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Viral Load Testing
Nucleic Acid-Based HIV-1 Viral Load Assays
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Introduction
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Amplicor HIV-1 Monitor v.15 Assays
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Versant HIV-1 RNA 3.0 Assay (bDNA)
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NucliSens HIV-1 Assay
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LCx HIV RNA Quantitative Assay
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Recent Advances in Diagnostic Testing: "Real-Time" Amplification and Detection
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COBAS AmpliPrep/COBAS TaqMan HIV-1 Test
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NucliSens EasyQ HIV-1 v1.1 Assay
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RealTime HIV-1 Assay
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Specimen Collection, Processing, and Storage
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transparent imageChoice of Anticoagulant
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transparent imageTime to Processing
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Viral Load in Tissues and Latent Reservoirs
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Summary
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References
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Tables
Table 1.Comparison of Current Commercially Available Nucleic Acid-Based HIV-1 Viral Load Assays
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Figures
Figure 1.Probability of Developing AIDS According to HIV-1 Concentration and CD4+ Lymphocyte Count
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Figure 2.Schematic Representation of the Branched DNA (bDNA) Assay for Quantifying HIV-1 (Versant HIV-1 RNA 3.0)
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Figure 3.Schematic Representation of the Nucleic Acid Sequence-Based Amplification (NASBA) Reaction
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Figure 4.Schematic Representation of the TaqMan Real-Time PCR
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Introduction
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Measurement of blood plasma HIV-1 RNA concentration ("viral load") using nucleic acid-based molecular diagnostic assays is the standard of care in many areas of the world with access to antiretroviral therapy (ART). Untreated HIV-1 infection is characterized by high-level viral production and CD4 T-cell destruction progressing, despite an often lengthy clinical latency period, to significant net loss of CD4 T cells and AIDS.(1,2,3) The absolute level of steady-state viral load is a strong predictor of the rate of disease progression and, by itself or in combination with CD4 T-cell counts, has great prognostic value (Figure 1).(4,5,6,7,8,9) As a result, current U.S. Department of Health and Human Services guidelines advocate the use of plasma viral load testing when considering ART initiation, monitoring response to therapy, and instituting a change in drug regimen.(10) To support the key clinical goal of achieving maximal viral load suppression, many sensitive and precise viral load assays have been developed to quantify HIV-1 RNA accurately. This chapter focuses on the commercially available assays that quantify viral nucleic acid, as well as the next generation of tests, based on real-time polymerase chain reaction (PCR) or nucleic acid sequence-based amplification (NASBA) (see below). These newer tests offer an expanded linear dynamic range of detection and increased automation, providing enhanced efficiency, reproducibility, and overall throughput.

To reach the sensitivity seen in today's commercial viral load assays, detection and quantification of viral RNA requires amplification of either the target nucleic acid or the signal chemistry used for detection. There currently are 4 major viral load assay manufacturers, 3 of which offer kits that have been approved by the U.S. Food and Drug Administration (FDA) for use as a diagnostic test within the United States. Each uses different strategies for the requisite steps of sample preparation, amplification, and detection, reaching limits of quantitation as low as 50-80 copies/mL. A comparison of assay characteristics and performance is shown in Table 1. Additionally, assay precision typically allows one to discern a 3-fold or greater change (0.5 log10 copies/mL) in viral load as biologically significant, although this ability may decline at the lower end of the linear range for some assays.

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Amplicor HIV-1 Monitor v.15 Assays
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There are 3 Amplicor HIV-1 Monitor v1.5 assays (Roche Diagnostics, Branchburg, NJ) currently approved for in vitro diagnostic use in the United States. The 3 viral load assays share a PCR-based target amplification strategy (11) but differ in the type of equipment used, degree of automation, and method of sample preparation. Following isolation of viral RNA, reverse transcription (RT) is performed to yield single-stranded complimentary DNA (cDNA). The cDNA is amplified exponentially in repeated cycles of heating and cooling by PCR with the v1.5 primers SK145 and SKCC1B, which are complementary to highly conserved regions in HIV-1 gag and are optimized to yield equivalent amplification of HIV-1 group M (subgroup A-H) viruses.(12,13) Viral RNA is quantified using a synthetic, noninfectious armored (protein-coated) RNA construct as the internal quantitation standard (QS), added to the specimen at a known concentration before RNA extraction. The internal QS can be differentiated from the viral target sequences and serves to compensate for variability in RNA extraction and to indicate substances in plasma that may be inhibitory to PCR amplification. External controls consisting of a high positive, low positive, and a negative control also are included in each assay. All Amplicor HIV-1 Monitor assays use 1 of 2 viral RNA extraction procedures with overlapping linear dynamic ranges: the Ultrasensitive (50 to 100,000 copies/mL) or the Standard (400 to 750,000 copies/mL).(14) The limit of detection is determined from serial dilutions of viral standards and represents the lowest viral concentration detected with a positivity rate >95% using either preparation procedure.(15)

