How can threshold values, such as Cycle Threshold (Ct) from real-time Polymerase Chain Reaction (PCR), be used in infectious disease diagnostics?

Utilizing Cycle Threshold (Ct) Values in Infectious Disease Diagnostics

Cycle threshold (Ct) values from real-time PCR should not be used alone to determine infectiousness or guide clinical decisions due to substantial interlaboratory variance and lack of standardization across different testing platforms. 1

Understanding Ct Values in Real-Time PCR

• Ct value represents the PCR cycle at which target-specific amplification signal becomes detectable, potentially inferring viral load in a sample 2
• Lower Ct values generally correlate with higher viral loads, while higher Ct values indicate lower viral loads 2, 3
• Ct values are inversely related to the amount of target nucleic acid present in the sample - the lower the Ct value, the higher the amount of target nucleic acid 1

Limitations of Ct Values in Clinical Decision-Making

• The Infectious Diseases Society of America (IDSA) concluded that data are insufficient to establish SARS-CoV-2 infectiousness based on nonstandardized instrument signal values such as Ct values 1
• Substantial interlaboratory variance in reported Ct values makes quantitative estimation of pathogen concentration unreliable for clinical decisions 1
• Ct values are often mistakenly considered as standardized quantitative metrics, when in fact they vary significantly between different test systems and laboratories 1
• IDSA specifically recommends against using Ct values to guide release from isolation for COVID-19 patients 1

Factors Affecting Ct Values

• Pre-analytical variables affecting Ct values include:

  • Collection technique and specimen type 3
  • Sampling time relative to infection onset 3, 4
  • Transport and storage conditions 3

• Analytical variables affecting Ct values include:

  • Nucleic acid extraction method 1
  • Different batches of the same reagent may perform differently 1
  • PCR platform and test design 1
  • Target gene selection 5

• Post-analytical variables:

  • Method of Ct value determination 3
  • Interpretation thresholds vary between laboratories 2

Potential Applications of Ct Values

• Monitoring individual patients over time to assess viral clearance or response to therapy, rather than comparing between patients 1
• In research settings, establishing specific cut-off thresholds through statistical modeling may help differentiate causative pathogens from incidental detection 6
• Ct values may provide supportive information when combined with other clinical and laboratory data, but should not be the sole determinant for clinical decisions 2

Best Practices for Ct Value Interpretation

• Consider Ct values as qualitative or semi-quantitative rather than truly quantitative measurements 1
• Interpret Ct values in conjunction with clinical presentation, timing of symptom onset, and other laboratory findings 3
• Be aware that the same Ct value may have different implications across different testing platforms and laboratories 1
• External quality assessment (EQA) schemes have demonstrated that Ct values cannot be reliably compared between different laboratories or test systems 1

Common Pitfalls to Avoid

• Assuming Ct values are standardized across different testing platforms or laboratories 1
• Using arbitrary Ct value cutoffs to determine infectiousness without validation 1
• Relying solely on Ct values for patient management decisions without considering clinical context 2, 3
• Failing to recognize that detection of pathogen nucleic acid does not necessarily indicate viable, infectious organisms 1
• Assuming a negative PCR result following a positive result proves a patient is no longer infectious 1