POSITIVELY AWARE JULY/AUGUST 2011
Undetectable by whose standard? New viral Load tests are more sensitive, but what does that really mean? By Tony Mills, MD.
When I first got involved in HIV in the mid 1980s, we hadn’t yet isolated the virus that causes AIDS. Although we didn’t know the etiology of the infection, we knew it destroyed the body’s immune system and the measurement of the CD4 T-cells was the most important number in the life of a patient with “GRID” (Gay-Related Immune Deficiency), the disease that would become known as AIDS.
Soon we isolated the virus, found a cause for HIV, and an antibody that told us who was infected.
In the HIV world, a popular analogy in the early years was that having HIV was like being on a train speeding towards a cliff: the distance to the cliff was the T-cell count and the speed of the train was the HIV viral load. It was a fatalistic analogy but, unfortunately, a somewhat apt one. The train speed (HIV VL) was impossible to control, but we fought tooth and nail to maintain the distance from that cliff (CD4 count). The T-cell count was the most important number in an HIV-positive person’s life.
From T-cells to viral load
Three decades have passed since the first reports of the epidemic, and how we measure success against the virus has changed in many ways. The T-cell count is certainly still important. It predicts who might be at risk of opportunistic infections and it monitors our progress in immune restoration as we move back towards the world of the living and contemplate the long and bright future ahead. But it is the HIV viral load that is truly the bellwether. A low viral load is a surrogate marker of health and it tells us that the medications are working well, that we are doing everything we can to fight the disease, and that we are winning the battle against our foe.
Assessment of HIV viral load is one of the best predictors of clinical disease progression and it is the main parameter used to evaluate treatment response in HIV-positive patients. Reproducible and ever more sensitive assays based on real-time PCR (polymerase chain reaction) technology have been developed to quantify HIV in the bloodstream of positive patients. Recent improvements have allowed reliable measurements of various strains of HIV, including the difficult-to-quantitate non-B subtypes. As we look towards the future, and certainly in cutting-edge research today, testing specimens other than serum or plasma for HIV has become important. This has been a challenging task, to measure HIV viral load without plasma, but it is an important goal, particularly for developing parts of the world.
In almost all cases, HIV infection results in a lifelong persistent presence of the virus, despite evidence for almost complete suppression of viral replication using highly active anti-retroviral therapy (HAART). Sensitive and reproducible means for quantifying plasma viremia (viral load) have been developed since the mid-1990s when a landmark study conducted on specimens collected from individuals enrolled in the Multicenter AIDS Cohort Study (MACS) demonstrated that plasma HIV-RNA was a better predictor of progression to AIDS and death than CD4+ T-cell counts.1,2Besides having prognostic value, soon thereafter measurement of plasma HIV-RNA was found to provide the most accurate information as to the efficacy of antiretroviral drugs and herein lies the key to its importance today.
When first attempts were made to measure HIV viral load, reverse transcriptase (RT)-PCR methodologies were modified to assess as accurately as possible the circulating HIV-RNA molecules in the plasma. In more recent years, the development of RT-PCR technologies has resulted in improvements in sensitivity and in the time required for the assay. All RT-PCR assays were originally designed to measure and quantify the replication of clade B virus. As the global initiative has grown and antiretroviral therapy is now more accessible in developing regions of the world, the challenge of testing HIV-1 non-B subtypes has become more and more of an issue and the need exists for novel technologies adept at measuring this strain of the virus.
The newer real-time viral load testing technologies produce results faster, display larger dynamic ranges, use fully automated extraction procedures, and allow for measurement of greater numbers of samples. All these characteristics are important in both resource-rich countries and in resource-limited environments. Unfortunately, in these resource-poor settings, the required laboratory infrastructure is often found only in major urban areas and isn’t available in the rural environments where much of the HIV epidemic is located. In resource-limited settings, a large emphasis has been placed on developing techniques to assess HIV viral load in some way other than using plasma. The use of dried blood spots has proven to be invaluable in these undeveloped areas for both the process of viral load monitoring and for drug resistance (genotype) testing and HIV sub-typing.
