Journal of Clinical Virology

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1 Journal of Clinical Virology 52S (2011) S77 S82 Contents lists available at SciVerse ScienceDirect Journal of Clinical Virology j ourna l ho mepage: Evolution in the sensitivity of quantitative HIV-1 viral load tests Bryan R. Cobb, Jeffrey E. Vaks, Tri Do, Regis A. Vilchez Roche Molecular Systems, 4300 Hacienda Drive, Pleasanton, CA, USA a r t i c l e i n f o Keywords: HIV-1 HIV viral load Lower limit of detection Lower limit of quantification Real-time PCR a b s t r a c t Significant advancements in molecular diagnostics have been made since the inception and application of PCR-based technologies in clinical diagnostic laboratories and the management of HIV-1 infected patients. More recently, real-time PCR has improved the overall performance of assays used for detecting and quantifying HIV-1 RNA viral load in patients undergoing antiretroviral treatment. The effects of these changes and the interpretations of the HIV-1 viral load results are discussed in this review in the context of the different assays used, the viral dynamics of the HIV-1 virus, and the recent changes to HIV-1 treatment guidelines Elsevier B.V. All rights reserved. 1. Background HIV-1 RNA viral load tests Real-time PCR tests are capable of detecting and quantifying small amounts of nucleic acid. This is especially the case with HIV-1 viral load monitor tests, where the analytical sensitivity of commercial tests is currently as low as 20 copies/ml. 1 Nearly two decades ago, Roche Molecular Systems (Pleasanton, CA) HIV- 1 viral load tests, the AMPLICOR HIV-1 MONITOR Test, version 1.5 (which used microwell plates) and later the semi-automated COBAS AMPLICOR HIV-1 MONITOR Test, version 1.5 (CA-HIM Test), became the gold standard for clinical monitoring of HIVinfected patients undergoing antiretroviral therapy (ART). Both tests were used in the registration trials of the first antiretroviral agents. Originally, these FDA-approved tests had a lower limit of detection and low end linear range of 400 copies/ml. A later update to the CA-HIM Test, in the form of an ultrasensitive test procedure, established an improved sensitivity of 50 copies/ml (Table 1). These early viral load tests were based on endpoint PCR technologies, where viral load results were determined using a spectrophotometer that measured a color change at the end of the PCR. Real-time PCR involves the measurement of fluorescence at each PCR cycle, and utilizes new enzymes, chemistries, and advanced PCR components. These changes enhance test performance, providing broader linear dynamic ranges, and increased sensitivity, Abbreviations: ART, antiretroviral therapy; CA-HIM Test, COBAS AMPLICOR HIV-1 MONITOR Test version 1.5; kpcr, kinetic polymerase chain reaction; LLoQ, lower limit of quantitation; LOD, lower limit of detection; PCR, polymerase chain reaction; TaqMan HIV-1 Test v1.0, COBAS AmpliPrep/COBAS TaqMan HIV-1 Test version 1.0; TaqMan HIV-1 Test v2.0, COBAS AmpliPrep/COBAS TaqMan HIV-1 Test version 2.0; WHO, World Health Organization. Corresponding author. Tel.: address: bryan.cobb@roche.com (B.R. Cobb). specificity, precision, and reproducibility. With real-time PCR, clinical laboratories have been able to move toward complete system automation, significantly reducing turnaround time and human error, and increasing workflow efficiencies. 2 Abbott Laboratories (Abbott, Chicago, IL) and Roche Molecular Systems both have commercialized, automated, FDA-approved, real-time PCR tests. Siemens Healthcare (Deerfield, IL) has developed kinetic PCR (kpcr) testing, but currently does not have an FDA-approved real-time PCR test. The Abbott RealTime HIV-1 assay targets a single region of the HIV-1 genome (the integrase gene) using a partially double-stranded probe that, once bound to its target, releases fluorescence. The Roche Molecular Systems COBAS AmpliPrep/COBAS TaqMan HIV-1 Test, version 2.0 (TaqMan HIV-1 Test, v2.0) uses a dual target approach (co-amplification and detection of sequences in both the LTR and gag regions of the genome) to optimize detection and quantification of HIV-1 subtypes 3 6 by targeting a region non-susceptible to mutations induced by drug pressure. The past decade has seen improvements in the comparability of results between commercial viral load tests through calibration to a higher order standard (i.e., traceability to the 1st HIV-1 WHO International Standard, NIBSC RNA 97/656). 7,8 For example, during the introduction of the bdna test, clinicians were required to divide results by a factor of 2 when evaluating patients who had been previously monitored by the CA-HIM Test. 9 Differences in the sensitivity between the CA-HIM Test and realtime PCR tests at the clinically relevant low end of the linear range have recently become apparent Discrepancies were initially reported with the Roche Molecular Systems first-generation realtime PCR test, the COBAS AmpliPrep/COBAS TaqMan HIV-1 Test, version 1.0 (TaqMan HIV-1 Test, v1.0) that yielded detectable results (and greater than the lower limit of quantitation [LLoQ]) where results had previously been reported as undetectable /$ see front matter 2011 Elsevier B.V. All rights reserved. doi: /j.jcv

