AEGiS-11IAC: HIV RNA copy number estimates by PCR based amplification assays that use serial dilution of Amplicon prior to detection.

11th International AIDS Conference

Vancouver, British Columbia — July 7-12, 1996

HIV RNA copy number estimates by PCR based amplification assays that use serial dilution of Amplicon prior to detection.

Int Conf AIDS 1996 Jul 7-12; 11:17 (abstract no. We.A.522)
Bremer JW, Brambilla D, Reichelderfer P; Rush Medical College, Chicago, IL, USA. Fax: 312-942-6787.

OBJECTIVE: To compare the results obtained from four algorithms that have been used to estimate HIV RNA copy number from the PCR based Roche Amplicor HIV Monitor assay.

METHODS: Twenty-one virology laboratories participating in the DAIDS Virology Quality Assurance Program used the Roche assay to estimate RNA concentration on a recent proficiency panel. The panel consisted of blinded replicates of plasma spiked with known copy numbers of HIV RNA at a sequence of five-fold dilutions. The Roche assay employs six five-fold dilutions of an amplified specimen and two five-fold dilutions of an internal control to estimate RNA copy number. Four algorithms were applied to the results from each laboratory to calculate copy number. Under the first two, the least diluted well (top-down) with an optical density (OD) between 0.2 and 2.0 is selected for estimating copy number. Under the second two, the most diluted well (bottom-up) with an OD in that range is selected. The same approaches are applied to the internal controls. The first and third methods selected a control well with an OD between 0.20 and 2.0, while the second and fourth selected a control well with an OD between 0.3 and 2.0

RESULTS: Among the 500 assays that were included in the analysis, there were five for which the estimates of copy number differed quite widely among algorithms. The first algorithm produced estimates that were about 1% of the theoretical RNA concentrations and the second produced estimates that were 10% of the theoretical concentrations. Both the third and fourth methods produced copy numbers that were very close to the theoretical concentrations. Reproducibility, which was assessed by comparing estimates of RNA copy number among replicates, was also better for the third and fourth methods across the entire panel. The third method produced fewer invalid results (all OD's out of range) than did the fourth method.

CONCLUSIONS: The third algorithm, which selected the most diluted well with an OD between 0.2 and 2.0 for both specimen and internal control, appeared to be the best for determining HIV RNA copy numbers for the Roche assay.

Keywords: AEGIS, HIV, Polymerase Chain Reaction, RNA, RNA, Viral, HIV-1, HIV Infections, HIV Core Protein p24, Laboratories, virology, ICA11

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WeA522