Ultrasensitive reverse transcription-PCR assay for quantitation of human immunodeficiency virus type 1 RNA in plasma

Abstract

With the recent introduction of combination therapy, human immunodeficiency virus type 1 (HIV-1) RNA levels in plasma have been dramatically reduced, frequently to below the limit of quantitation (400 copies/ml of plasma) of the AMPLICOR HIV-1 MONITOR Test (Roche Diagnostic Systems). To achieve enhanced sensitivity of the AMPLICOR HIV-1 MONITOR Test, a modified specimen preparation procedure that allows input of RNA from 10-fold more plasma per amplification reaction was developed. This "ultrasensitive" method allows the accurate quantitation of plasma HIV-1 RNA levels as low as 50 copies/ml. A precision study yielded average within-run and between-run coefficients of variation (CV) of 24.8 and 9.6%, respectively. A multicenter reproducibility study demonstrated that the laboratory-to-laboratory reproducibility of this assay is good, with an average CV of 32%. The linear range of this test is between 50 and 50,000 copies/ml of plasma. RNA concentrations measured by the ultrasensitive and standard HIV-1 MONITOR tests exhibited good agreement within the shared linear range of the two methods. The two measurements were within a factor of 2 for 91% of the specimens tested, with the concentration measured by the ultrasensitive method being only slightly lower (median, 22% lower). Preliminary studies suggest that this assay will prove to be useful for predicting the stability of viral suppression in patients whose RNA levels drop below 400 copies/ml in response to highly active antiretroviral therapy.

FIG. 1

Linear range for the ultrasensitive AMPLICOR HIV-1 MONITOR Test. Six replicate tests were performed for each input viral RNA concentration. For input RNA concentrations below 50 copies/ml, some of the replicates gave negative results and were excluded from the data analysis. At each input RNA concentration, the mean of the log₁₀ calculated RNA concentrations and the standard deviation were determined (circles). The open circles indicate the results for concentrations that were used for linear regression analysis. The closed circles indicate the results for concentrations that were not included in the regression analysis. Linear regression analysis was performed for input RNA concentrations of from 50 to 50,000 copies/ml (solid line and equation). The regression analysis was performed with the individual results for each replicate at each input RNA concentration.

FIG. 2

Correlation between the ultrasensitive and standard AMPLICOR HIV-1 MONITOR Tests. The log₁₀ calculated RNA concentration was determined by each method for a set of 100 specimens. Specimens are grouped by calculated RNA concentration (○, 250 to 9,999 copies/ml; ×, 10,000 to 49,999 copies/ml; ▵, 50,000 to 99,999 copies/ml; □, 100,000, to 499,999 copies/ml). Separate linear regressions were performed for each group of specimens (see Table 2). The regression line for specimens with <10,000 copies/ml is shown (solid line).

FIG. 3

Specificity of the ultrasensitive AMPLICOR HIV-1 MONITOR Test. A set of 50 HIV-1-negative specimens was tested by the ultrasensitive and standard methods. The HIV-1 and QS signals are shown. The dashed line indicates the test cutoff (A₄₅₀, 0.2).

FIG. 4

Detection of HIV-1 RNA in clinical specimens with low titers of HIV-1 RNA. RNA titers were measured by the ultrasensitive AMPLICOR HIV-1 MONITOR Test in a set of 24 specimens that gave negative results by the standard AMPLICOR HIV-1 MONITOR Test. The solid line indicates the lower limit of the linear range. Filled bars indicate the RNA titer in specimens that gave positive results, and open bars indicate specimens that gave negative results.