2 S78 B.R. Cobb et al. / Journal of Clinical Virology 52S (2011) S77 S82 Table 1 Characteristics of currently used FDA approved real-time PCR tests. Abbott Realtime HIV-1 TaqMan test v2.0 Test parameter CA-HIM, v1.5 a HIV-1 TaqMan test v1.0 Target Single target (gag) Single target (gag) Dual target (gag, LTR) Single target (Integrase) Sample volume (input) b 0.5 ml 1.0 ml 1.0 ml 0.2 ml 0.6 ml 1.0 ml LLOQ (=LOD) 50 copies/ml 48 copies/ml c 20 copies/ml 150 copies/ml 40 copies/ml 40 copies/ml ULOQ 100,000 copies/ml 10,000,000 copies/ml 10,000,000 copies/ml 10,000,000 copies/ml Assay range ,000 copies/ml; 48 10,000,000 copies/ml 20 10,000,000 copies/ml 40 10,000,000 copies/ml 400 1,000,000 copies/ml a Quantitation standard Yes Yes Yes Generic Internal Control, Pumpkin Gene Fluorescence, Partially Double Stranded Probe Fluorescence, Hydrolysis Probes Fluorescence, Hydrolysis Probes Detection Colorimetric change, alkaline phosphatase Plastic seal required for 96-well plate, 2 area requirement Amperase (UNG), Sample Processing Unit Contamination control Amperase (UNG) Amperase (UNG), Sample Processing Unit LOD = Limit of Detection, LLOQ = Lower limit of Quantification, ULOQ = upper limit of quantification. HIV-1 TaqMan test, v1.0 and v2.0 = COBAS AmpliPrep/COBAS TaqMan HIV-1 Test, v1.0 and v2.0. a CA-HIM, v1.5 = COBAS AMPLICOR HIV-1 MONITOR Test, v1.5; 50 copies/ml LOD/LLOQ determined using the ultrasensitive method (standard procedure requires 200 L of sample and provides for LOD of 400 copies/ml, ULOQ, 1E6 copies/ml.) b Sample input is amount of material required to add to the system. Abbott RealTime HIV-1 test sample input volume can require up to 1.8 ml, depending on rack size and primary tube diameter. c US FDA approved LOD. Table 2 Fold-change and comparison between copies/ml and log 10 values. Viral load (copies/ml) Log 10 (copies/ml) Log 10 Reference fold fold fold fold (<50 copies/ml) by the CA-HIM Test (referred to here as low-level viremia ) Patients, whose viral load was previously thought to be suppressed on ART, were diagnosed with the new tests as failing treatment, posing a new challenge to clinicians and laboratories in interpreting results and managing patients treatments. 13 These differences were brought about by technological improvements in test design and variations in the performance of the HIV-1 tests at the lower range Laboratory results and interpretations The higher sensitivity of the second-generation real-time PCR viral load test (the TaqMan HIV-1 Test, v2.0) has been driven by improvements in test design and analytical performance, rather than the clinical need for a more sensitive test. In general, most studies show fairly similar variability between the real-time PCR tests for HIV-1 RNA viral load monitor tests used today Since current real-time PCR tests have similar precision, low-level viremia is unlikely to be explained by minor differences in precision between tests. To test the true measure of differences in assay performance, head-to-head comparison studies should be performed that measure the same viral material (typically cultured virus and clinical specimens) on two different platforms, rather than comparing two different studies where different material or sample panels are used. Throughout the linear range of any given real-time PCR test, biological variability (short-term, within-subject random variability) can in general contribute as much as ±0.3 log 10 (copies/ml) to variability in HIV-1 viral load results 21 ; and test variability (sample independent variability) typically contributes up to ±0.2 log 10 (copies/ml) 22 when independently assessed (Table 2). At a HIV-1 RNA titer of 50 copies/ml, a 0.3 log 10 difference equates to a 2-fold variation with a range in results from 25 to 100 copies/ml. Similarly, a 0.5 log 10 difference equates to a 3-fold variation giving a range from 16.7 to 150 copies/ml (Table 3). Table 3 The four categories of HIV-1 viral load results and interpretations (HIV-1 TaqMan Test, v2.0). Specimen results are interpreted as follows: Titer result Interpretation Target Not Detected Ct value for HIV-1 above the limit for the assay or no Ct value for HIV-1 obtained. Report results as HIV-1 RNA not detected. <2.00E+01 copies/ml Calculated copies/ml are below the Limit of Detection of the assay. Report results as HIV-1 RNA detected, less than 20 HIV-1 RNA copies/ml. >2.00E+01 copies/ml and <1.00E+07 copies/ml Calculated results greater than or equal to 20 copies/ml and less than or equal to 1.00E+07 copies/ml are within the linear range of the assay. >1.00E+07 copies/ml Calculated copies/ml are above the range of the assay. Report results as greater than l.00e+07 HIV-1 RNA copies/ml. If quantitative results are desired, the original specimen should be diluted 1:100 with HIV-1-negative human EDTA-plasma and the test repeated. Multiply the reported result by the dilution factor.

