RT-qPCR Testing and Performance Metrics in the COVID-19 Era
Abstract
:1. Introduction
2. Diagnostic RT-qPCR
2.1. Developmental-Stage Performance Measures
2.1.1. Limit of Detection or Analytical Sensitivity
2.1.2. Analytical Specificity
2.2. Real-World Performance Measures
- A positive result, meaning that the target infectious agent is detected:
- A negative result, meaning that the target infectious agent is not detected:
- Early or late in infection, viral load is low, and extracted RNA levels may be below the detection threshold.
- Improper sample collection technique, storage, and delays in transportation or handling can result in too low an RNA yield or degradation.
- Suboptimal reagents, inhibitors in the sample or instrument malfunction can compromise the amplification process.
2.2.1. Diagnostic (or Clinical) Sensitivity
2.2.2. Diagnostic Specificity
2.2.3. Accuracy
2.2.4. Positive and Negative Predictive Values (PPV and NPV)
- PPV answers the question: What is the probability of someone with a positive test result really having the disease? It considers both true positives and false positives, and a high PPV means that a positive test result is highly indicative of that person actually having the disease. PPV = TP/(TP+ FP).
- NPV addresses the question: What is the probability of someone with a negative test result truly not having the disease? It considers both true negatives and false negatives, and a high NPV means that a negative test result is highly indicative of that person actually being disease-free. NPV = TN/(TN + FN).
2.2.5. Likelihood Ratios (LR)
- Positive likelihood ratios (PLRs) are used to measure how much a positive test result changes the odds of a condition being present compared to the odds without the test. It provides information about how well a positive test result can discriminate between individuals with the target condition and those without. PLR = true positive rate/false positive rate, i.e., Sensitivity/(1 − Specificity)
- Negative likelihood ratios (NLRs) give the change in the odds of having a diagnosis in patients with a negative test. It quantifies how much a negative test result reduces the likelihood of the target condition being present compared to the likelihood without the test result. NLR = false negative rate/true negative rate, i.e., (1 − Sensitivity)/Specificity
2.2.6. Confidence Intervals
3. Limitations of RT-qPCR Testing and Performance Measures
4. RT-qPCR Testing in the Context of the COVID-19 Pandemic
5. Conclusions
Funding
Institutional Review Board Statement
Informed Consent Statement
Data Availability Statement
Conflicts of Interest
References
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Bustin, S.A. RT-qPCR Testing and Performance Metrics in the COVID-19 Era. Int. J. Mol. Sci. 2024, 25, 9326. https://doi.org/10.3390/ijms25179326
Bustin SA. RT-qPCR Testing and Performance Metrics in the COVID-19 Era. International Journal of Molecular Sciences. 2024; 25(17):9326. https://doi.org/10.3390/ijms25179326
Chicago/Turabian StyleBustin, Stephen A. 2024. "RT-qPCR Testing and Performance Metrics in the COVID-19 Era" International Journal of Molecular Sciences 25, no. 17: 9326. https://doi.org/10.3390/ijms25179326