An interesting preprint by Doshi from the University of Maryland School of Pharmacy, Baltimore looked at this issue. It was published by the Oxford University Press for the Infectious Diseases Society of America.

Efforts to control the spread of contagious disease have historically focused on separating the sick, i.e. symptomatic patients from those who are not sick. The logic behind this is straightforward; sick people carry infectious potential while those not sick do not. With the advent of laboratory testing, it is now possible to culture live organisms from those potentially infectious individuals.

Today, multiple new forms of testing other than culture are in widespread use, particularly the test for SARS-CoV-2, the so called PCR test. It is presumed to identify current SARS-CoV-2 infection. It is seen as an essential tool to identify patients who are infected but not showing symptoms at the time of testing and also amongst those who are symptomatic to identify COVID-19 patients from those with other aetiologies.

However, any test is only useful when it is used at the right time in the right place with the right information.

Positive COVID-19test results are interpreted as indicating active infection with replicating virus whilst negative results are used to rule this out. So long as tests are used in ways to reduce false negatives and false positives, the role of PCR tests has seemed essential in mitigating the epidemic spread of SARS-CoV-2.

In the current issue of Clinical Infectious Diseases, Jefferson and others presented systematic review providing information on interpreting PCR results in relation to viral cultures.

Their report highlights the necessity to understand PCR tests in the context of the distinct concepts of infection versus infectiousness, and viable viruses versus non-infectious RNA

In their systematic review of studies, all case series in which investigators evaluated PCR positive results and compared them to viral culture, Jefferson and others found many specimens despite being PCR positive failed to grow SARS-CoV-2 in culture.

Culture medium presents an ideal condition for viruses to grow and may detect virus that is not present in the quantities required to initiate infection in a human host.

Logically, if a specimen is culture negative, one might conclude it does not contain viable virus, which might indicate decreased infectious potential to others. On the other hand, the presence of genetic material detected in PCR might be able to reflect live virus below the level of detection culture or it may represent a false positive test i.e. no viral RNA or non-infectious viral RNA from a SARS-CoV-2 infection in the recent past.

The existence of people who are PCR positive for SARS-CoV-2 yet not infectious, makes the. interpretation of PCR tests complicated.

During this pandemic there was evidence presented that there were two types of tests, those testing for active infection such as PCR and rapid antigen tests and those used for diagnosing past infection such as antibody tests.

However, the studies that Jefferson and others review suggests that the facts are more complicated. PCR tests for the presence of specific RNA sequence reflecting SARS-CoV-2 does not test for whole viruses and indeed cannot distinguish between live virus and RNA fragments with no infectious potential.

However, does this mean that the PCR test is worthless? Not really. The longer after symptom onset a specimen is taken, the less likely it is to contain viable virus.

In particular, Jefferson and others noted that infectious potential declined after day eight following symptom onset, even amongst cases with ongoing high viral loads. Specimens were also less likely to culture from patients with milder symptoms versus more severe symptoms. This was consistent with the interpretation of other authors who reviewed some of these same studies.

An inverse relationship was also observed between ability to grow virus and culture and cycle threshold. PCR tests noted for their ability to detect genetic target sequences even when only present in minuscule amounts is enabled by the core features of the technology, which amplifies genetic target sequences through repeated cycles.

There is an inverse relationship between cycle threshold and amount of genetic material in a specimen; the fewer the number of cycles needed to detect genetic material, the greater the amount of genetic material present in the specimen. The findings from Jefferson and others therefore indicate the cycle threshold, a value that may not be reported to clinicians or patients could have utility in determining infectious potential of the individual, a point that has previously been made.

The question is not whether people who are PCR positive yet culture negative ever transmit virus, but how often this occurs and in whom? How early is early infection? How late is so late one need not worry about transmission? What environmental characteristics impact transmission even when tests are positive?

Do asymptomatic people really have no symptoms or just fewer symptoms missed because we do not evaluate symptoms systematically? Even more importantly, what are the consequences for themselves and for others? There is still a lot to know about the context of testing, who to test and what it all means.

Importantly, language needs to change. PCR tests do not detect the virus, they detect the presence of known genetic sequences from which inferences are drawn. The common purveys viral shedding does not measure how much virus or even non infectious rare RNA fragments is being actively dispersed by a person, a phrase that only indicates PCR positivity. We have allowed seemingly benign short hands like detecting the virus to obscure what is actually being measured, leading to potentially erroneous conclusions with serious consequences, quarantining non-infectious persons and various aspects on other parts of people’s lives and health.

Dr Paul Ettlinger

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