Over the years, many diagnostic companies have been involved in the creation of assays to accurately assess HIV viral load. As a result of all of this research, several methods of quantifying HIV viral load have been developed. Four different commercial viral load assays based on distinct principles are currently approved by the regulatory agencies in the U.S. and/or Europe, and are widely used for antiretroviral treatment monitoring. The most recent tests are based on nucleic acid amplification by PCR and real-time detection. The four assays are: COBAS TaqMan (Roche; Pleasanton, CA);3 NucliSENS EasyQ HIV-1 v2.0 (bioMérieux; Boxtel, Netherlands);4,5 RealTime HIV-1 m2000rt (Abbott; North Chicago, IL);6,7 and VERSANT HIV-1 RNA 1.0 kinetic PCR (kPCR; Siemens; Berkeley, CA).8 Besides real-time detection, all these assays have an initial step of automated nucleic acid isolation, in most cases based on the use of magnetic particles, which generally improves their performance. Although commercial HIV viral load assays differ in terms of principle and performance, they have all been optimized and their current sensitivity, specificity, and reproducibility are generally excellent.
All viral load methods described have provided an overall reproducibility above 95%, with co-efficient variations below 10%. They are comparable in the range of lower HIV-RNA detection limit (less than 50 copies/mL on average, though the newest Roche assay goes down to less than 20 copies/mL), as well as upper limit (greater than 107 copies/mL). In comparison studies, there is excellent correlation between the various assays. Also of great interest is the similarity in cost between the different assays. In general, there is a less than a 10% difference in direct reagent and consumable costs between assays.9, 10 Providers are faced with a variety of choices of HIV viral load assays to order for any given patient. Often providers opt to utilize whichever technology has been used in the past to monitor a specific patient. This reduces confusion and minimizes inter-assay variability in results. Conversely, providers might opt to utilize the newest technology or the most sensitive technology in an effort to be on the cutting edge of HIV diagnostic development.
It is worth noting here that with the newer and more sensitive assays, patients who were once always “undetectable” may now show variable low-level viremia. While this can be disconcerting to the patient and to the physician, it truly is a result of the increased sensitivities of the assays at low viral loads and it is most often not an indication that the medications are not working and that resistance is developing. There was so much concern in the HIV provider community about this low level viremia that the DHHS recently addressed it directly. In the latest version of the Guidelines for the Use of Antiretroviral Agents in HIV-1-Infected Adults and Adolescents, released in January of 2011, the DHHS has this to say about the new phenomenon of low-level detectable viremia:
“Optimal viral suppression is generally defined as a viral load persistently below the level of detection (<20–75 copies/mL, depending on the assay used). However, isolated “blips” (viral loads transiently detectable at low levels, typically <400 copies/mL) are not uncommon in successfully treated patients and are not thought to represent viral replication or to predict virologic failure. In addition, low-level positive viral load results (typically <200 copies/mL) appear to be more common with some viral load assays than others, and there is no definitive evidence that patients with viral loads quantified as <200 copies/mL using these assays are at increased risk for virologic failure. For the purposes of clinical trials the AIDS Clinical Trials Group (ACTG) currently defines virologic failure as a confirmed viral load >200 copies/mL, which eliminates most cases of apparent viremia caused by blips or assay variability. This definition may also be useful in clinical practice.”11
The newest HIV viral load assays have improved their sensitivity and reproducibility, and require less time to process and to provide results. It was not uncommon in the early 1990s for an HIV viral load assay to take two weeks to produce results. Now, turnover can be as quick as overnight. Another important advancement in the latest technologies is the minimization of the risk of contamination, mainly as a result of the advancements of automated RNA isolation and real-time amplification and detection. In the past, large volumes of plasma were often needed to quantify HIV viral loads. Today, the required volume of the specimen for testing is generally much lower than that required by earlier assays. In addition to being more economical, the sensitivity of the assays continues to improve. HIV viral load tests are often batched and run together for economic reasons. The number of tests performed in each run on most systems is 96, allowing large workflow for laboratories with a high number of patients. A further benefit of the new assays is that many of them have incorporated the option of testing specimens other than plasma/serum. Work is currently being done looking at seminal plasma viral loads to learn more about transmission risks in patients on and off of medications. In truly resource-limited areas where refrigeration, transportation, and easy access to local RT-PCR technology are the exception rather than the rule, dried blood spots (blots of blood on paper) are often used to assess the viral load. These DBS’s, as they are called, are more stable in unfavorable environments and, compared to tubes of blood, they are more easily batched and shipped to the nearest HIV clinical lab.
