Nucleic acid amplification tests (NAATs) are the current gold standard for the determination of viral load (VL) in plasma or serum through quantitative measurement of RNA or DNA targets. The presence of extracellular viral nucleic acid may indicate that the virus is actively replicating and thus capable of infecting new cells and increasing the disease burden of the patient. Uncontrolled viral replication without therapeutic intervention can then lead to disease progression with various consequences ranging from severe sequela to death. Thus, quantitative NAATs are often recommended for patients receiving treatment for viral infections such as HIV-1 and HCV.
VL monitoring in HIV-1 infection management
HIV-1 VL is the most important indicator of response to antiretroviral therapy (ART) and has become the standard of care in the clinical management of HIV-1 infected individuals. International treatment guidelines recommend that VL be measured in all patients at entry into care, initiation of therapy, and every three to six months thereafter.1-4 Optimal treatment outcome is a sustainable viral suppression—persistently undetectable HIV-1 RNA, or viral load below 50 copies/mL.1,2,5-7 Patients with levels below this threshold have the lowest probabilities of morbidity and mortality. The threshold for virologic failure is more varied; however, an analysis of AIDS clinical trial group studies indicated that a threshold of 200 copies/mL eliminated most cases of apparent viremia caused by viral load blips or assay variability.8
When assessing an HIV-1 viral load assay for implementation in the clinical setting, it is important to consider performance features of diagnostic assays used for VL measurements—for example, lower limit of quantitation (LLoQ) and linear range. Since viral load continues to be an important factor in clinical decision making, the assay chosen to monitor HIV patients must be accurate, precise, and reliable for all relevant subtypes and circulating recombinant forms. High precision and accuracy is particularly important at the level of detection where treatment failure or poor adherence may be considered if viral loads break through a low-level threshold.
A variety of methods are used for viral load monitoring in a clinical sample. The most commonly used methods in clinical laboratories include polymerase chain reaction (PCR), nucleic acid sequence–based amplification (NASBA), branched DNA (bDNA), and real-time transcription mediated amplification (TMA) assays. PCR requires a thermal cycler to produce a series of temperature changes between 50 and 95 degrees, allowing specific primer and probes to anneal to nucleic acid targets and drive amplification. In contrast, TMA is an isothermal technique that utilizes biochemical reactions, rather than mechanical thermocycling, to drive amplification for reliable and robust target amplification. That technique, along with target-specific sample purification based on target capture, ensures high sensitivity and specificity as evidenced by its wide application in blood screening.9-11
There are various commercial viral load assays based on these distinct chemistries on automated platforms available for antiretroviral treatment monitoring, approved by regulatory agencies around the world. Studies indicate that currently available PCR-based viral load monitoring assays offer similar performance and correlation in the higher quantification range but decreased concordance at low viral load.12-16 As therapies improve for HIV-1, higher scrutiny on the precision and accuracy will be expected at low viral loads to distinguish true clinically significant changes in viral load from background noise that may result from assay variation. Thus, precision and accuracy at critical thresholds need to be thoroughly evaluated prior to an assay’s clinical use.
For example, in a study comparing commercial HIV-1 PCR assays, variability in assays’ detection rates was seen at the 50 copies/mL threshold.17 This variability in detection confirmed previous findings: good correlation at relatively high viral loads, e.g. at 100 copies/mL (CV of 33 percent), and poor interassay concordance at low level viremia, e.g. at 25 copies/mL (40.7 percent to 83.1 percent).18 A more recent study compared the performance of the NASBA, PCR, and TMA viral load assays in 404 plasma samples.19 Although overall agreement among the four viral load assays was good, the PCR and TMA assays demonstrated the highest correlation. Further, they exhibited excellent precision (< 9 percent at 56 cp/mL), were equally sensitive in the clinically relevant viral load range, and detected the major HIV-1 group M subtypes. Consistent with these findings, another study found similar high precision with the TMA assay (9 percent CV at 60 copies/mL) even when compared among instruments, operators, lots, and runs.20
Application of VL monitoring to other diseases
Viral load monitoring plays a key role in various other diseases, including hepatitis (HCV RNA and HBV DNA). Quantitating levels of HCV RNA is the standard of care for the diagnosis and monitoring of HCV-infected patients during treatment and has been incorporated into treatment guidelines in the United States and Europe.21,22 The goal of HCV therapy is to cure the infection as judged by a sustained virologic response (SVR), which is defined as undetectable HCV RNA plasma/serum concentration at 12 (SVR12) or 24 (SVR24) weeks after treatment completion using a sensitive HCV RNA quantitation assay with a LLoQ less than 25 IU/mL.22
As is the case with HIV RNA assays, the performance of commercially available HCV assays differs in precision, limit of detection, and limit of quantitation that could potentially impact treatment decisions.23 The utility of viral load monitoring for patients under treatment with the new direct acting agents is an area of active investigation.24,25 In a recent study comparing two commercially available PCR-based assays, HCV-RNA levels measured by either assay at week 2 in genotype 3 patients on sofosbuvir/ribavirin therapy were predictive of treatment outcomes. The slightly more sensitive assay was also shown to be predictive of outcomes at weeks 1 and 4.25 It has yet to be seen if measurement of very early viral kinetics with a TMA-based assay may be useful to identify those patients who might benefit from very short treatment durations with highly potent antiviral therapies. Sensitivity may prove increasingly important as treatments evolve to include only a single measurement to predict when treatment is effective.
The importance of viral load monitoring continues to expand as new technologies enable the detection and quantitation of extremely low levels of virus with high precision and accuracy. NAAT remains the standard of care for monitoring the progression of infection in global epidemics such as HIV and HCV. New technologies have enhanced our knowledge of these infections and will hopefully lead to better long-term care for those undergoing treatment. Further, some technologies facilitate efficient processing and workflow management necessary to meet healthcare mandates for expanding services and reducing cost while maintaining the highest levels of quality.
Scott Hauenstein, PhD, serves as senior manager of scientific affairs for Hologic, Inc., developer and manufacturer of the Aptima HIV-1, HCV, and HBV assays, currently CE-marked (not available for sale in the United States) for viral load monitoring, and the
Panther automated system for molecular diagnostics.