3 B.R. Cobb et al. / Journal of Clinical Virology 52S (2011) S77 S82 S79 Given these differences, it is likely that with most quantitative HIV-1 RNA tests, results within 0.5 log 10 would vary between a result <LLoQ (a number would not be reported) and up to 150 copies/ml. Similarly, a 2-fold variation would mean that some proportion of results would be less than the LLoQ of the test (versus above the LLoQ and therefore a number). Aside from the biological and test-dependent contributions to variability, there are other sources of error that can explain discrepant results. Errors can arise from pre-analytical steps, especially improperly processed specimens Freezing of tubes, inadequate centrifugation speed, and centrifugation before versus after shipment can all lead to over-quantification of results when using BD Vacutainer PPT TM Plasma Preparation Tubes (PPTs). In these cases, over-quantification can be the result of a detection of HIV-1 RNA or proviral DNA sequestered within white blood cells and possibly other cellular components presumably due to cellular leakage into the gel barrier from improper handling. 25 Therefore, the correct handling of samples and processing of tubes are important steps to take into consideration when evaluating and resolving discrepancies in viral load test results. As tests have progressed to real-time PCR, the ability to detect HIV-1 RNA more accurately and with better precision has made differences in results at the low end more noticeable when compared to tests that use endpoint PCR. 1,17 There are both clinical and analytical factors to consider when evaluating an undetectable HIV-1 RNA viral load result. A package insert provides the results interpretation for a given viral load monitor test, derived primarily from the analytical performance characteristics of the given test (Table 3). For example, the CA-HIM Test results are reported as HIV-1 RNA Not Detected (less than 50 copies/ml) for the ultrasensitive test procedure when the results are below the linear range of the test based on absorbance (and a calculation cannot be determined), and HIV-1 RNA detected, less than 50 copies/ml when a calculation could be made but is below the linear range of the test. Both results often translate collectively as less than 50 copies/ml, HIV-1 RNA undetectable. In contrast, the TaqMan HIV-1 Test, v2.0 reports results Target not Detected in the absence of amplification and detection, interpreting this as HIV-1 RNA, not detected (Table 3). This is different from results where HIV-1 RNA is detectable but not quantifiable (below the LLoQ) and is reported, as HIV-1 RNA detected, less than 20 HIV-1 RNA copies/ml. Likewise, Abbott s RealTime HIV-1 assay reports results as either Target not Detected which signifies no target was detected or Detected (if <LLoQ) which indicates that the target was detected but less than the lower limit of quantitation. 30 It is important to note that the LLoQ and the limit of detection (LOD) are assessed differently. These assessments are guided by the Clinical Laboratory Standards Institute (CLSI). 31 The LOD (also referred to as analytical sensitivity) can be defined as the lowest viral load level tested in a given experiment at which at least 95% of tested samples will be reported as detected. For all HIV-1 viral load tests currently used, the LODs coincidentally are equal to the LLoQ. For other viral targets, the LLoQ can be equal or greater than the LOD. This is the case for hepatitis C viral load tests and several other tests where the LODs happen to be lower than the respective LLoQs. Some scientific and regulatory authorities also recognize that the 95% detection rate may be extrapolated by fitting a cumulative normal distribution (PROBIT) model to the observed detection rate against the nominal (log 10 ) concentration data. The LOD is estimated as a prediction from the fitted model with the concentration corresponding to 95% detection rate, along with the 95% confidence intervals. In contrast, the LLoQ is defined as the lowest target concentration expected to be quantifiable with the total analytical error not exceeding a specified goal. 31 Total analytical error at a given Table 4 Differences in HIV-1 group M limit of detection by subtype (HIV-1 TaqMan Test, v2.0). Subtype Isolate designation Lowest concentration level 95% hit rate (copies/ml) A 92UG A 4237A/98 20 B 92TH B 8E5/LAV 20 C 92BR C 3777A/97 11 D 92UG D 92UG E 92TH E 92TH F 93BR G ARP173/RU H HIVV Source: COBAS AmpliPrep/COBAS TaqMan HIV-1 Test, version 2.0 [package insert]. Branchburg, NJ: Roche Molecular Systems, Inc concentration level is defined as the magnitude of the bias at that level + 2 standard deviations (estimated from within the laboratory precision study). 