RT-PCR Viral Presence Testing And Related

PCR viral presence (nasal / throat mucous swab) tests, when positive, indicate that the viral RNA, but not necessarily viable/infectious virus, is present in the mucous swab. When symptoms and a positive test are found, it likely means the person is infected. It is also possible they are fortunate enough to just have a random virus in their mucous but not actually in their body (not likely, but may occur). Sometimes people who have recovered will have mucous remnants that contain viruses which may be the cause of some “had COVID, recovered, tested negative, then later retested positive again” cases. False positives can also be caused by non-viable viruses that cannot infect but do contain RNA. Positive/negative results, the Ct count, and patient physiology should interpreted by a professional.

The PCR viral/antigen presence test uses an “amplification” technique that enables the most sensitive equipment to detect fewer than 50 viruses in a sample. “During the exponential amplification phase, the quantity of the target DNA template (amplicon) doubles every cycle… However, the efficiency of amplification is often variable among primers and templates.”. The viral sample is placed in a reagent chemical that, when temperature cycled, results in a new copy for (nearly) every RNA/DNA copy in the sample, thus doubling the population with each cycle. The reagent also adds markers to the copies such that, at some point, the markers fluoresce with a light intensity bright enough to be detected by the equipment. The cycle where this occurs is known as the Ct. There is also a closely related measurement known as Cq. Lower values mean more RNA was present because it took fewer cycles of doubling to reach the point where fluorescence was detected.

While standards for determining positivity thresholds are not strongly developed, one study stated it’s Ct interpretations: “A cycle threshold value (Ct-value) less than 35 was defined as a positive test result, and a Ct-value of 39.2 or more was defined as a negative test. A medium load, defined as a Ct-value of 35 to less than 39.2, required confirmation by retesting.”. The amount of RNA present in the sample is also dependent upon the sample taken and how close the sample was to an actual site of infection and the number of cells infected in that area and fluid movement between the infected area and sample collection location. The luck of the swab does impact Ct.

Every PCR test result provided to clinicians and patients should include not only a positive/negative indication, but also the actual Ct count. Additionally, “It is proposed that Ct counts be grouped into categories of “strongly shedding”, “moderately shedding”, “borderline positive / lightly shedding”, and “negative”. These categories could identify a clinically useful spectrum of PCR “positive-ness”. Further, it maybe reasonable to assume that viable virus is likely when Ct counts are low, but not necessarily with borderline positive Ct counts. PCR tests detect viral RNA presence, but do not indicate virus viability”

The resources below provide a more complete and accurate understanding.

PCR Test Equipment Manufacturer ThermoFisher:
PCR Basics
Real-Time PCR: Understanding Ct

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“Viral culture was positive only in samples with a cycle-threshold value of 28.4 or less. The incidence of culture positivity decreased with an increasing time from symptom onset and with an increasing cycle-threshold value (Table S3).”

Cycle threshold (CT) values and their reference ranges, as applicable, must be reported by laboratories to FDOH via electronic laboratory reporting or by fax immediately.

Inactivated viruses most often cause positive RT-PCR indications

An inactivated virus, incapable of causing infection and symptoms, can fool a PCR viral presence test into a false positive infection indication because the RNA still exists even though the virus is inactivated / non-viable.

“A positive PCR result does NOT prove active replication of a virus. It does NOT prove infectious virus is present.”

“PCR methods do have some serious limitations for environmental viral analysis, including small sample volumes, the presence of PCR-inhibitory substances, and an inability to differentiate between infective and noninfective viruses.”

“Enteric viruses that are important causes of human disease must often be detected by reverse transcription polymerase chain reaction (RT-PCR), a method that commonly yields positive results with samples that contain only inactivated virus.”

Science Direct: RT-PCR amplification detects inactivated viruses in water and wastewater