The Amplicor HIV-1 Monitor is the least automated of all the assays. The test consists of independent steps for RNA isolation, reverse transcription and PCR (RT-PCR) amplification, and detection using a colorimetric readout.(11) Viral RNA is released from the virions with guanidine isothiocyanate, and nucleic acid from the relatively impure lysate is precipitated with isopropanol. RT-PCR amplification occurs in a single tube using the thermostable recombinant enzyme Thermus thermophilus DNA polymerase (rTth pol), which has both RT and DNA polymerase activities. The PCR products are serially diluted and denatured, and single-stranded DNA is bound to microwells coated with HIV-specific oligonucleotide probes. An avidin-horseradish peroxidase (HRP) conjugate is added, binding to the biotin-labeled amplicon. The amount of bound amplicon is determined using an enzyme-linked immunosorbent assay (ELISA) plate reader after the addition of an HRP-specific colorimetric substrate.

The COBAS (comprehensive bioanalytical system) Amplicor HIV-1 Monitor test requires manual RNA extraction but uses the COBAS Amplicor analyzer for RT-PCR amplification, dilution, and detection via magnetic particles coated with oligonucleotides specific for the target or QS amplicon. Detection is as described above by colorimetric determination using target- or QS-specific oligonucleotide detection probes. This assay version offers the advantage of decreased sample manipulation and potentially higher throughput as well as increased consistency and reproducibility due to automated dilutions and detection. Also, being a closed system, it allows greater flexibility when considering equipment placement within the laboratory.

The COBAS AmpliPrep/COBAS Amplicor HIV-1 Monitor kit further automates the PCR-based viral load assay with the COBAS AmpliPrep for sample processing. The AmpliPrep uses streptavidin-coated magnetic particles that bind either the viral RNA or QS RNA via biotinylated capture probes, allowing automated nucleic acid isolation and washing following virion lysis. The processed specimen, linked to the magnetic beads, undergoes RT-PCR, dilution, and detection with the COBAS Amplicor analyzer.

These end-point PCR-based assays are sensitive, highly reproducible, and specific for group M subtypes. However, contamination of high-copy amplified product in low-copy specimens and blood plasma-based PCR inhibitors are major concerns. High-copy amplicon contamination is minimized by incorporating 2'-deoxyuridine 5'-triphosphate (dUTP) during PCR and AmpErase (uracil-N-glycosylase) in the amplification mix, for selective digestion of contaminating products in the subsequent PCR cycles. Compared with the branched DNA (bDNA) and NASBA assays, however, the linear dynamic ranges of the Standard and Ultrasensitive extraction procedures are limited, potentially requiring more frequent retesting of specimens outside the assays' dynamic range.

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Versant HIV-1 RNA 3.0 Assay (bDNA)
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The Versant HIV-1 RNA 3.0 assay (Bayer, Tarrytown, NY) is a bDNA sandwich nucleic acid hybridization method that quantifies plasma HIV-1 by amplifying the signal rather than the target RNA. The bDNA assay is performed in a 96-well microtiter plate format using the System 340 bDNA Analyzer.(16,17,18,19) It is FDA approved for in vitro diagnostic use and has a wide linear dynamic range (75 to 500,000 copies/mL within the United States, 50 to 500,000 copies/mL outside the United States). The bDNA assay does not require viral RNA purification or PCR amplification steps. Instead, virions are concentrated by centrifugation and disrupted by detergent and proteinase K, releasing viral RNA. This lysate is incubated with 2 sets of oligonucleotides. The first set captures viral RNA, hybridizing both to conserved regions of the HIV-1 pol gene and to oligonucleotides bound to the microwell (Figure 2). The second set of oligonucleotides provides signal amplification. This set consists of 4 components: oligonucleotides with homology to both the target RNA and to preamplifier oligonucleotides (target probe), preamplifier oligonucleotides, amplifier oligonucleotides, and oligonucleotide probes bound to alkaline phosphatase (AP) for detection. Each of these components binds by hybridization to the next at multiple sites. In this way, the signal is amplified without copying the target RNA. Detection is by chemiluminescence using an AP-specific substrate. The amount of light detected is directly proportional to the amount of bound nucleic acid. The absolute quantity of HIV-1 RNA is determined from an external standard curve run on the same plate. The assay does not incorporate an internal QS.

