466 Persistence of Stable, Quantifiable Viremia in Patients on Antiretroviral Therapy Despite Suppression of Plasma HIV-1 RNA to less than 50 Copies/ml F. Maldarelli*1, A. Wiegand1, S. Palmer1, M. Kearney1, V. Boltz1, M. Polis2, J. Falloon2, J. Mican2, R. Davey2, D. Rock2, S. Liu3, A. Planta3, J. Metcalf3, J. Mellors4, J. Coffin1 1HIV Drug Resistance Prgm, NCI, NIH, Frederick, MD; 2Lab of Immunoregulation, NIAID, NIH, Bethesda, MD; 3Natl Inst of Allergy and Infectious Diseases/CCMD Clin NIH, Bethesda, MD; and 4Univ of Pittsburgh, PA
Background: Potent antiretroviral therapy is effective in suppressing but not eradicating HIV-1 infection. Antiretroviral drug resistance leading to treatment failure can evolve despite initial suppression of HIV-1 RNA to below the limit of detection of current assays. HIV-1 RNA assays of greater sensitivity and precision are needed to detect and quantify persistent viremia in patients (pts) on therapy.
Methods: We developed a more sensitive and precise assay than has previously existed to quantify HIV-1 RNA in plasma. HIV-1 is pelleted from plasma by ultracentrifugation and RNA is extracted using guanidinium isothiocyanate. Recovery of HIV-1 is monitored using an avian retrovirus as internal standard spiked into the plasma samples. Reverse transcription and real time PCR are used to amplify and quantify a specific, conserved HIV gag sequence. This single copy assay has a dynamic range of 106 to 1 copy of HIV RNA/ml.
Results: We applied this assay to detect and quantify persistent viremia in 15 pts with HIV-1 RNA suppressed to < 50 copies/ml (as determined by standard methods) for at least 131 days. The single copy assay detected HIV-1 RNA in plasma from 14 of the 15 pts. In one pt, no HIV-1 RNA could be detected in the assay (< 0.25 copies/ml). HIV-1 RNA levels ranged from 1-40 copies/ml with a mean of 9.3 and a median of 3.9 copies/ml. No HIV-1 RNA was detected in plasma from 10 seronegative controls. To investigate whether persistent viremia represented a therapeutic steady-state, we analyzed longitudinal samples from 5 of the pts. HIV-1 RNA levels remained stable (mean cv for pts = 0.34) over the period of observation (up to 7 months). We investigated baseline and on treatment factors for associations with the level of persistent viremia. Higher levels of persistent viremia were associated with higher pre-treatment HIV-1 RNA levels. The lowest levels of persistent viremia were present in patients on > 3 antiretroviral agents. No obvious association was found between the level of persistent viremia and the duration of antiretroviral therapy, baseline CD4 cell count, or change in CD4 cell count in response to therapy.
Conclusions: In 14 of 15 pts, the single copy HIV-1 RNA assay revealed stable, persistent viremia in pts on suppressive antiretroviral therapy (< 50 copies/ml by standard assays). The apparent relation between regimen potency and the level of viremia suggests that viremia may be sustained by ongoing HIV-1 replication.
