RT-PCR: Cycle threshold value does not hold clinical relevance

A survey of 700 labs in the U.S. found that the Ct values could vary by up to 12 cycles for the same target gene across labs

February 05, 2022 08:47 pm | Updated 08:47 pm IST

How it works:  When the processed sample is placed in an RT-PCR machine, the viral genetic material is amplified. Two copies of genetic material become four, four become eight in the next cycle, and so on.

How it works: When the processed sample is placed in an RT-PCR machine, the viral genetic material is amplified. Two copies of genetic material become four, four become eight in the next cycle, and so on.

The art of medicine is taught by the bedside. It involves detailed history-taking, followed by a physical examination which employs the senses of the physician. Humans, however, prefer concrete numbers to abstract skill and intuition. Unfortunately, most of these numbers do not have any clinical relevance. Yet, they continue to be used indiscriminately to drive irrational and often expensive treatments, including unnecessary hospitalisations. At present, the most often repeated number appears to be the cycle threshold value (Ct value) reported on an RT-PCR at the time of diagnosis.

RT-PCR test

An RT-PCR test detects genetic material in a sample (for COVID, the sample used is a swab from the throat and nose). The sample, after processing, is placed in an RT-PCR machine where the viral genetic material is amplified. So two copies of genetic material become four, four become eight in the next cycle and so on. There are fluorescent marker labels present in the mixture, which attach to the genetic material and release a fluorescent dye, which is measured by the machine as it happens (“real-time”). When the fluorescence crosses a certain threshold, the machine labels the sample as testing positive, and the number of cycles that the sample goes through before reaching this threshold is called the cycle threshold (or Ct value). It follows, therefore, that greater the quantum of genetic material (or virus) in the sample, fewer the cycles to reach the threshold of detection. For approximately every 3.3 rise in the Ct value, the quantum of starting genetic material is likely to be tenfold lower.

When a swab is done in a symptomatic individual at the peak of early symptoms, the Ct value is more often than not low. This leads patients to believe that they are at a high risk of having severe disease. The consequent fear and anxiety lead to frequent blood tests, scans and a gamut of unproven therapies. This is unscientific for several reasons.

Dynamic measure

Firstly, the Ct value is a dynamic measure and can evolve rapidly. A low Ct value at the time of diagnosis does not mean that it will stay low the next day (as the body’s immunity kicks in). Similarly, a swab done very early in the infection may reveal a high Ct value, which if repeated a day or two later, may reveal a lower Ct value. It is possibly for this reason that Ct values have not been convincingly correlated with disease severity, and serve no role in predicting the trajectory for a patient (yet, this is commonly used as an argument to prescribe tests and medicines). Secondly, several technical and logistical factors influence the value: the way specimens are collected (a quick superficial swab versus an aggressive swab), the type of specimen (nasal versus pharyngeal versus nasopharyngeal), the medium in which the swab is transported, the time lag between collection of the specimen and processing. All of this can influence the quantum of viral genetic material present, and subsequently, the Ct value. In a letter published in C linical Infectious Diseases, the College of American Pathologists (CAP) Microbiology Committee reports how a survey of 700 laboratories in the U.S. using standardised proficiency testing material from the same batch found a variability in Ct values by 14 cycles. Even within the same test at the same lab the Ct values could vary by 3 cycles for different target genes, and up to 12 cycles for the same target gene across labs. The letter reiterated the fact that these RT-PCR assays were not approved to be used quantitatively, but as a positive/negative test.

The caveats

Does the Ct value have any real value at all? Studies suggest that when used early in the disease, it may correlate with transmissibility at the time of testing. This, again, needs to be viewed with the caveats of the dynamic nature of the value, and the unfeasibility of serial testing, in addition to the technical variability alluded to above. When used to decide when a person can come out of isolation in high-risk settings such as hospitals, the Ct value may be a guide in older patients, those who are immunocompromised and those who have severe disease, all of which are patient groups in whom prolonged viral shedding has been reported.

The knowledge of the Ct value offers no insight to the treating clinician or the patient, and amounts to “noise” without a meaningful clinical “signal”. It causes panic, and the value is being used to drive irrational testing and treatment measures. Labs should consider not reporting Ct values on a routine basis.

(Lancelot Pinto is a Consultant Respirologist at P.D. Hinduja National Hospital and Medical Research Centre, Mumbai)

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