On November 13th, Elon Musk underwent four rapid antigen tests for COVID-19, yielding two positive and two negative results. This outcome prompted a concerned reaction from Musk, who expressed his bewilderment on Twitter, stating, “Something extremely bogus is going on.”

The notion of a “bogus” situation arises from the apparent lack of understanding regarding the inherent behaviour of these diagnostic tools. The technical characteristics of the tests, when properly considered, render Musk’s experience entirely logical. Here’s a breakdown of why his results are entirely plausible.

When the FDA authorized the initial rapid antigen COVID-19 tests in August, they drew a parallel to at-home pregnancy tests. These diagnostics share common traits: they are economical, do not necessitate laboratory processing, and deliver results within a brief timeframe.

However, a critical distinction lies in their accuracy, which is constrained by a specific temporal window. Rapid antigen tests operate by identifying proteins present on the surface of the virus. The higher the viral particle concentration within the body, the greater the probability of detection.

The peak viral load typically coincides with the onset of symptoms. In the hours or even days preceding or following this peak, the likelihood of obtaining a false negative increases.

COVID-19 is most transmissible approximately two days prior to the emergence of symptoms. Consequently, a rapid antigen test might yield a false negative result precisely when an infected individual most requires accurate isolation guidance.

The extensive reliance on rapid antigen testing by certain entities may have contributed to instances of widespread transmission. Elon Musk’s discordant test results align with the established performance limitations of this testing modality.

Following his queries about rapid antigen testing, Musk shifted his focus to PCR (polymerase chain reaction) testing, enquiring, “Is it possible to generate a false positive if you simply run enough cycles?”

The answer to this question is no.

PCR is a laboratory-based technique employed for the amplification of specific DNA sequences.

The process involves combining a biological sample with the necessary reagents for DNA replication, followed by the addition of DNA primers designed to target the sequence of interest.

While PCR testing offers superior accuracy over a prolonged period, it presents certain disadvantages compared to rapid antigen testing. These include higher costs, the necessity of laboratory facilities, and a longer experimental duration.

PCR’s efficacy hinges on the presence of the target DNA within the sample. False positives, in this context, are typically a consequence of contamination. To mitigate this risk, a negative control is routinely incorporated.

In the negative control, pure distilled water is used instead of the patient’s nasal swab DNA sample. Should a positive result emerge from the negative control, it unequivocally indicates sample contamination.

Regarding the “cycles” to which Musk refers, DNA replication at ambient temperatures would be an exceedingly protracted process. To accelerate this, the reaction mixture is subjected to controlled heating and cooling cycles. Each such cycle contributes to the amplification process.

Depending on the specific assay design, experiments may involve 30 to 40 thermal cycles. However, it is crucial to understand that the total number of PCR cycles does not unilaterally determine the test outcome. Instead, the quantity of target DNA is quantified at a predetermined cycle threshold.

This cycle threshold remains consistent across all PCR experiments conducted by a laboratory for a particular DNA target. The explanation provided by @c0nc0rdance offers a lucid perspective on this matter:

“Number of cycles” doesn’t matter. Where you set the threshold does. Saying “we’re running too many cycles” is like saying “we’re recording too much security video after the crime was committed”. It doesn’t matter.

A third category of COVID-19 testing involves antibody testing.

Rather than directly detecting the virus, antibody testing ascertains the presence of an immune response mounted against the virus.

When the human immune system encounters a novel pathogen, such as a virus or bacterium, it initiates a defense mechanism to neutralize it. Concurrently, a subset of immune cells generates antibodies that specifically recognize the invading agent.

These antibodies function akin to a biological “wanted poster,” marking the pathogen for elimination.

Antibody testing specifically searches for antibodies indicative of prior COVID-19 infection. The detection of these antibodies confirms that the individual has been previously infected by the SARS-CoV-2 virus.

Antibodies persist in the bloodstream for an extended duration following infection, rendering antibody testing a reliable retrospective diagnostic tool. However, the immune system requires a period to synthesize these antibodies in response to a new threat, thus making antibody testing an ineffective method for diagnosing acute infections in the initial stages.

The three primary types of COVID-19 tests currently available—PCR, antibody, and rapid antigen—each possess distinct limitations. They address the query, “Is there evidence of COVID-19 in this sample?” through different mechanisms. A comprehensive understanding of these limitations and differences is paramount for accurate interpretation of test results.

Emma Bell is a bioinformatician engaged in research at the Princess Margaret Cancer Centre in Toronto, Canada. Her work utilizes computational programming, statistical analysis, and machine learning methodologies to deepen the understanding of cancer biology.

This article was originally published by Emma Bell Ph.D. on Medium. The original publication can be accessed here.

References

Rubin, R. JAMA. (2020) The Challenges of Expanding Rapid Tests to Curb COVID-19

Centers for Disease Control and Prevention (2020) Test for past infection