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. 2003 Oct;41(10):4531-6.
doi: 10.1128/JCM.41.10.4531-4536.2003.

New real-time reverse transcriptase-initiated PCR assay with single-copy sensitivity for human immunodeficiency virus type 1 RNA in plasma

Sarah Palmer  1 , Ann P Wiegand, Frank Maldarelli, Holly Bazmi, JoAnn M Mican, Michael Polis, Robin L Dewar, Angeline Planta, Shuying Liu, Julia A Metcalf, John W Mellors, John M Coffin
Affiliations

New real-time reverse transcriptase-initiated PCR assay with single-copy sensitivity for human immunodeficiency virus type 1 RNA in plasma

Sarah Palmer et al. J Clin Microbiol. 2003 Oct.

Abstract

More sensitive assays for human immunodeficiency virus type 1 (HIV-1) RNA are needed to detect, quantify, and characterize persistent viremia in patients who are receiving antiretroviral therapy and whose plasma HIV-1 RNA levels are suppressed to less than 50 to 75 copies/ml. We therefore developed an internally controlled real-time reverse transcriptase-initiated PCR assay that quantifies HIV-1 RNA concentrations down to 1 copy per ml of plasma. This assay with single-copy sensitivity (the single-copy assay) generates a reproducible linear regression plot of input copy number versus threshold cycle by using HIV-1 RNA transcripts at copy numbers ranging from 1 to 10(6) per reaction mixture. The single-copy assay was compared to the ultrasensitive AMPLICOR HIV-1 MONITOR assay and a more sensitive modification of the ultrasensitive assay by repeatedly testing a low-copy-number panel containing 200 to 0.781 copies of HIV-1 RNA per ml of plasma. This comparison showed that the single-copy assay had a greater sensitivity than the other assays and was the only assay that detected HIV-1 RNA at levels as low as 0.781 copies/ml. Testing of plasma samples from 15 patients who were receiving antiretroviral therapy and who had <75 HIV-1 RNA copies/ml revealed persistent viremia in all 15 patients, with HIV-1 RNA levels ranging from 1 to 32 copies/ml (median, 13 copies/ml). The greater sensitivity of the single-copy assay should allow better characterization of persistent viremia in patients who are receiving antiretroviral therapy and whose HIV-1 RNA levels are suppressed to below the detection limits of present assays.

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Figures

FIG. 1.
FIG. 1.
(A) Plot of input control template (RNA transcript) copy number versus Ct value; (B) plot of input control template copy number versus measured copy number per reaction mixture. The hashed lines represent regression plots of the measured data, and the solid lines represent the 95% confidence intervals for the measured values.
FIG. 2.
FIG. 2.
Comparison of bDNA assay values versus single-copy assay values for 22 individual patients with bDNA assay values of >1,000 copies/ml. Twenty individual patient plasma samples (open circles) were analyzed by both assays at a single time point. For two of the patients, levels were measured at four times (one time before and three times after treatment initiation) (filled symbols). For all patient samples, 1 to 2 ml of patient plasma was analyzed by the single-copy assay.
FIG. 3.
FIG. 3.
Single-copy assay measurements of HIV-1 RNA loads in 15 patient plasma samples found to contain <75 copies/ml by the bDNA assay. Each point represents the mean and standard deviation of at least triplicate values for the sample from each patient. Pretherapy samples were available for patients 1 to 4.
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