32 The LLoQ is set as the lowest concentration that is not lower than LOD and has the total analytical error not exceeding a preset specification. The current FDA-approved quantitative HIV-1 viral load tests report only <LLoQ, detected regardless of whether the LOD is less than or equal to the LLoQ (Table 3). Furthermore, the LOD or overall sensitivity of a given test is typically determined using the most conservative estimate. Different HIV-1 Group M subtypes can have different detection rates when the LOD is verified, as assessed by using a positivity rate of >95% for each subtype (Table 4). 33 Thus, the analytical performance characteristics should not be expected to be inherently the same across heterogeneous samples and this is true of any PCR-based assay. Additionally, some variability in test performance can be expected when used in the clinic environment where conditions may be different from those that exist in clinical trials. 2. Analytical considerations In a given plasma sample from an HIV-1-infected patient, the volume drawn will have various proportions of numbers, X, of HIV-1 RNA copies that generate three types of reported test results (Table 3 and Fig. 1). These proportions can be approximated using a Poisson probability distribution describing the probability of having X = 0, 1, 2,... copies in a test sample as a function of the mean number of copies per sample volume in the patient s blood. The random variation of the number of copies in a test sample randomly drawn from the bulk of the patient s blood is explained by that it is equally likely for each copy of viral nucleic acid to be present in any part of the bulk of blood during the sample collection. This allows, with various probabilities, any number from 0 to the total number of copies in the patient s blood to be captured in the test sample, though the closer that the number of the copies is to the mean number of copies per test sample, the more often it is captured in the randomly drawn test sample. The described variation of the number of copies in a test sample and the corresponding uncertainty in the reported results does not depend on the test s design and implementation. It is completely defined by the nature of random variation of the number of viral targets captured in the test sample drawn randomly from the bulk of patient blood. Understanding such uncertainty helps to optimally interpret the reported patient sample results from a PCR viral load monitor test.

4 S80 B.R. Cobb et al. / Journal of Clinical Virology 52S (2011) S77 S Concentration (x LoD) Target not Detected <LLoQ, Detected Quantitative Fig. 1. Proportions of three types of reported test results vs. target concentration. The proportions of the three types of reported results as functions of the mean concentration in LOD units for the typical case when LLoQ = LOD. When the patient concentration tends to 0, the proportion of Target not Detected results tends to 1, while the proportions of <LLoQ and quantitative results both tend to zero. As the concentration tends to LLoQ, the proportion of <LLoQ results peaks. When the concentration tends to infinity, the proportion of quantitative results tends to 1, while the proportions of Target not Detected and <LLoQ results both tend to zero. At any concentration, the sum of three proportions of the three types of reported results is always 100% Viral dynamics and current clinical guidelines In the past, the validation of new viral load assays focused on quantitative comparisons to earlier version viral load predecessors as the clinical utility of testing was primarily to evaluate and monitor the relative changes in viral load to assess treatment efficacy (e.g., ensuring a 1.0 log 10 decrease by week 8 on therapy). 34 These quantitative comparisons include linear regression analyses, mean bias and variance. Subsequent treatment guidelines have increased the emphasis on whether ART has achieved maximal suppression, 35,36 and that a new viral load method can classify a patient as having succeeded or failed their ART regimen, a determination best ascertained with concordance testing at medically relevant decision points. Understanding the relationship between a test s quantitative performance and its clinical interpretation is critical for successful patient management. The way in which diagnostic test manufacturers report results is often confusing. The guidelines have historically equated an undetectable result with a <50 copies/ml result, while the test reports these as detectable results (but not quantifiable) and below the LLoQ (Table 3). The term undetectable which has been used for describing results <LLoQ, detected in the context of newer tests may not be appropriate. Rather, a more appropriate description is unquantifiable versus undetectable, and this has already been proposed for interpreting hepatitis C viral load results. 