Discordance between Viral Loads Determined by Roche COBAS AMPLICOR Human Immunodeficiency Virus Type 1 Monitor (Version 1.5) Standard and Ultrasensitive Assays Caused by Freezing Patient Plasma in Centrifuged Becton-Dickinson Vacutainer Brand Plasma Preparation Tubes

Abstract

The Roche COBAS AMPLICOR human immunodeficiency virus type 1 (HIV-1) Monitor (version 1.5) standard and ultrasensitive viral load assays often gave discordant results, with viral loads from the standard assay exceeding those from the ultrasensitive assay by more than 0.5 log 10 for approximately 20% of specimens received. We began studies to determine the extent, magnitude, and reproducibility of the discordance between the assays and to discover and eliminate the cause of this discordance. Until then, we revised our standard operating procedure to include both standard and ultrasensitive testing on all specimens submitted for viral load determinations. Discordant results usually recurred on retesting. They were most prevalent for specimens with ultrasensitive viral loads of <1,000 and rare for specimens with viral loads of >10,000. Often, standard assay results exceeded those of the ultrasensitive assay by 50-to 100-fold. At higher viral loads, the difference between the standard and ultrasensitive assays persisted, but the percent difference was smaller and rarely caused discordance. The proportion of discordant results was significantly higher in specimens from pediatric patients than in specimens from adults. The ultrasensitive viral load determinations generally agreed with the results of the B-DNA (Bayer) viral load assays. If the plasma was transferred from the centrifuged plasma preparation tubes before freezing, standard and ultrasensitive results were concordant with each other and with values determined on plasma from lavender-topped EDTA tubes.Human immunodeficiency virus (HIV) viral load determinations represent the standard of care in the initial assessment of HIV-infected persons and in the subsequent management of the disease (4,11,13). Spurious high viral loads may cause patients to be started unnecessarily on antiretroviral therapy. For the patient on therapy, such elevated results could prompt unnecessary repeat testing, HIV genotyping, or medication changes. Pregnant women could receive unnecessary caesarean sections. Providers may wrongly conclude that there is nonadherence to a prescribed antiretroviral regimen. Erroneous conclusions may be drawn if the patient is participating in a clinical trial. Thus, accuracy and reproducibility of the viral load results are important considerations. The Roche standard and ultrasensitive assays, available in manual and automated (COBAS) versions, are the most common tests for viral load quantification, and the Bayer Versant human immunodeficiency virus type 1 (HIV-1) RNA 3.0 (B-DNA) assay (Tarrytown, New York) is also widely used. The standard and ultrasensitive assays have been reported to correlate well both within versions and between versions 1.0 and 1.5 (2, 3, 5, 6), although at very low viral loads version 1.5 is the more sensitive (2, 9).Our laboratory offered both (COBAS) standard and ultrasensitive assays (version 1.5) and performed the ordered test. When standard and ultrasensitive assays were performed with the same patient sample, the ultras...

Overestimation of Human Immunodeficiency Virus Type 1 Load Caused by the Presence of Cells in Plasma from Plasma Preparation Tubes

“…Freezing of centrifuged PPTs in situ has previously been reported to cause an artificial elevation in HIV-1 RNA levels (11,17,19,21). We found elevated levels of HIV-1 RNA in plasma from PPTs that had not been frozen but that were shipped after centrifugation.…”

Coamplification of HIV-1 Proviral DNA and Viral RNA in Assays Used for Quantification of HIV-1 RNA

“…The issue that needs to be addressed relates to the contribution of residual proviral DNA to HIV-1 quantification in the assay. As stated by Salimnia et al (11), "A recent study by the Viral Quality Assurance Laboratory (NO1-AI-85354) determined that a single copy of HIV-1-specific proviral DNA could increase the detected viral load by 100 to 200 RNA copies (Cheryl Jennings [Rush Medical Center], personal communication)." Keeping this in mind, it is reasonable to assume The Roche COBAS Amplicor HIV-1 Monitor version 1.5 assay that is used for quantification of HIV-1 amplifies a 143-bp segment in the conserved gag (group antigen) region of the HIV-1 genome.…”

“…Integrated proviral DNA of the HIV-1 viral genome contains the gag gene and is therefore amplified when present in the extracted RNA specimen. In fact, the Amplicor assay for the detection of HIV-1 proviral DNA uses the very same primers to target the same 143-bp region of the gag gene of the HIV-1 genome and can potentially amplify any virus-specific template, either DNA or RNA, that may be present (11). Yet another factor to consider in this analysis is the method of extraction of nucleic acid.…”

“…Some reports attribute the observed VL blips to random biological and statistical variations, with limited clinical significance (6), while others have implied that the elevated HIV-1 VL may interfere in the assessment of virological suppression. Subsequent studies show that aspiration and transfer of plasma from a PPT to a storage tube after centrifugation or recentrifugation of the PPT after transportation reduces or eliminates the artificial elevation of VL observed in the PPT (9,10,11). Salimnia et al (11) imply that the elevation may be due to nonparticulate genetic material released from cells associated with the gel in the PPT after the plasma was thawed.…”

“…Subsequent studies show that aspiration and transfer of plasma from a PPT to a storage tube after centrifugation or recentrifugation of the PPT after transportation reduces or eliminates the artificial elevation of VL observed in the PPT (9,10,11). Salimnia et al (11) imply that the elevation may be due to nonparticulate genetic material released from cells associated with the gel in the PPT after the plasma was thawed. Additional investigations report that samples collected in PPTs can be centrifuged up to 6 h after phlebotomy and stored in situ at 4°C for up to 72 h without any effect on the measured HIV-1 VL (9).…”