The role of cycle threshold values in infectious disease diagnostics

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LEARNING OBJECTIVES

Upon completion of this article, the reader will be able to:

1. Recall the strengths and limitations of RT-PCR tests.

2. Describe the definition of cycle threshold (Ct) value and how it is used in infectious disease diagnostics.

3. Discuss research into the utility of Ct values in assessing COVID-19 disease progression.

4. Discuss research into the utility of Ct values in assessing disease progression for other respiratory diseases as well as gastrointestinal illnesses.

Effective interventions in the management of infectious diseases rely on rapid and specific detection of causative pathogens, driving patient care decisions and outcomes. Successful pathogen detection is facilitated by reliable, accurate diagnostic tools; and among the variety of testing tools available, molecular assays such as polymerase chain reaction (PCR) have become dominant. Sensitivity and specificity are paramount in diagnostics and molecular testing provides both. In theory, PCR can detect the presence of a single copy of nucleic acid from a single organism. The use of sequence-specific primers and probes in these assays yields unparalleled specificity and allows for testing to be multiplexed to detect multiple pathogens in one test run. This specificity allows not only for detection, but also for determining the presence of specific genes for typing different strains and antimicrobial resistance profiling.¹ Alternatively, primers and probes can be designed to be less specific, or target highly conserved regions, to provide broad coverage and detect divergent pathogen genomes.²

PCR testing is successful in identifying organisms that cannot be grown in vitro or in situations where current culturing techniques lack sensitivity and/or require prolonged incubation periods.² Other testing methods, such as culture or serology, often do not match the level of sensitivity or timeliness of molecular testing. In some cases, alternative methods may increase the risk of false negatives or incorrect results in critical testing situations where fastidious pathogens may be difficult to grow or immune responses difficult to detect.

Utility of cycle threshold values

Effective PCR tests have now been developed for a wide variety of pathogens. However, these PCR tests, specifically real-time PCR (RT-PCR) tests, can reveal more than just the simple presence of a pathogen. Results from molecular tests are typically reported in a binary, positive or negative manner; however, this approach may not recognize the full benefits these assays have to offer. In addition to reporting pathogen detection, RT-PCR reports cycle threshold (Ct) values. The cycle threshold is defined as the thermal cycle number at which the fluorescent signal exceeds that of the background and, therefore, passes the threshold for positivity. Ct value is inversely related to the quantity of target nucleic acid in the sample, with lower Ct values reflecting higher viral loads, which can allow for comparison of relative levels of pathogen. Using this principle, a more specific quantification may be obtained with the use of a standard curve of known DNA quantity. This quantification can help establish pathogen burden and distinguish between what are normal, commensal levels and what are pathogenic levels of a microbe.

In addition to quantification, Ct values can provide additional information that may be useful when complex or unexpected results are received. Analysis of amplification curves and Ct values can be helpful in assessing sample quality and/or potential contamination. For patients with co-infections, the Ct values from a multiplex panel can help clarify which pathogen is most likely the causative agent in an illness. For patients on antimicrobial therapy, comparison of Ct values over time can be used as an indicator of the response to therapy.¹

While PCR testing and Ct values can offer a deeper insight during the diagnostic process, they are not without limitations; perhaps the most important of which is the inherent inability to distinguish between live and dead organisms. The exquisite sensitivity of PCR allows for the detection of minute quantities of nucleic acid; however, this does not always correlate with the presence of live organisms. The assay will amplify nucleic acid from dead microbes as readily as from living.¹ As dead organisms can be shed for weeks after recovery, a recovering patient might have the same positive test result as an actively infected patient. It is important to be able to distinguish between these two patients, as the positive test from the recovering patient may have no clinical relevance, while the actively infected patient may require treatment. In this situation, utilizing Ct values for relative quantification of the pathogen may help the clinician distinguish between these two patients.³

Ct values in COVID-19

The ongoing COVID-19 pandemic, with its clinical unknowns and a lack of standardized quantitative assays for Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2), has brought on an unprecedented level of interest in the clinical utility of Ct values. Patients infected with SARS-CoV-2 can display an array of clinical presentations, ranging from an absence of symptoms, to the requirement of intensive care and respiratory support, to an eventually fatal outcome.⁵

In fact, the proposed disease pathogenesis of COVID-19 indicates two overlapping disease states: an initial viremic phase where signs and symptoms are attributable to viral replication, and a subsequent inflammatory phase where the severity and critical illness is attributable to a “cytokine storm.” ⁶ While these two phases have different management implications from a treatment choice perspective – antiviral/antibody therapy versus anti-inflammatory therapy – a clear prognostic approach is still missing. The ability to predict prognosis and infectiousness of patients during the viraemic phase could fundamentally impact triage and management of patients and, potentially, of their contacts.

Early in the COVID-19 pandemic it was suggested that evidence from the 2002 SARS-CoV epidemic indicated that higher viral loads were associated with increased need for intensive care and overall worse outcomes. While there are differences between SARS-CoV and SARS-CoV-2, it has emerged that, similar to SARS-CoV, the viral load of SARS-CoV-2 could also play a key role in determining COVID-19 disease severity and prognosis, as well as infectiousness.^{3, 7-11}

^{Caption: The ability to recover infectious virus is strongly related to Ct value, as the estimated odds ratio of finding infectious SARS-CoV-2 virus decrease by 0.67 for every unit increase in Ct value. All five samples with Ct >35 from which virus was successfully propagated were from symptomatic patients and none had severe illness. Source: Centers for Disease Control and Prevention. Wide confidence levels is due to small sample size.
Source: CDC Library: COVID-19 Science Update: 09/04/2020}     

A recent systematic review assessed evidence to establish whether SARS-CoV-2 Ct values correlate with clinical outcomes and, therefore, whether they could provide valuable patient management information to clinicans.¹² The findings indicate that lower Ct values may be associated with adverse outcomes and, in several instances, a correlation with clinical course and prognosis was identified. While the review concluded that, in general, higher Ct values correlate with lower viral loads, limitations have been highlighted around the fact that Ct values and lower viral load may not be directly proportional due to the presence of inhibitory factors within samples as well as the linear dynamic range of the assay.⁴

While the results of SARS-CoV-2 PCR diagnostic assays remain – in the majority of cases – reported as positive or negative results in the clinical setting, a discussion has emerged about the possibility of reporting and interpreting Ct values to aid clinicians in patient management. The increasing evidence supporting the utility of Ct values has led public health authorities, such as Public Health England and the Centers for Disease Control and Prevention (CDC), to develop guidance and recommendations regarding their use. Experts agree that if Ct values are to be reported, linearity, limits of detection and reference quantification curves must be standardized and validated. Interpretations of a single positive Ct value may be of limited use in the clinic.^{13, 14} However, indications are emerging that serial Ct values may offer greater utility for the purpose of clinical management.¹³