The Versant HIV-1 RNA 3.0 assay demonstrates a high degree of precision and can discern 2- to 3-fold changes in HIV-1 RNA,(17,19) even at the low end of the linear dynamic range. In multiple comparative studies including predominantly subgroup B virus from patient plasma, there is excellent correlation between the Versant and COBAS Amplicor v1.5 assays.(20,21,22,23,24) For example, in the largest comparative study (24) of 1,000 specimens from an equal number of patients, with a predominance of specimens in the lower region of the collective dynamic range (50-250 RNA copies/mL), the correlation (R2) was 0.957 with a slope of 1.004, and the mean difference between values from the two assays was low (0.10 log10). In the examples where discordance was observed at the lower limit of detection (50 copies/mL), there was no evidence of bias for one assay or the other. There are, however, reports that show higher discordance between the two assays.(25-27) These results may be attributed to a significantly smaller sample size and a greater proportion of non-B subtypes. Measurement of non-B subtypes and recombinant forms with different assays results in comparatively greater variation.(28,29)

Of special note, nominal viral load values from the previous bDNA assay version (Quantiplex 2.0) are not directly comparable to the current version (3.0), and are lower by a factor of 1.75.(30)

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NucliSens HIV-1 Assay
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The NucliSens HIV-1 QT assay (bioMerieux, Boxtel, Netherlands) is based on target amplification using NASBA technology. The assay selectively and directly amplifies HIV-1 RNA in an isothermal, 1-step sandwich hybridization procedure using 2 oligonucleotide primers, 3 enzymes, nucleoside triphosphates and the appropriate buffers (Figure 3).(31) First, RNA is extracted and highly purified using guanidine thiocyanate and silicon dioxide particles. The RNA is amplified by repeated cycles of synthesis and transcription off a double-stranded DNA intermediate. An oligonucleotide primer (P1) specific to a region in HIV-1 gag is used to synthesize cDNA from the specimen RNA template using avian myeloblastosis virus reverse transcriptase. The RNA strand is degraded by RNAse H, allowing the oligonucleotide primer P2 to bind and initiate second-strand DNA synthesis. Antisense RNA is then transcribed off the double-stranded DNA via a T7 polymerase promoter (originally incorporated by P1). This cycle is repeated, resulting in exponential amplification (106- to 109-fold). The amount of nucleic acid is determined directly by electrochemiluminescence, which is characterized by very high sensitivity and a broad dynamic range. Quantitation of HIV-1 viral load is accomplished using the NucliSens Reader by coamplification of 3 internal RNA quantitation standards, or calibrators. The calibrators are spiked into the original specimen and are coextracted and coamplified with the sample RNA.

The NucliSens HIV-1 QT assay is compatible with plasma collected in tubes using ethylenediaminetetraacetic acid (EDTA), citrate, and heparin as anticoagulant. The nucleic acid extraction and NASBA steps can be performed manually with standard laboratory equipment, with the automated NucliSens Extractor, or with the NucliSens miniMAG. These methods yield relatively low throughput, accommodating 10-sample batches, but the NucleiSens easyMAG assay, with higher sample throughput, is under development. The assay is sensitive, with a 4-log10 dynamic range (176 to 3,470,000 copies/mL), and shows excellent correlation with the Amplicor HIV-1 Monitor (Standard and Ultrasensitive) tests using subgroup B viruses (95% confidence interval [CI]: 0.945 and 0.977).(32)

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LCx HIV RNA Quantitative Assay
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The LCx HIV RNA Quantitative Assay (Abbott Laboratories, Abbott Park, IL) uses competitive RT-PCR for target amplification of blood plasma HIV-1 pol sequences followed by microparticle enzyme immunoassay. The assay uses an internal standard for each specimen that is carried through specimen preparation, amplification, and detection to control for sample preparation and amplification inhibitors, and a set of 6 calibrator/standards for quantitation. It has an extensive linear dynamic range, 50 to 1x106 copies/mL for a 1 mL plasma input volume and 178 to 5x106 copies/mL for a 0.2 mL plasma input volume.(33)

