in studies, though comparisons between studies are complicated by differences in approach and interpretation of findings. Both saliva and nasal swabbing are included in the CDC Interim Guidelines for Collecting, Handling, and Testing Clinical Specimens for COVID-19.
Antigen Testing (Rapid)
Antigen tests detect the SARS-CoV-2 spike protein, and are used to diagnose active viral infection in symptomatic persons within the first 5 to 12 days of infection. Like RT-PCR tests, these tests use nasopharyngeal or nasal swab specimens, and like antibody testing, use immunoassays to achieve their result. Antigen tests have the benefits of low cost, point-of-care application and rapid results (usually within 15-30 minutes). The tests are generally less sensitive than RT-PCR for the detection of viral nucleic acid, however, and false positives can occur. A negative result in a symptomatic patient may thus require confirmation via RT-PCR.
Antibody Immunoassay (Serological) Testing
The third common test for SARS-CoV-2, antibody immunoassays are used to determine the presence of an adaptive immune response in individuals who may have been exposed to the virus. These tests cannot be used to diagnosis the active presence of disease.
Penn Medicine uses an advanced chemiluminecent micro-particle immunoassay (CMIA) from Abbott Laboratories that measures human SARS-CoV-2 IgG antibodies generated as part of the adaptive immune response to COVID-19.
The primary serum antibody (75 percent) in the blood; immunoglobulin G (IgG) binds to specific antigens on the surface of foreign particles in the blood (e.g., bacteria, viruses and other invaders) to prime them for phagocytic recognition and ingestion. In so doing, IgG creates recognizable antibodies unique to a specific disease. The presence of these unique antibodies indicate that a person has been exposed to, and had an immune reaction, to the disease.
Chemiluminescence, an immunoassay technique that has the benefit of ultra-sensitivity to detect small amounts of a biological molecule, is a key facet of the SARS-CoV-2 IgG Assay. In a chemiluminescent immunoassay, the indicator of the analytic reaction is a luminescent molecule that reacts to emit visible or near-visible radiation in the form of light. This reaction can then be measured as a relative light unit (RLU). There is a direct relationship between the amount of IgG antibodies to SARS-CoV-2 in a sample and the RLU detected by the assay system optics.
Applications for the SARS-CoV-2 IgG Immunoassay
For practical use, the SARS-CoV-2 IgG assay can be used to detect COVID-19 in persons whose viral load is below the detection limit for RT-PCR tests.
The test can also be used to identify convalescent plasma donors and to perform epidemiological studies of COVID-19 prevalence in a community. A two-part research initiative on convalescent plasma therapy for COVID-19 patients is now underway in the Division of Infectious Diseases and the Division of Transfusion Medicine and Therapeutic Pathology at Penn Medicine.
Efficacy
The capacity of the SARS-CoV-2 IgG Immunoassay to correctly identify non-infected persons (specificity) appears to be ~99 percent. According to Abbott validation studies, the SARS-CoV-2 IgG assay correctly identified infection in 25 percent of individuals from 3-7 days after onset of symptoms, 86 percent of those between day 8-13, and ~100 percent of persons 14 days after symptom onset.
Validation studies at Penn Medicine performed on serum samples from patients are consistent with those from Abbott performance data. Using patient serum samples collected at least 14 days after a positive SARS-CoV-2 molecular test, there was 98 percent agreement with molecular results. Penn researchers observed approximately 99 percent specificity in pre-COVID-19 era banked serum samples. Additional validation studies are currently in progress to further verify the specificity of the assay using sera from patients with non-SARS-CoV-2 coronavirus infections.
Who Should Be Tested for COVID-19?
The Centers for Disease Control and Prevention offers guidelines for COVID-19 screening in individuals and populations, including:
Additional Resources from Penn Medicine