To Interpret the SARS-CoV-2 Test, Consider the Cycle Threshold Value

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Salient features:

This article focuses on:

  • Role of cycle threshold score in the interpretation of SARS-CoV-2 Test

Detailed summary:

  • To prevent the spread of COVID-19, it is important to identify and isolate people who are infectious.
  • It is important to ensure that those with a high viral load are isolated and not able to transmit to others.
  • Currently, diagnosis, screening, and surveillance depend on a SARS-CoV-2 reverse transcriptase quantitative PCR (RT-qPCR) test and results are generally reported to the ordering physician as positive or negative.
  • However, the test does cycle threshold (Ct) value.
  • Reporting the Ct value, or a calculate viral load, can aid in interpretation and clinical decisions.
  • Ct values provide valuable information about the amount of viral RNA in the samples and particularly later in the course of infection.
  • The Centers for Disease Control and Prevention (CDC) suggest one of two approaches for discontinuing isolation, one based on time since symptom onset and resolution, and the other centred on two negative tests at least 24 hours apart.
  • The first approach assumes a person is generally no longer transmitting virus 10 days after symptom resolution.
  • This approach is test-sparing and is particularly useful when resources are scarce or in the outpatient setting where repeat testing is onerous.
  • When testing is available, decisions to lift isolation rely heavily on negative PCR tests to define a patient as no longer infectious.
  • However, if a positive PCR test is intended to mean infectivity, then this approach may not be optimal. Closer examination of the test results from RNA quantities near the lower limit of detection of the assay could help guide clinical and public health strategies.
  • The SARS-CoV-2 RT-qPCR test provides real-time quantification by first reverse transcribing SARS-CoV-2 RNA into DNA (RT step), and then performing qPCR where a fluorescence signal increases proportionally to the amount of amplified nucleic acid, enabling accurate quantitation of the RNA in the sample.
  • If the fluorescence reaches a specified threshold within a certain number of PCR cycles (Ct value), the sample is considered a positive result.
  • The Ct value is inversely related to the viral load and every ~3.3 increase in the Ct value reflects a 10-fold reduction in starting material. Many qPCR assays involve a Ct cutoff of 40 to consider the test positive, allowing detection of very few starting RNA molecules.
  • This high sensitivity for viral RNA can be helpful for initial diagnosis.
  • High Ct value represents presence of very low copies of viral RNA.
  • In the cases, where viral RNA copies in the sample may be fewer than 100, results are reported to the clinician simply as positive. This leaves the clinician with little choice but to interpret the results no differently than for a sample from someone who is floridly positive and where RNA copies routinely reach 100 million or more.
  • We propose that for inpatients whose symptoms have fully resolved and two tests over 24 hours apart are either negative or close to the Ct cutoff (i.e. Ct >34), they likely do not have meaningful or transmissible disease, and thus do not need to be retested.
  • This would conserve valuable testing capacity, reagents, and PPE.
  • The clinician could take the Ct results in context and determine when the patient can discontinue isolation.
  • This could shorten duration of isolation and for healthcare workers and other essential workers would provide a more evidence-based, testing-informed pathway for more rapid return to work.
  • Taking the Ct value into account also help justify symptom-based strategies recommended by the CDC including time-since-illness-onset and time-since-symptom-resolution based approaches.
  • The Ct value could be high as a result of early disease and the Ct value would have to be considered in clinical context.
  • A person with a high Ct value tested early in the disease course might be or become infectious and this would present as a significant decrease in Ct value 24 hours following the first test.
  • A patient with resolved symptoms and two Ct values both close to the cutoff is likely recovering and no longer infectious. Evidence from both viral isolation and contact tracing studies support a short, early period of transmissibility.
  • By accounting for the Ct value in context, RT-qPCR results can be used in a way that is personalized, highly sensitive, and also more specific.
  • To implement this, the actual Ct values could be reported along with reference ranges or converted to viral load and or categorized as high, medium, or low.
  • Repeat testing over 24 hours is not always feasible and is always resource heavy when testing is limited.
  • Time since symptom onset or time since symptom resolution based approaches may be as or more useful in many situations.
  • Xiao et al. showed that older patients were more likely to have prolonged positive results and presumably longer infectivity.
  • Severe disease is also a risk factor for longer viral shedding.
  • Prolonged positive SARS-CoV-2 RT-qPCR results raise questions about the sufficiency and sustainability of current isolation guidelines.
  • The Ct value from positive test results, when interpreted in context, can help to refine clinical decision-making.

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