The LCx HIV RNA Quantitative Assay can be performed with varying degrees of automation. Sample preparation may be performed manually using modified Qiagen sample preparation kits (QIAGEN, Hilden, Germany),(33) or with automation using the MagNA Pure LC instrument. Both approaches demonstrate equivalent performance characteristics.(34) Following sample preparation and reverse transcription, the cDNA is amplified in a competitive PCR reaction, in which primer concentration is limiting. Primers are labeled with the hapten carbazole, allowing subsequent capture via anticarbazole labeled microparticles using the LCx Analyzer. HIV- and QS-specific detection probe-conjugates are present during the PCR cycles, allowing colorimetric distinction and quantification after amplification in a microparticle enzyme immunoassay. Copy number is calculated by the LCx Analyzer, which determines the signal ratio for the HIV and QS products, and based on the independent calibration curve, the concentration is determined.

Of interest, the LCx assay is the only one among the 4 commercial assays described here that can specifically amplify and quantify group O viruses.(35,36) It also demonstrates superior performance in measuring group M subtype C and some circulating recombinant form (CRF) viruses.(36) Numerous other studies comparing the performance characteristics of viral load assays in genetically diverse strains (36) underscore the challenges of designing amplification strategies to equally detect all viruses. With continuous generation of diversity and recombinant forms in combination with global migration, discrepant viral load results should be reevaluated in the context of group and subtype involved.

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Recent Advances in Diagnostic Testing: "Real-Time" Amplification and Detection
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An important advancement in diagnostic testing for HIV-1 viral load is the implementation of "real-time" detection of accumulating product during the exponential phase of NASBA or PCR amplification, as opposed to the traditional end-point detection assays described above.(37) This approach alleviates the need to perform serial dilutions of the amplified product necessary in traditional end-point PCR reactions, reducing the number of steps, sample manipulation, and risk of contamination. Most importantly, real-time detection maintains the sensitivity and specificity of end-point amplification assays, but with a greatly expanded linear dynamic range, typically on the order of 5 log10. Figure 4 shows a schematic of one strategy for probe design (TaqMan), detection, and quantification using an external standard curve. Strategies used for probe design and detection, reliance on internal vs external standards, and computation methods for quantification differ among the assays, as described below, but the basic principle of detection during amplification remains.

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COBAS AmpliPrep/COBAS TaqMan HIV-1 Test
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The AmpliPrep/COBAS TaqMan HIV-1 test combines automated sample preparation for HIV-1 RNA purification using the AmpliPrep, and real-time PCR amplification and detection using the COBAS TaqMan or the COBAS TaqMan 48 analyzer. Similar to the Amplicor HIV-1 Monitor, this RT-PCR assay targets a conserved region in HIV-1 gag and uses armored RNA as an internal standard for quantification to normalize for sample preparation and possible PCR inhibition. Also similar to the Amplicor HIV-1 Monitor assays, dUTP and AmpErase are incorporated in the PCR for selective amplification of the target sequences. However, because the relative fluorescence level at an early stage in PCR amplification is used as a basis for quantification, amplicon dilutions performed for quantitation in end-point PCR assays are not necessary.

RT-PCR is performed with the recombinant enzyme Z05, which has both reverse transcriptase and DNA polymerase activity under the proper buffer conditions.(38,39) During the PCR amplification, the target probes, designed to independently hybridize either to the HIV-1 gag target sequence or to the QS, are both present and bind to their respective targets. These primers are labeled with both a fluorescent moiety and a quencher so that, when the primer is coupled to an oligonucleotide, the quencher is in close proximity to the fluorescent moiety and fluorescence is quenched. During PCR, the 3'-5' exonuclease activity of Z05 digests the target-bound oligonucleotide probe, releasing the reporter and quencher dyes from close proximity. Fluorescent signal is then emitted and detected by the analyzer.

