Evaluation of a second-generation nucleic acid sequence-based amplification assay for quantification of HIV type 1 RNA and the use of ultrasensitive protocol adaptations

Accurate assessment of plasma HIV RNA levels at low concentrations is clini cally important. We evaluated a second-generation quantitative HIV RNA assa y (NucliSens HIV-1 QT), and three simple adaptations of the NucliSens stand ard protocol to lower the lower cutoff level. The assays were evaluated in constructed panels with known HIV RNA concentrations and in clinical sample s. Results were compared with those obtained with the first generation (NAS BA HIV-1 QT) and with two other commercially available assays: the Amplicor HIV Monitor test and the Quantiplex assay. In a constructed panel, results obtained by NASBA QT were on average 0.13 log(10) copies/ml (SD 0.15) high er than those of NucliSens. The NucliSens assay could quantify HIV RNA in a t least 50% of the samples down to 518 (2.71 log(10)) copies/ml and NASBA Q T to 5.80 x 10(3) (3.76 log(10)) copies/ml). Both assays correlated well wi th the known input (R NucliSens = 0.99; R NASBA QT = 0.996), but results we re more variable at lower input levels. With the three different ultrasensi tive NucliSens adaptations, HIV RNA could be quantified in at least 50% of the samples down to 100 (2.00 log(10)), 46 (1.66 log(10)), and 10 (1.00 log (10)) copies/ml, respectively. In patient samples, Amplicor results were on average 0.11 (SD 0.20) log(10) copies/ml above, NucliSens 0.02 (SD 0.29) c opies/ml above, and Quantiplex 0.13 (SD 0.19) copies/ml below the mean of t he three assay results per sample. The variation remained the same over the range of RNA levels with all three assays. The NucliSens assay can quantif y HIV RNA at lower levels than the NASBA QT and is comparable to other comm ercially available assays. The lower cutoff of the NucliSens can be lowered down to 10 copies/ml.