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HIV & AIDS
The Abbot Molecular, a molecular diagnostic branch of the Abbott the thermocycling process could potentially lead to a large variation in the final
Laboratories, recently launched an m2000 RealTime system, which is quantification of amplified products. To circumvent this problem, a TaqMan-
another realtime PCR-based assay that can be used to quantify HIV-1 viral based method was developed and validated for quantification of HIV-1
load and has recently been approved by the FDA. This is a fully automated pro-viral DNA.
30
This assay is based on the principle of realtime PCR.
29
Because
system with the m2000sp for sample preparation and m2000rt for realtime of the inherent advantage of realtime PCR over classic PCR, this new assay
PCR. It has an ID tracking system that recognizes bar-coded tubes and the provides a highly accurate and reproducible detection method for HIV-1
shortest turnaround time—about seven hours. In contrast to the TaqMan pro-viral DNA with a broad linear range of detection.
30
realtime PCR, however, Abbott Molecular has designed a unique partial
double-stranded oligonucleotide probe that allows broader coverage of In contrast to the quantitative HIV DNA PCR methods described above, a
detection, including rare HIV-1 subtypes. Among the group M viruses, the commercial qualitative DNA detection test is available. The Roche
Abbott RealTime HIV-1 assay appears to have the higher capacity to detect Amplicor™ HIV-1 DNA PCR assay version 1.5 is used to measure pro-viral
other HIV subtypes, including some of the circulating recombinant forms DNA in whole blood.
31
This assay is used primarily to determine the
(CRFs).
15,16
Besides the group M viruses, the Abbott RealTime HIV-1 assay can infection status of infants born to HIV-infected mothers. Serological testing
also detect the group O HIV strains. With the increasing incidence of non-B in HIV-exposed infants is of no value in the first six months of life because
or CRF infections in the US,
17
the Abbott assay could potentially be useful, of the presence of large amounts of maternal HIV antibody. HIV-exposed
if clinically demonstrated, in assisting physicians to treat patients accordingly infants are typically assessed using HIV-DNA PCR at least three times in the
depending on the subtype of HIV-1 infection. first four months of life (within seven days of birth and at one and four
months) to rule out active HIV disease. This assay, when used in an
In addition to the above commercial viral load assays, a number of other experienced molecular laboratory, can reliably detect HIV sequences in
methods are used internationally or are under development. These methods samples with as few as five copies of HIV in one million mononuclear cells.
include the ligase chain reaction (LCR, Abbott Laboratories), Qβ-replicase A positive HIV DNA PCR test should be confirmed with a second sample
(Gene Track Inc.), strand displacement amplification (SDA, Becton Dickinson with another HIV DNA PCR assay or a quantitative plasma RNA PCR assay.
Co), hybrid capture system (Digene Co.), transcription-mediated The HIV DNA PCR assay is sometimes used to rule out active HIV infection
amplification (TMA, Gen-Probe Inc), and the HIV RT viral load test (ExaVir in patients who have repeatedly reactive serological tests (EIA) with
Load, Cavidi Tech). Each of these methodologies is based on different indeterminate confirmatory tests (IFA or Western Blot).
scientific principles, but they all have the potential to provide similar and
competitive assays for measuring HIV-1 viral load. Detection of HIV-1 Drug Resistance
HIV is a highly variable and active replicating virus that has the ability for
Another reported method is immuno-PCR for the detection of ultra-low multiple genomic mutations, making it prone to developing drug resistance.
levels of HIV p24 antigen.
18
In this technique, a serological antigen capture Drug resistance can be developed early in the latent phase even before or at
EIA method is coupled to realtime PCR, where the detector antibody is the time of HAART.
8
Mechanisms of resistance may vary from mutation to
indirectly labeled with a 500 base pair strand of DNA. The DNA is mutation depending on the kind of drug regimen used. For example, drug-
subsequently used as a template for amplification, with the detection of resistant mutations to the nucleoside RT inhibitors (NRTIs) block incorporation
amplicons using fluorescence-generating probes. This method was shown of NTRI into pro-viral DNA;
7
drug-resistant mutations to the non-nucleoside RT
to detect lower levels of HIV virions than the NAT methods. Another method inhibitors (NNRTIs) prevents binding of the NNRTIs to RT; and mutations to the
is a boosted EIA method to detect p24 antigen, a method that is much PR inhibitors (PRIs) resist PRI binding to the binding pocket of the protease.
easier to perform than NAT methods and has been reported to have a Genotypic and phenotypic resistance assays can be used to identify these
sensitivity equivalent to or even surpassing that of NAT.
9,20
For more mutations and viral resistance or susceptibility to drugs. These techniques
extensive reviews of these technologies see references 18–22. allow clinicians to choose or change specific therapeutic regimens based on
the patient’s drug-resistance profile or to treat naïve patients who are infected
Measurement of HIV-1 Pro-viral DNA with drug-resistant viral strains, thus improving outcomes of patient
Treatment of HIV-infected patients with highly active retroviral therapy management.
34,35
Genotype analysis compares a patient’s viral genotype with
(HAART) or antiretroviral therapy (ART) often achieves non-detectable HIV-1 wild-type or known mutant oligonucleotides using direct sequencing of the
viral RNA levels. However, questions remain as to whether or not the pro-viral amplified HIV genome or hybridization techniques. The assays target specific
DNA, either integrated or un-integrated, is still present or responds to HAART. genes involved in drug mechanisms such as RT or PR. Phenotypic assays, on
It is not known whether relative changes in pro-viral DNA levels precede the other hand, measure drug inhibitory performance or the degree to
changes of viral RNA levels post-HAART introduction. Therefore, one of the which the drug inhibits viral replication by determining shifts in viral
current challenges in HIV disease management is to detect the presence of susceptibility. Results of the phenotypic assay can then be detected with any
low-level pro-viral DNA in latently infected CD4 T-lymphocytes and other of the viral assays such as p24 antigen, or an RT assay, reported as a fold-
reservoirs, especially because these hidden reservoirs can replenish and revive change between the isolates. Comparative studies can be performed for the
viral infection upon activation.
23–25
Several research-based assays have been patient’s genotype and phenotype results to document a pattern of resistance
reported previously for quantification of HIV-1 pro-viral DNA, all of which were to the drug and others in that class. Algorithms have been created for
based on the principle of conventional PCR.
26–29
The potential limitation phenotypic interpretation of genotypic mutations. Another method used for
associated with the traditional quantitative PCR is that the DNA copy numbers resistant testing is a virtual phenotyping system. This system uses a patient’s
are calculated based on the final amplified gene products. As DNA is amplified pre-determined genotype assay result and compares it with a larger database
exponentially during PCR, a small variation in amplification efficiency early in of paired genotype and phenotype patterns.
36
22 US INFECTIOUS DISEASE 2007
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