ACMT COVID-19 Web Series FAQs

Viral Test Issues:

In the setting of this epidemic, a “viral test” attempts to answer the question: “Am I infected with SARS-CoV-2?” This type of testing is a real-time, polymerase chain reaction (RT-PCR) laboratory process that takes a sample from a person (nasopharyngeal or throat swab are most commonly used, but saliva and even stool samples can potentially be processed), chemically deactivates any virus present in order to liberate its genetic material (RNA), then uses (usually) a heat-mediated process to tag and rapidly replicate and detect the virus’ genetic material. If a specific sequence characteristic of SARS-CoV-2 is present, at some point in the rapid (seconds) “cycle time”, a positive signal will be generated. Each developing laboratory and manufacturer use some variation in sequencing, amplification, or detection technology that influence the level of detection, rapidity of signal generation, and total testing time. 

However, there are important issues related to the sensitivity of these tests (the number of true positive results obtained; limiting the number of false negatives). Problems can occur at any point in the testing process, including the adequacy of sampling done way back at the beginning of the testing process, the stability of transport medium to get the specimen to the laboratory and the ability to remove virus material from the swab [although transport media and nature of swab is more important when one is trying to establish or identify a virus in culture (allowing it to replicate)], and the ability to detect as few as 1 genome copy in a microliter of fluid [the RT-PCR testing process itself].

Another issue is when was the person sampled – if a person has been exposed, but not yet infected (the virus multiplying and infecting cells), it is unlikely that a test will be positive. Where the specimen was obtained can also make a difference: nasopharyngeal swabs are high yield sampling sites early on in infection, but drop off in the subsequent week while throat swabs, sputum, and stool are more likely to persist positive for a couple of weeks.

There have also been reports of viral tests being negative and then “turning positive” on repeated testing – often without the patient becoming symptomatic, and without evidence that they infect anyone else. There are many potential explanations for this unsettling pattern, and the relative contribution of those that actually occur, whether repeated low-grade infection, variable cut-off value issues for the test, test specificity (e.g., shared target gene material with other coronavirus species), or even specimen or laboratory contamination, is not known at this time.

Antibody Test Issues:

An “antibody test” attempts to answer the question: “Was I exposed in the past to SARS-CoV-2?” This type of test is a blood test that attempts to identify a person’s immune response to SARS-CoV-2 by measuring IgA, IgM, or IgG antibody proteins that have been generated by lymphocytes (B cells) that have been responded to exposure to SARS-CoV-2. This kind of testing could be used to indicate very recent (within a week or so) infection if the test identifies IgA (generally thought of as mucosal release of antibody) or IgM (acute phase antibody response); but most antibody tests are reporting total virus-targeted immunoglobulin or specific IgG antibodies (usually requiring a couple of weeks to be generated, but persisting for an indeterminate period of months); thus, these tests indicate past exposure/infection.

At this point, scientists do not know if these antibodies are “neutralizing antibodies” and thus confer immunity to repeat infection, or are just a marker of past infection. This situation is not much different than antibody testing for a number of other viral infectious diseases for which we routinely obtain “titres”, such as measles, mumps, rubella – one could still be immune even without a measurable titre. (Nonetheless, we re-immunize high-risk people such as healthcare workers for these communicable diseases.) In contrast to “viral tests” where the biggest problem is sensitivity (minimizing false negatives e.g., by adequate sampling of the back of the nasal cavity), the biggest problem with “antibody tests” is specificity of the results (“true negative” results, minimizing the number of false positives). Because SARS-CoV-2 is a coronavirus and there are many coronaviruses in circulation (one of the common causes of the common cold), antibody response to some of these related virus species could be reflected by the test. In order to make sure that the tests are as specific for the SARS-CoV-2 as possible, tests need to be run on blood specimens obtained before the SARS-CoV-2 was circulating, which means testing of a lot of old “blank” serum specimens against the test.

Even if tests appear “pretty good” in terms of sensitivity and specificity, the accuracy when applied to a population who may or may not have been infected is very dependent on the prevalence of the disease in the tested population. As detailed below, the predictive value of a test may not be very good, even if the test is very “accurate” (high specificity and sensitivity), when it is applied to a low-risk population. These are the two major reasons (adequacy of testing of “negative specimens” – test result specificity; and interpretation of positive results in a low prevalence situation) that “antibody tests” have been delayed in their release and the source of concern as they have now been rapidly released under the FDA’s Emergency Use Authorization (EUA). An upcoming ACMT webinar will explore this issue in more detail.

As an example, even if a test is 99% accurate in those with or without the disease (99% sensitivity, 99% specificity), positive test results can be very misleading when there is a low prevalence of infection. In an area with 1% of the population infected/exposed (left-hand 2x2 table), a negative test is both very likely and likely to be a “true negative”, indicating non-exposed. However, it is just as likely that a positive test (in isolation) is a false positive as it is a true positive. As shown, it isn’t until the disease is very widespread (in the example on the right, 10% of the population) that a positive test becomes additionally helpful in any given person (again, this is looking at the test result in isolation from any clinical symptoms or other features that suggest exposure):