37 For results interpretations below a test s LLoQ for an HIV-1- positive patient, a result of <LLoQ, detected may fluctuate with a Target Not Detected result if repeated with frequencies of the two types of results, dependent on how close to zero the actual viral load titer is. Technically, a result of <LLoQ, detected may be observed, but not consistently. In this case, most experts would consider that HIV-1 is suppressed and optimal viral suppression is generally defined as a viral load persistently below the level of detection of the test. 38 Similarly, a Target Not Detected result does not mean that the patient is negative for HIV-1 RNA as evidence indicates that HIV- 1 persists in latently infected, resting, and activated CD4+ cells in the blood and lymphoid tissue of patients receiving ART The only results that would be expected to be consistently Target not Detected are from plasma samples from HIV-1-negative donors. This is due to the fact that tests require acceptable analytical specificity where HIV-1-negative samples achieve Target Not Detected close to 100% of the time. Detection of virus at low levels should be considered in the context of the viral kinetics of HIV-1, which is thought to involve different phases of viral decay in patients suppressed with ART. These phases are (1) a rapid initial phase of decay followed by (2) a longer turnover from a different population of infected cells, and (3) latent reservoirs in resting CD4+ cells stably producing virus. 43 HIV-1 phases of decay might provide a clinical context for the viral load results of some patients with very low but detectable levels, i.e., detectable below the published LOD, but not quantifiable. In addition, some scientists have postulated that detectable viremia is permissible below a virologically relevant threshold, based on the observation of intermittent and in some cases persistent low-level viremia in patients on therapy whose viral loads often do not exceed copies/ml, which may represent a threshold barrier to virological evaluation and breakthrough Furthermore, it has been proposed that residual viremia may originate from latently infected, resting CD4+ T cells upon occasional and/or spontaneous cellular activation, a theory supported by studies using assays with single copy sensitivity that HIV-1 viremia is persistently detectable in patients who are reported to be durably suppressed and undetectable using standard test methods. 36,43,48 In January, 2011 the US Department of Health and Human Services HIV-1 treatment guidelines were updated to state that there is no definitive evidence that patients with viral loads quantified as <200 copies/ml are at increased risk for virologic failure. 35 This change was due to the acknowledgement of the higher sensitivity of newer assays, the persistence of HIV-1 even with adherent ART, and the lack of clinical or virological progression in the face of residual low-level viremia. These updated guidelines help clinicians and laboratories interpret HIV-1 low-level viremia and provide a new threshold for the clinical management of patients receiving

5 B.R. Cobb et al. / Journal of Clinical Virology 52S (2011) S77 S82 S81 antiretroviral therapy. 35 In addition, these recommendations may have important implications for future drug trials that use HIV-1 RNA viral load as a primary efficacy endpoint. 3. Conclusions Advances in technologies will continue to bolster improvements in molecular diagnostics applications, potentially providing a diagnostic tool before it can be well understood clinically. This has been the case with more sensitive tests for HIV-1 RNA viral load monitoring and detecting low levels of HIV-1 RNA without a clear context or supporting data to determine clinical significance. However, more sensitive HIV-1 RNA tests have proved to be useful in research studies to help better understand the viral dynamics of HIV-1 and the impact of low-level HIV-1 viral load to the immune system and its long term clinical implications. 42 As technologies evolve, HIV-1 RNA viral load clinical thresholds should be evaluated in the context of test performance characteristics to define treatment endpoints that would best assess drug efficacy and reduce the risk for drug resistance. HIV-1 lowlevel viremia should be interpreted based on changes to recent guidelines, which have been quick to take into consideration the performance of newer real-time PCR tests. Focus at the low end of the test will likely shift toward therapeutic approaches that eradicate HIV-1, which will require efforts to target viral reservoirs or low-level replicating virus. Funding source None. Ethical approval Not required. Conflict of interest Bryan R. Cobb, Jeffrey E. Vaks, Tri Do, and Regis A. 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