Though it is true that the newest HIV viral load assays offer many advantages, several limitations must also be addressed. The first and most important limitation is their cost. The cost of antiretroviral drugs in the developed world continues to escalate with the drive for newer, simpler, and more effective medications fueling the rising cost of the newer drugs. In the developing world, much effort has been made to decrease the costs of drugs and thereby make them affordable and within reach for the general population. While the ethical commitment to provide treatment to those who need it in these resource-limited areas has forced reduced medication costs, there has not been a parallel reduction in the costs of laboratory monitoring tests, especially in HIV viral load assays.
The cost of viral load testing using the newest commercial real-time assays is more expensive than it ever was before. In many countries and in many settings, the increased cost per test is difficult, if not impossible, to afford. Moreover, logistical and technical aspects represent a further limitation for the use of the tests. Correct use of the technologies is complex and requires a high level of education and skill. The diagnostics manufacturers often provide skilled specialist support, but this level of support is often very expensive. The supply of reagents, additional expenses due to consumables, constant air-conditioning requirements, regular electricity supply and power back-up, and availability of protocols and package product inserts in a variety of languages further limit the optimal implementation of the newest HIV viral load assays and further elevate the costs.
In today’s HIV world, the viral load assay is the most important indicator of the success of a patient’s HIV regimen. Maintenance of an undetectable viral load is fundamental: it’s how we know we are keeping the virus in check and it’s how we know we are moving in the right direction towards long-term HIV suppression and long-term survival in the battle with HIV. The currently available real-time PCR assays for HIV viral load measurements are extremely accurate and reproducible in their quantification of the level of HIV activity. They provide advantages in terms of time consumption, cross-contamination, and sample volume needed to obtain results.In addition, these tests allow an improved workflow, reliable detection of non-B variants, and permit samples other than serum/plasma to be tested. These newest HIV viral load tests are not without limitations, however. They are expensive and they require a complex laboratory infrastructure that is not usually available in many resource-poor countries. In addition, these tests allow an improved workflow, reliable detection of non-B variants, and permit samples other than serum/plasma to be tested. These newest HIV viral load tests are not without limitations, however. They are expensive and they require a complex laboratory infrastructure that is not usually available in many resource-poor countries. In resource-limited settings, efforts to provide access to adequate laboratory facilities where HIV viral load can be tested with the newest assays has to be considered as a priority, since the success and the benefit of the rapid scaling-up of first-line HAART seems to be largely dependent on adequate laboratory monitoring, particularly when access to second-line therapies is still limited. If viral load cannot be accurately monitored and durable high level virologic suppression maintained, then infection by and accumulation of drug-resistant viruses and their subsequent transmission within the population could be an inevitable consequence that could significantly impact the life expectancy of all of us living with HIV.
This is the last of a three-part series of articles sponsored by Abbott Molecular.
Tony Mills, MD is a leading clinician in HIV disease and men’s health in Los Angeles. He has been principal investigator on more than 50 clinical trials, is a nationally known speaker, and has most recently formulated a line of natural supplements for men. Visit www.tonymillsmd.com.
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