Virus Load Testing


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Virus Load Testing

Measurement of plasma HIV-1 RNA levels (virus load) can be used to monitor the course of disease and the response to antiretroviral therapy in patients with HIV-1-infection. Assays based on different methods for quantifying plasma HIV-1 RNA assay have been developed. These include reverse transcription followed by polymerase chain reaction (RT-PCR) (Amplicor HIV-1 Monitor, Roche Diagnostic Systems), nucleic acid sequence-based amplification (NASBA; HIV-1 RNA QT, Organon-Teknika), and nucleic acid hybridization and branched DNA (bDNA) signal amplification (Quantiplex HIV-1 RNA, Bayer Nucleic Acid Diagnostics). A fourth assay, based on DNA hybridization and colorimetric detection (Digene assay; Digene Diagnostics) has also been developed, but is not widely available at this time.

In PCR-based assays, HIV RNA is converted into DNA by reverse transcription followed by PCR amplification of the DNA. The PCR product is detected by hybridization with an enzyme-conjugated probe specific for HIV-1, and quantified by reacting bound probe with a substrate that undergoes a color change, as in an ELISA. The branched DNA assay uses non-enzymatic means to amplify the signal from HIV RNA. In this assay, viral RNA is "captured" by hybridization to complementary oligonucleotides that are bound to the wells of a microtiter plate. The captured viral RNA target is then hybridized to branched oligonucleotides (hence the name "branched" DNA assay), which in turn are hybridized to enzyme-conjugated oligonucleotides that can be quantified as above. The NASBA assay is similar in concept to the RT-PCR assay except that reactions occur at one temperature. At present the Roche Amplicor HIV-1 Monitor (RT-PCR) assay is the only one approved by the U.S. Food and Drug Administration, and is the most widely used in clinical practice.

Results of the three commercially available quantitative HIV-1 RNA assays are highly correlated (42; 43). All three assays have a lower limit of quantification of approximately 50-80 copies/mL (Table 3). (Although the lower limit of the standard Amplicor HIV-1 Monitor assay is 400 copies/mL, the range of the assay can be extended by pelleting virion particles prior to RNA extraction, a modification commonly referred to as the "ultrasensitive" HIV-1 Monitor assay.) These assays are much less precise at plasma HIV-1 RNA titers below 200 copies/mL (44). Serial testing of clinically stable patients not on antiretroviral therapy (or on a stable failing regimen) has shown the relative stability of plasma HIV-1 RNA levels over the short term (weeks to months), with a biological variation of approximately 0.3-0.4 log10 copies/mL (45; 46). Given these factors, changes of greater than 0.5-0.7 log10 (3- to 5-fold) are likely to reflect significant changes in HIV-1 replication (47).

Although most strains of HIV-1 that circulate in North America belong to subtype B, more than 10 different subtypes are found around the world. The HIV-1 Monitor 1.0 (RT-PCR) assay is significantly less sensitive for detecting HIV-1 from subtypes A, E, and F as compared to the Quantiplex version 3.0 (bDNA) assay (48). Plasma HIV-1 RNA levels that appear to be lower than expected in a patient with advanced disease can be a clue to infection with a non-subtype B strain. Incorporation of alternative primer sets in the new version of the HIV-1 Monitor assay (version 1.5) has improved the ability of this assay to diverse HIV-1 subtypes.

4/15/01