Viral load monitoring: shifting paradigms in clinical practice

To earn CEUs, visit www.mlo-online.com/ce
LEARNING OBJECTIVES
Upon completion of this article, the reader will be able to:

1. Describe the importance of viral load monitoring in patient outcome and drug therapy.
2. Explain the difference between no detectable level of HCV RNA and LLOQ results.
3. Define the terms SVR, RGT, HAART, and DAA.
4. Describe the different classes of drugs used for HIV treatment.
5. Explain the importance of HCV genotyping as an aid to improve patient treatment and drug therapy.

In the past year or two, though, there have been notable changes in the viral load testing landscape for three key viruses—hepatitis C (HCV), HIV-1, and cytomegalovirus (CMV). The primary driver behind the shift has not been new technology per se but rather the availability of new therapies or other significant developments in clinical guidelines and practice.

The changing HCV landscape

For chronic HCV infection, viral load monitoring to measure HCV RNA levels in plasma or serum has played a critical role in assessing the efficacy of antiviral therapy for several years. Achievement of a sustained virologic response (SVR)—traditionally defined as undetectable serum or plasma HCV RNA 12 to 24 weeks after completion of treatment—is the primary goal of anti-HCV therapy and is typically interpreted as a clinical “cure.”^1-3

But recently introduced treatment options for HCV genotype 1 infection are bringing changes to therapy. A new standard of care has emerged with the regulatory approval of the protease inhibitors boceprevir and telaprevir. Used in combination with pegylated interferon and ribavirin, these direct-acting antiviral (DAA) drugs  can significantly improve patient outcomes vs. peginterferon/ribvirin alone.^1,4-9 They can also shorten therapy duration from 48 weeks to 24 to 36 weeks in many patients with HCV genotype 1 infection.^10-11

The use of viral load monitoring of HCV RNA levels during treatment to guide treatment duration decisions, known as response-guided therapy (RGT), has become an important part of patient management protocols.^12-14 Clinicians typically base treatment duration decisions on the viral genotype as well as the rate of change in RNA levels—i.e., shorter treatment if declining rapidly and achieving non-measurable levels at defined timepoints, or longer if declining slowly, with possible cessation of treatment if declining little or not at all. Despite the complexities that RGT can add to patient management, it represents a personalized approach that can help optimize treatment safety and outcomes while minimizing the duration of periods when patients suffer drug side effects.

At the same time that the new drugs have revolutionized hepatitis C treatment, they have necessitated refinements in therapy approaches in relation to viral load levels and medical decision points. Both direct-acting antivirals act differently in terms of their viral kinetics, and it has become evident that measurement distinctions at the lower end of the dynamic range can have an impact on therapy decisions and treatment efficacy.

Refining HCV medical decision points

With the traditional standard of care, the goal of suppressing viral load to achieve SVR was relatively modest. Clinical cure was defined as achieving a goal of < 50 lu/mL, or assay readout below the lower limit of quantification (BLOQ). But recent studies have demonstrated that for hepatitis C, there is a clinically significant difference between nondetectable HCV RNA and low amplification when applied to the novel triple therapy approach of a DAA protease inhibitor plus pegylated interferon and ribavirin.

In the latter, some residual HCV RNA is detected but is reported by the software as “LLOQ [Lower Limit of Quantification], HCV RNA detected” because the level is below the test’s labeled LLOQ (BLOQ). This means that the result is outside of the linear range where the manufacturer can make a highly reproducible and accurate measurement. But the residual HCV RNA can still be detected, even at very low levels, between about 1 and 15 IUs per milliliter. Assessing detectable levels of viremia below the LLOQ can make a difference in clinical decision points and outcomes, according to a recent study.

A reanalysis of boceprevir and telaprevir clinical trials by the FDA’s Harrington and others revealed that HCV RNA measures reported as detectable/BLOQ were common during treatment and tended to peak in their frequency before or near key RGT decision timepoints. They also noted that subjects with on-treatment detectable/BLOQ HCV RNA consistently had lower SVR rates compared with subjects with undetectable HCV RNA at the same timepoint. Their conclusion was that detectable/BLOQ HCV RNA should not be considered equivalent to undetectable HCV RNA for the purposes of making boceprevir and telaprevir RGT decisions.¹

Reporting HCV viral load results

As a result, while the 2011 American Association for the Study of Liver Diseases (ASLD) guidelines have not been revised to reflect it, HCV therapy in practice is beginning to reflect new medical decision points. The critical takeaway of this for laboratorians is how to report results for HCV viral load tests. When the instrument provides a readout of “Less than the Lower Limit of Quantification, HCV RNA detected,” many labs combine that with the “Target Not Detected” result, and in their LIS system it simply indicates the result is either negative or less than the LLOQ, suggesting that nothing was measured.