The TaqMan HIV-1 test has a linear dynamic range of 40 to 1x107 copies/mL, allowing a single assay to quantify most specimens. The limit of detection was determined as the lowest concentration detected with a positivity rate of >95% using a dilution series of a subtype B laboratory isolate (95% CI: 34.7-48.0 copies/mL).(40) Performance analyses using group M subtypes A-H quantified by the COBAS AmpliPrep/COBAS HIV-1 Monitor Test v1.5 were correlative (R2 = 0.99), with greatest deviation at the highest and lowest ends of the linear range and within 0.3 log10 of the nominal reference value. The limit of detection using group M subtype A-H reference panels with concentrations determined by the Versant HIV-1 RNA 3.0 assay was determined as >=50 copies/mL (range: 15-46). In direct comparative studies using >150 patient samples for each comparison (TaqMan vs Versant HIV-1 RNA 3.0, LCx HIV RNA, or COBAS Amplicor HIV-1 Monitor v1.5) across a wide viral load range, there was excellent overall agreement in samples identified as positive (>94.6%) with strong correlations of viral loads between any two methods (r >0.91).(41)

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NucliSens EasyQ HIV-1 v1.1 Assay
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The NucliSens EasyQ HIV-1 v1.1 (bioMerieux) assay consists of nucleic acid extraction and target NASBA as described above for the NucliSens HIV-1 QT assay. Real-time detection of amplicons occurs during the NASBA reaction following primer exhaustion and continued production of antisense RNA from the T7 promoter. During the exponential phase of production, amplicons are detected using molecular beacon oligonucleotide probes that fluoresce upon binding to the specific target sequence.(42) A single-calibrator RNA species is added to the original specimen and is carried through sample preparation, amplification, and detection. Two detection probes labeled with distinct fluorophores differentiate the target and calibrator amplicons, thus allowing quantification of the HIV-1 RNA in the original specimen based on the RNA formation rates in the transcriptional phase. This assay platform offers automated sample preparation and single-tube amplification and detection using the NucliSens EasyQ Analyser. HIV-1 viral load is determined using the NucliSens EasyQ Director software.(43)

The published performance characteristics of the NucliSens EasyQ HIV-1 v1.1 assay show that it is comparable to the NucliSens HIV-1 QT, the Versant HIV-1 RNA, and the COBAS Amplicor HIV-1 Monitor v1.5 assays in linearity, specificity, and reproducibility.(44,45) In the larger and more comprehensive of the 2 studies evaluating >1,000 clinical specimens and virus panels of multiple subtypes at 4 testing sites,(45) the correlation coefficient between the NucliSens HIV-1 QT and the other assays ranged from 0.85 to 0.91. The assay specificity was 99.3% (n = 441) with a linear range between 50 and 3x106 IU/mL. All reference panel subtypes (group M, A-H) were recognized and the nominal values were similar to the other assays, with the Amplicor assay yielding the highest values. Of note, both studies demonstrated underrepresentation of viral loads in subgroups A and C in the assays tested.(44,45)

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RealTime HIV-1 Assay
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The RealTime HIV-1 Assay (Abbott Laboratories) measures PCR product amplification in real time with a unique partially double-stranded probe design to minimize inefficient binding due to sequence mismatch at the probe binding site. The probe strands are labeled with a fluorophore (reporter) at the 5' end, and a quencher moiety at the 3' end of the shorter, complimentary strand.(36,46) In the presence of target pol sequences, the reporter probe preferentially binds, and upon release of the shorter quencher probe, fluoresces. The RealTime HIV-1 Assay uses an armored RNA internal standard introduced during the specimen preparation step, which is differentiated from the amplified target sequence by a fluorescent single-stranded oligonucleotide probe. Because the exonuclease activity of rTth polymerase is not required in the RealTime assay, as it is for TaqMan-based assays, the annealing temperature of the probe binding step is lower than that of the amplification steps, minimizing the impact of mismatches at the probe binding site. Sample preparation and amplification/detection steps can be automated using the m2000sp and m2000rt instruments.

