The Truth about SARS-CoV-2 Cycle Threshold Values Is Rarely Pure and Never Simple

Abstract

Nucleic acid amplification tests (NAATs) are the reference standard methods for severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) detection because of their high analytical sensitivity and specificity. Many NAATs, which use reverse transcription real-time PCR (RT-PCR), are clinically validated, technically validated, and authorized by the US Food and Drug Administration (FDA) to be interpreted qualitatively as “detected” (positive for SARS-CoV-2 RNA) or “not detected” (negative for SARS-CoV-2 RNA). As of this writing there are over 250 SARS-CoV-2 molecular diagnostic tests that have obtained emergency use authorization from the FDA. The primary results generated by RT-PCR are fluorescent light emissions; serial detection of this fluorescence is plotted and the amplification curves visualized. Positive or negative interpretation depends on whether or not the curve exceeds a specified signal threshold. Part of the resulting process includes determination of the number of cycles needed before the fluorescent signal crosses this threshold (Ct value). In general, the more viral RNA in the initial specimen, the fewer the number of amplification cycles required to generate a positive signal; thus, the lower the Ct value, the higher the viral burden in the primary sample. Though all current SARS-CoV-2 NAATs are authorized only for qualitative interpretation, as of December 10, 2020, the FDA explicitly states that the Ct value results may be reported by the clinical laboratory in addition to the qualitative interpretation. Throughout the pandemic, many scientists, physicians, politicians, and public citizens have attempted to emphasize the importance (or unimportance) of certain pandemic-related interventions, mitigation strategies, the disease itself, and testing approaches. Some have advocated that a specific variable is most important in a testing approach and should be maximized to the potential detriment of the others: detection limit and specificity, cost, turnaround time, sample type, or accessibility of collection. If the truth was obvious, then there would be little debate, but the debate has been important and earnest. As Oscar Wilde wrote, “The truth is rarely pure and never simple.” We suggest that this quote describes the current situation on the debate over the relevance of Ct values, and we will explore the clinical utility of quantitative SARS-CoV-2 testing here. Though NAAT methods remain the reference standard for the qualitative detection of SARS-CoV-2 in clinical samples, there are important nuances to consider regarding Ct values. The College of American Pathologists has urged caution in using SARS-CoV-2 Ct values for the purpose of clinical decision-making (1). Several important points were raised that illustrate the difficulty of interpreting Ct values without additional information that is typically not readily available or may not be routinely considered: (a) The amount of detectable viral RNA in a clinical specimen is impacted by multiple variables, including the specimen collection method, specimen source, transport media type and volume, duration from specimen collection to analysis, and days from infection or onset of symptoms to specimen collection; (b) Ct values can vary substantially between and within RT-PCR methods. This variability is evidenced by SARS-CoV-2 proficiency testing data, in which Ct value differences of 3 to 12 cycles were observed from the same proficiency material (1); (c) no international, commutable quantitative reference standard material that can be used to harmonize assays across laboratories exists at this time. These concerns would largely be moot, at least for individual medical centers or hospital systems, if the number of collection variables were limited and the same RT-PCR was used for all patients. However, from a practical standpoint, supply chain issues have made such a locally standardized approach difficult to achieve. Throughout the pandemic, clinical laboratories relied on multiple collection methods, specimen types, swabs, transport media, and NAATs including RT-PCR, as well as other non-Ct–generating amplification methods, to achieve adequate testing capacity and clinically actionable turnaround times. Therefore, to use Ct values, or more generally, quantitative SARS-CoV-2 NAATs for clinical purposes, it is critical to address these challenges. Though a limited number of clinical laboratories offer quantitative NAATs to monitor influenza and other respiratory viruses, viral load testing is standard of care for numerous blood-borne viruses, such as HIV and the hepatitis viruses, as well as viruses important in transplantation, such as cytomegalovirus, Epstein-Barr virus, and BK virus. Lessons learned from the study of these viral load assays include the identification of critical variables, such as amplicon size, genomic target, primer/probe design, and nucleic acid extraction method, that contribute to differences in quantitation. Nevertheless, improvement in the inter-laboratory agreement of these virus load assays is typically observed when a common calibrator is used. For SARS-CoV-2, global efforts are underway to develop