But the two results clearly mean something different from the standpoint of clinical guidance to treatment decisions. And assays from different manufacturers cannot be substituted without clinical validation in drug trials. So the best practice for labs in reporting HCV viral load is to transfer unmodified test results directly to the LIS and not bundle them together or include interpretations. Individual assay manufacturers can work with labs to educate physicians and pharmaceutical colleagues about how the test works and what the results mean, to minimize confusion and help optimize the use of the new DAA drugs in clinical practice.

The end of response-guided therapy?

There is also a very dynamic drug pipeline in hepatitis C, and the day is approaching when HCV therapy will probably be an interferon-free regimen in which an injectable is not needed and the entire treatment is administered orally. Clinical trials are ongoing, but theoretically this could be a reality as soon as 2015.

Thus response-guided therapy could become a thing of the past, along with the current drug-specific rules and different cut points that are unpopular with physicians. The new approach will likely incorporate combination therapy for a fixed period of perhaps 12 weeks and then a follow-up period. This should not impact HCV viral load test design, as current PCR assays have a cutoff of the linear range around 10 to 15 IU/mL, which is probably about as good as it is going to get and sensitive enough for clinical needs.

HIV: assessing community viral load

One of the most significant changes in the HIV field is a growing interest in expanding viral load testing throughout the developing world, which was strongly recommended in the 2013 WHO guidelines.¹⁵ In addition, there is a trend emerging related to the concept of community viral load, which involves the use of viral load testing not simply for guiding patient treatment decisions, but also for public health analysis and planning.

The idea of community viral load is based on a two-part premise: (1) the goal of HIV treatment is to repress replication and to get viral loads as low as possible; and (2) the higher a patient’s viral load, the greater the chance of that person transmitting HIV. Community viral load is a measurement of how well the public health system and clinicians are doing at finding and treating patients and suppressing viral load, and in turn reducing the risk of new infections in the community. Of course, like any simplifying summary statistic, it does not tell the whole story and should be complemented by other measurements, particularly the HIV prevalence rate.¹⁶  The impetus for the trend came from initial reports out of Vancouver and San Francisco, but there is likely to be more widespread interest as viral load testing becomes more widely available and adopted in the developing world.^17,18

Detecting low-level viremia

Similar to HCV viral load testing, there is increasing discussion about the clinical utility of detectable HIV virus below the LLOQ of an assay. For highly active antiretroviral therapy (HAART) naïve patients, the treatment goal is to have an undetectable viral load within 12 to 24 weeks of the onset of therapy.¹⁷ So as a starting point, clinicians need to use an assay that can detect HIV-1 at levels low enough for them to monitor the patient’s response to therapy according to their cutpoints and adapt the therapeutic regimen accordingly. (Current FDA-approved HIV-1 viral load tests have LLOQs that range from 20 cp/mL to 75 cp/mL.)

However, a “target not detected” or “undetectable” result for a real-time PCR test could either mean that HIV-1 is not present in the sample or that the target is present but at a level below the LLOQ. Some tests are able to detect concentrations lower than the LLOQ with lower positivity rates. For example, instead of a 95% positivity rate for copies/mL concentrations above the LLOQ, the test may offer a 50% positivity rate for a lower concentration. That would mean that when a patient’s sample at that concentration is tested 10 times, it will generate a detectable result five times and an undetectable or “target not detected” result five times.

Some recent HIV-1 studies have suggested that there is clinical value in detecting very low-level viremia in HAART patients.^20-22 Doyle et al reported that for patients with a viral load 20 The results indicated that time to virologic failure was significantly shorter for patients with a viral load of 20-39 cp/mL and 40-49 cp/mL when compared to patients with a baseline viral load of

The rise of integrase inhibitors

As HIV evolves and mutates, researchers have also gained a better understanding of the biology of the virus. This has led to the development of integrase inhibitors, a new class of efficient antiretroviral drugs. The integrase inhibitors are playing an increasing role as the “backbone” of antiretroviral regimens, replacing protease inhibitors or non-nucleoside reverse transcription inhibitors. In particular, raltegravir is being used successfully for the first line of therapy in combination with tenofovir and emtricitabine.^19,23 Additional integrase inhibitors, such as elvitegravir and dolutegravir, are now available, and more compounds in this class are being developed.

