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Update on the Diagnosis and Monitoring of HIV-1 Infection
confirmed diagnosis of HIV infection. Therefore, laboratory methods that required to measure the amplified products. The method requires a
provide accurate information about the patient’s existing infection and separate RNA extraction step, making it more labor-intensive, although a
immunity status would allow the physician to make a realistic prognosis and new pre-amplification procedure (NucliSens extractor) using silica for
to initiate or change the therapy accordingly. The viral load (viral RNA levels) extraction is rapid and efficient. The bioMerieux company also has a
and cluster of differentiation 4 (CD4) T-cell counts have been the mainstay of realtime NASBA system—NucliSENS EasyQ—for investigational use (not yet
accessing patients’ infection status, disease progression, and response to FDA-approved). This system uses the same principle as the NASBA system,
therapy. Typically, the increasing viral load with continuing decline of the CD4 with a molecular beacon detection method.
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T-cell count indicates a poor prognosis with a high likelihood of rapid
progression to AIDS, although this may vary with individuals and gender.
4,5
The Amplicor Monitor assay also utilizes a gene-template-based RT-PCR
The viral load, measured as viral RNA copies/ml, can be determined in plasma amplification process to convert the target viral RNA template to a DNA
by several commercial amplification-based nucleic acid tests, including form that can be amplified and detected, i.e. the method combines RT and
reverse transcriptase-polymerase chain reaction (RT-PCR), nucleic acid DNA amplification reactions. The enzyme RT converts viral RNA into cDNA,
sequence-based amplification (NASBA), and branched DNA (bDNA). Viral which is amplified using PCR. This is performed in one step with a specific
load testing has been shown to be a most important means of managing RT (rTth polymerase isolated from thermophilic bacterium, Thermus
HIV-infected patients. To determine the baseline viral load, experts suggest thermophilus), which has both RT and DNA amplification properties. The
measuring viral load twice in a two-week interval. The same viral load test for RT-PCR reaction is carried out in an instrument called a thermocycler that
a given patient should be used prospectively every several months thereafter allows for the rapid changes in temperature required for cycling through
to determine disease progression.
6
A typical response of a drug-naïve patient extension and denaturation steps. Oligonucleotide primers that are biotin-
after initiation of antiretroviral treatment should expect a viral reduction in labeled initiate the replication of DNA, and the DNA sequence products
the order of 0.7–0.8 log with the use of reverse transcriptase (RT) inhibitors, (amplicons) become biotinylated. These products are subsequently captured
or 2–3 log viral reduction with protease (PR) inhibitors. The use of RT–PR by complementary oligonucleotides immobilized on a microtiter plate. The
inhibitor combinations often results in a significant reduction in viral load to products are detected by an avidin–horseradish peroxidase conjugate that
a non-detectable level by the current molecular methods, i.e. fewer than binds to the biotinylated captured amplicons; a substrate is subsequently
40–50 viral RNA copies/ml.
7
Clinical data further suggest that early treatment added to form color, and a microtiter plate spectrophotometer is used to
in acutely infected patients may have additional benefits.
8,9
measure the level of signal (color) generated by amplified products. The
region amplified is a highly conserved region of the HIV-1 gag gene with a
There are four FDA-approved and commercial test kits available for the sequence of 142 bases.
measurement of HIV-1 RNA viral load in plasma: two versions of the
Amplicor HIV-1 Monitor™ (Roche Diagnostics systems) (RT-PCR method); An automated system called the COBAS AmpliScreen is also available for
the Versant™ HIV-1 RNA 3.0 (Bayer Inc), which is known as bDNA; the HIV RNA testing, and is FDA-approved. It offers full automation for the
NucliSens™ HIV-1 QT system (bioMerieux Inc.), also known as NASBA; and RT-PCR method. Roche has also developed a realtime PCR-based system,
the RealTime HIV-1 Viral Load Test (Abbott Laboratories). All of these the COBAS AmpliPrep/COBAS TaqMan HIV-1 test. It is a combination of the
methods are based on nucleic acid amplification strategies to amplify small AmpliPrep purification system with a realtime PCR amplification and COBAS
amounts of viral RNA to measurable quantities. RT-PCR requires reverse TaqMan analyzer. These assays have the advantage of extended dynamic
transcription of the RNA to complementary DNA (cDNA) before ranges, but require an investment in specialized testing equipment.
amplification, and NASBA requires replication similar to retroviral Technically, the RT-PCR assay is moderately difficult to learn and requires a
replication before amplification.
10
The bDNA method, alternatively, is a skilled user; a relatively slow learning curve should thus be expected. One of
direct signal-based amplification process.
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All three assays have the disadvantages of PCR-based assays is cross-contamination. Cross-
comparable results with a typical detectability of 400–500 viral RNA contamination by amplicons can be minimized with the addition of the
copies/ml of plasma. Ultra-sensitive versions of these assays are also enzyme uracil-N-glycosilase (UNG, commercially known as AmpErase) to the
available, which further extend the detection limit to 40–50 copies/ml by reaction to eliminate previously amplified amplicons; here, deoxyuridine
using a larger volume of plasma.
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triphosphate (dUTP) are intentionally incorporated into DNA.
Specifically, the NASBA method uses a gene-target-based amplification Unlike the Amplicor and NASBA assays that amplify the target gene, i.e. the
principle that involves the addition of avian myeloblastosis virus (AMV) RT, viral nucleic acid, the bDNA technique is a sandwich hybridization assay that
a bacteriophage (T7) RNA polymerase, and ribonuclease (RNase) H into one directly captures viral RNA in plasma by binding to a pol gene site and,
tube with repetitive steps of reverse transcription from viral RNA template together with pre-amplification probes, binds to a microwell coated with
with subsequent RNA amplification from cDNA. The amplified single- specific oligonucleotide probes. Although a larger number of assays can be
stranded RNA is detected by hybridization to complementary wild-type or performed per day, the turnaround time for results is significantly longer
labeled with electrochemiluminescence (ECL) probes. The amount of than for the other assays. This may be an important factor in situations
chemiluminescence detected is proportional to the quantity of the where rapid turnaround times are required. The detection limit has recently
amplified product. The NASBA system, unlike the RT-PCR method, has the been reduced to 50 copies/ml in the 3.0 version. The requirement of larger
advantage of performing all of the reaction steps at constant (isothermal) volumes of plasma may be of concern when testing infants and newborns.
temperature (40–41ºC) and thus does not require thermocycling. Another A pediatric assay that requires only 50ml of plasma was available through
advantage is that NASBA can be performed on non-plasma samples. A Chiron. However, the assay design was based on the first generation of
disadvantage is that a semi-automated ECL detection instrument is bDNA method; thus, the detection limit is high.
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