Clinical Applications of Quantitative Real-Time PCR in Virology

Abstract

Since the invention of the polymerase chain reaction (PCR) and discovery of Taq polymerase, PCR has become a staple in both research and clinical molecular laboratories. As clinical and diagnostic needs have evolved over the last few decades, demanding greater levels of sensitivity and accuracy, so too has PCR performance. Through optimisation, the present-day uses of real-time PCR and quantitative real-time PCR are enumerable. The technique, combined with adoption of automated processes and reduced sample volume requirements, makes it an ideal method in a broad range of clinical applications, especially in virology. Complementing serologic testing by detecting infections within the pre-seroconversion window period and infections with immunovariant viruses, real-time PCR provides a highly valuable tool for screening, diagnosing, or monitoring diseases, as well as evaluating medical and therapeutic decision points that allows for more timely predictions of therapeutic failures than traditional methods and, lastly, assessing cure rates following targeted therapies. All of these serve vital roles in the continuum of care to enhance patient management. Beyond this, quantitative real-time PCR facilitates advancements in the quality of diagnostics by driving consensus management guidelines following standardisation to improve patient outcomes, pushing for disease eradication with assays offering progressively lower limits of detection, and rapidly meeting medical needs in cases of emerging epidemic crises involving new pathogens that may result in significant health threats.

Keywords: Clinical laboratory; Continuum of care; Diagnostics; Molecular; Quantitative; Real-time PCR; Virology.

Figure 1

Example of amplification and detection of target nucleic acid by real-time PCR.

Figure 2

Quantitation of viral target using competitive quantitation standard (QS). The QS compensates for effects of inhibition and controls the preparation and amplification processes, allowing a more accurate quantitation viral target in each specimen. The competitive QS contains sequences with identical primer binding sites as the viral target to ensure equivalent amplification efficiency and a unique probe binding region that distinguishes the two amplicons. The competitive QS is added to each specimen at a known copy number and is carried through the subsequent steps of specimen preparation, reverse transcription (when applicable), simultaneous PCR amplification, and detection. Viral target concentration in the test specimens is calculated by comparing the viral target signal (solid line) to the QS signal (dashed line) for each specimen and control (A, B). In the presence of inhibitors, both QS and viral target are equally suppressed and yield accurate viral load calculations (C).

Figure 3

Likelihoods of different test results given different viral concentration. When the viral concentration tends to 0, the proportion of ‘Target not Detected’ increases to 1 (dotted line), increasing the likelihood of ‘Not Detected’ results. As the concentration tends to LLOQ (dashed line), the likelihood of ‘Detected but < LLOQ’ results peaks. When the concentration tends to infinity, the proportion of quantitative results tends to 1 (solid line), resulting in a continual increase in the likelihood of ‘Detected: Quantitative’ results. At any concentration, the sum of the three types of reported results is always 100% and throughout the concentration continuum, variations in result reporting exist. As the concentration of viral levels approaches the LLOQ, near equal likelihood of ‘Detected: Quantitative’ and ‘Detected but < LLOQ’ results are possible, as are ‘Not detected’ results. Decreasing concentrations will further shift the likelihoods and increase the chance of ‘Detected but < LLOQ’ or ‘Not Detected’ results. Diagram assumes LLOQ = LLOD.

Figure 4

Applications of real-time PCR in the continuum of care and patient management.

Figure 5

Biomarker appearance timeline for HIV-1 infection. Diagnostic testing for HIV-1 infection is dependent on the time from infection. HIV RNA levels begin to rise immediately after infection (solid line) but do not reach detectable levels until approximately 10 days post-infection, signalling the start of the acute HIV infection. Only during the process of seroconversion do p24 antigen (dashed line) and HIV antibody (dotted line) reach detectable levels.

Figure 6

Monitoring HCV viral loads during treatment. Despite advances in treatment for HCV patients, failure to achieve SVR is still a reality. Patients who do not achieve SVR fall into four categories: (1) null responders (black line) achieve less than 2-log decrease in hepatitis C viral load upon treatment; (2) partial responders (red line; light grey in the print version) experiences at least a 2-log decrease in hepatitis C viral load during HCV treatment but fail to proceed to an undetectable viral load level; (3) breakthrough patients (orange line; light grey in the print version) have an undetectable HCV viral load, but the virus rebounded during treatment; (4) relapsers (blue line; dark grey in the print version) have had an undetectable HCV viral load, but the virus rebounded after they completed HCV treatment.

Figure 7

On-treatment HIV patient monitoring. (A) HIV viral loads will fluctuate as patients are on treatment, and, in most instances, will remain ‘undetectable’ (at or below dotted line); viral ‘blips’ are not uncommon and will result in transient ‘detectable’ and even quantifiable results (above the dashed line). (B) Virologic failure will lead to a sustained high-level viral titre that, without intervention, will increase with time.