and evaluate quantitative standards, including work by the Coronavirus Standards Working Group, a consortium organized by the Joint Initiative for Metrology in Biology. The availability of a commutable, quantitative reference standard is an important step to facilitate assay harmonization and enable quantitative validation of existing qualitative NAATs, including the experiments required to characterize assay linearity, precision, and quantitative accuracy. Once assays are validated for quantitative testing, potential clinical applications for respiratory SARS-CoV-2 RNA concentrations exist, as described below. Viral burden may be useful as a prognostic marker for COVID-19 severity. For example, one study demonstrated that low Ct values (high viral burden) from nasopharyngeal swabs collected upon hospital admission at a single institution were associated with an increased patient risk of mechanical ventilation and in-hospital mortality (2). Though it remains unclear whether these findings are generalizable to other patient populations or different phases of the pandemic, one could imagine incorporating viral burden information into a risk score with additional clinical information and other common laboratory biomarkers to help assess the need for hospital admission and/or intensive care. The utility and impact of adding quantitative SARS-CoV-2 testing to these existing, widely available parameters has not yet been prospectively evaluated and would likely require multi-site, randomized controlled studies. Determining SARS-CoV-2 RNA concentrations in a sample may help to identify individuals with active infection and who are high risk for transmission. Such information may be especially useful when SARS-CoV-2 RNA is detected in asymptomatic individuals, with or without a previous COVID-19 diagnosis. Most agree that a high viral burden (low Ct value) is indicative of active infection, as viruses from these samples are more likely to be recoverable via cell culture. However, low viral burden (high Ct value) specimens can be more challenging to interpret. The differential diagnosis in cases where the Ct value is high and the individual is asymptomatic includes assay false positivity, persistent post-infection shedding of viral nucleic acid, asymptomatic infection, pre-symptomatic infection, or active infection. To date, a specific, commutable Ct threshold that differentiates active infection from a recovered infection has not been convincingly justified. Ct values associated with patients with COVID-19 span the detectable range, vary by specimen source, and can sometimes be recovered by culture even when the Ct value is high (3, 4). It therefore remains a major challenge to interpret the clinical significance of high Ct values in asymptomatic individuals. High Ct values are often at or near the limit of detection, which further complicates the interpretation. While reproducible RNA detection from the same primary specimen may help rule out a technical false positive, the absence of reproducibility does not necessarily confirm a false positive result or preclude the remaining options on the differential diagnosis list. Testing a second specimen collected 12 to 24 h later may be useful to ensure that the original specimen was not collected early in infection while the viral burden was increasing. Unless clinically validated for a specific use case, routine reporting of Ct values for SARS-CoV-2 is not recommended at this time, as it offers limited value and creates the potential for misinterpretation of the results. In the specific situation of an asymptomatic individual testing positive for SARS-CoV-2, Ct values in combination with repeat testing of a new specimen can provide useful information. As with all of laboratory medicine, it is imperative that the laboratory findings and the clinical scenario are interpreted as a whole, in order to maximize the likelihood of arriving at the truth. All authors confirmed they have contributed to the intellectual content of this paper and have met the following 4 requirements: (a) significant contributions to the conception and design, acquisition of data, or analysis and interpretation of data; (b) drafting or revising the article for intellectual content; (c) final approval of the published article; and (d) agreement to be accountable for all aspects of the article thus ensuring that questions related to the accuracy or integrity of any part of the article are appropriately investigated and resolved. Upon manuscript submission, all authors completed the author disclosure form. Disclosures and/or potential conflicts of interest: D.D. Rhoads, vice chair of the College of American Pathologists’ Microbiology Committee. D.D. Rhoads, former scientific advisor for Luminex and Talis Biomedical; B.A. Pinsky, former scientific advisor for GenMark Diagnostics. None declared. None declared. D.D. Rhoads has received support through contracts with Cepheid, Cleveland Diagnostics, and Luminex; B.A. Pinsky has been a co-investigator of National Institutes of Health (NIH) grant 1U54CA260517-01, received support through contracts with Agilent and PathogenDx, and received equipment and reagents from MesoScale Diagnostics. None declared. None declared.

Topics & Concepts