Assay performance characteristics from Abbott Laboratories show that the assay has a 5-log10 linear range, assay specificity of 100% (n = 259), 95% probability of detecting samples with a viral load of 25 copies/mL, and recognition of subtype panels from group M (A-H), group O, and group N.(46) In one comparative study using clinical specimens from Brazil consisting of subtypes B, C, and F and recombinant forms, viral loads from the RealTime HIV-1 Assay correlated with those derived from the LCx HIV-1 assay (R2 = 0.908). When viral load in 89 specimens was quantified by both assays, the results of the two assays were within 0.5 log10 of each other for 95% of the specimens.(36)

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Specimen Collection, Processing, and Storage
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Choice of Anticoagulant
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The choice of anticoagulant used in blood collection tubes can alter viral load results significantly by affecting either the virion decay rate ex vivo or the detection efficiency of the assay used. Plasma treated with sodium heparin is not appropriate for PCR-based assays, but is compatible with bDNA- or NASBA-based assays. In cases where RT-PCR must be used to quantify viral load in heparin-treated plasma, heparinase may be added directly to the sample.(47,48) In addition, the virion decay rate is significantly faster in heparin-treated blood when compared with sodium EDTA- or acid citrate dextrose (ACD)-treated blood.(47,48,49,50) Given identical blood processing protocols, plasma collected in sodium EDTA (lavender top tubes) yielded significantly higher viral loads than plasma collected in ACD (yellow top) or heparin (green top), and EDTA plasma thus is the preferred choice of specimen. Artifactually high viral loads determined by the Amplicor HIV-1 Monitor v1.5 assay using the standard extraction procedure were reported recently in approximately 20% of plasma specimens collected, spun, and stored frozen in plasma preparation tubes (PPTs).(51) This discordance was evident especially in low-viral-load pediatric samples, but also was seen in adult samples. Such discordant results were not evident in blood plasma aliquots that were placed in separate PPTs immediately after centrifugation.

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Time to Processing
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HIV-1 virions have a rapid turnover in vivo with an average half-life of approximately 6 hours.(1) Rapid degradation of HIV-1 in unprocessed, whole blood in vitro also has been reported,(52) with the fastest rate occurring within 6-8 hours postcollection.(47,49) Other studies, however, report minimal decay during this time period,(50,53) with one report showing a loss of <0.5 log10 RNA copies/mL at 30 hours postcollection.(54) To accommodate these varied results, most plasma processing protocols require separating cells and plasma within 6 hours. In situations where this is difficult or impossible, loss of plasma HIV-1 is minimal when plasma separator tubes (cell preparation tubes or PPTs) are used, centrifuged immediately, and temporarily stored at room temperature or 4º C for up to 24 or 30 hours, respectively, until long-term storage at -80º C can occur.(49,55)

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Viral Load in Tissues and Latent Reservoirs
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The standard use of viral load assays is for determining the concentration of virions in blood plasma that is readily accessible for clinical and research testing. Replication of HIV, however, occurs not in plasma, but within cells. In addition, only about 2% of the body's T cells are present in the blood, with the majority residing in lymph nodes, spleen, and other lymphoid organs. In research settings, the viral load assays have been used to quantify virus in a wide variety of sites including cerebrospinal fluid, semen, vaginal washings, lymph nodes, and blood cells (rather than blood plasma).(56,57,58,59,60,61) These studies have indicated only rough correlations between blood plasma viral load and the viral load in tissues of untreated persons. During antiretroviral treatment, however, the decay of HIV-1 viral load in tissues typically corresponds to virologic responses in plasma, making blood plasma a useful sentinel site for virologic responses in general. There currently is no clinical indication for viral load testing of tissues other than blood plasma.

It is important to note that a plasma viral load less than the limit of detection of available assays does not mean that the virus is absent from the body. Replication-competent HIV-1 persists in resting T cells of treated subjects with "undetectable" plasma viremia.(62,63,64,65,66,67,68,69) These latent reservoirs of HIV-1 likely contribute to viral persistence during therapy and virologic rebound when therapy is discontinued. Modifications to existing commercial viral load assays allowing lowered detection limits (<50 copies/mL) have revealed intermittent and continuous residual viral replication.(68,69) The clinical relevance of residual viremia with respect to disease progression, viral evolution, and treatment response is an active area of investigation.

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Summary
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A number of sensitive and robust commercial assays are available to accurately quantify HIV-1 RNA in blood plasma. Recent advances in assay automation have increased throughput and improved performance characteristics. The development of assays incorporating simultaneous target amplification and detection in real time has expanded linear dynamic ranges to as large as 5 log10. Although the increasing global heterogeneity of HIV presents a challenge to universally quantitative measurement of viral subtypes and quasispecies, continued improvement in probe design and chemistries have expanded the breadth of detectable and quantifiable viruses.

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