As pharmaceutical companies continue to focus on the integrase region of the HIV-1 genome, viral load test manufacturers are working to ensure the capability of assays and specifically primer/probe designs to withstand the rapid and constant genomic changes the virus undergoes. A naturally occurring mutation event or the increased pressure of selection from antiretroviral therapy drugs can alter the target sequence of primers and probes and result in reduced efficiency or the assay not being able to detect HIV-1 viruses.

One assay design strategy to mitigate the potential effect of mutation-induced changes in the HIV-1 nucleic acid sequence is to look at other (conserved) regions of the virus genome for a test target, to avoid regions that mutate in response to antiretroviral drugs. This approach is designed to add redundancy to the test and minimize the potential for mismatches and under-quantification of the virus.

On the HIV horizon

Looking ahead, there are two trends that promise to help shape the direction of HIV testing. One is the increasing use of PCR testing instead of traditional serologic testing for the diagnosis of HIV. The CDC already has new guidelines that suggest a role for HIV viral load testing in diagnosis. It’s clear that the key patients to diagnose are the ones who have very early infection, and viral load testing could identify these patients before they’ve mounted the immune response required for detection through a serologic test. Early detection may help these individuals, who are unaware they have HIV, and typically have very high viral loads, to prevent spreading the virus to other people. Additionally, early treatment may offer these patients a better prognosis and, hypothetically, better chances for a cure, because treatment may reduce the size and genetic diversity of their HIV reservoir.

The other key trend is the renewed interest in the HIV reservoir; that is, cells in the body that still carry latent HIV integrated into the host genome, even after antiretroviral treatment. This reservoir is really the barrier to curing individuals with HIV. Researchers are trying to better understand the number and types of cells carrying HIV, where these cells are in the body, and the proportion of these virus genomes which are able to replicate if induced. Other important questions are the mechanisms that create and maintain the reservoir, and the extent to which viral replication is continuing when patients are on an effective antiretroviral therapy. One important research goal will be the development of PCR-based tests that more accurately predict how patients are likely to respond if treatment is stopped or interrupted and identify which patients are effectively cured of HIV. In addition to someday facilitating the route to a cure, in the near term this kind of data might enable clinicians to make informed decisions about when to reduce the intensity of a patient’s antiretroviral therapy.

A new standard for CMV

In the specialized field of cytomegalovirus viral load testing for transplant patients, there is one notable development that is expected to transform clinical practice: the establishment of a WHO international unit standard and revised solid organ transplant guidelines that reflect the change.²⁴

While it is still too early to see the benefit of an international standard for reporting viral load results, the impact will likely be felt in three to five years. Most transplant patients are cared for in academic settings, where there is a great incentive to have clinical data harmonized with that of the rest of the world to permit the pooling of datasets across institutions. So labs and caregivers are expected to universally change to using international units for CMV viral load results. The 2013 consensus guidelines acknowledge that there is a lack of data at this point about which viral load thresholds trigger management changes in patients, but as more labs use the same international unit standard for measuring CMV viral load, it will be possible to accumulate data tying these measurements to clinical outcomes and to develop more precise guidelines for the management of CMV disease in transplant patients.

Impact on patient management

The anticipated clinical benefits can be estimated by looking at a good analogy in anticoagulation management: the establishment of the International Normalized Ratio, or INR, standard for the prothrombin time test. That standardization process has enabled cardiologists and other healthcare professionals to take what used to be very disparate results from different laboratories and physician offices and have the results be comparable. In turn, that has made it possible to establish evidence-based guidelines for patient management.

In addition, the revised guidelines make it clear that CMV monitoring has moved into the PCR era, with quantitative PCR viral load testing as the standard of care for monitoring patients. Last year, the FDA approved the first CMV viral load IVD test kit, with traceability to the WHO International CMV standard. The development now gives labs the choice of standardizing their own lab-developed test or adopting an IVD test that reports viral load results in international units per mL calibrated to the WHO standard.

In either case, there is significant optimism in the CMV field. Ultimately, the gathering of data with similar units and similar measurement scales will make it possible to have universal clinical recommendations for CMV patient management and, in the long term, a positive impact on outcomes.