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Journal of Clinical Microbiology, October 2003, p. 4531-4536, Vol. 41, No. 10
0095-1137/03/$08.00+0     DOI: 10.1128/JCM.41.10.4531-4536.2003
Copyright © 2003, American Society for Microbiology. All Rights Reserved.

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,1 Frank Maldarelli,1 Holly Bazmi,2 JoAnn M. Mican,3 Michael Polis,3 Robin L. Dewar,3 Angeline Planta,3 Shuying Liu,3 Julia A. Metcalf,3 John W. Mellors,2 and John M. Coffin1

HIV Drug Resistance Program, National Cancer Institute, National Institutes of Health, Frederick, Maryland,1 University of Pittsburgh, Pittsburgh, Pennsylvania,2 Laboratory of Immunoregulation, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Bethesda, Maryland3

Received 27 March 2003/ Returned for modification 19 May 2003/ Accepted 10 July 2003

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 106 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.

* Corresponding author. Mailing address: HIV Drug Resistance Program, National Cancer Institute at Frederick, 1050 Boyles St., Building 535, Room 109, Frederick, MD 21702-1201. Phone: (301) 846-5599. Fax: (301) 846-6013. E-mail: spalmer{at}ncifcrf.gov.

Journal of Clinical Microbiology, October 2003, p. 4531-4536, Vol. 41, No. 10
0095-1137/03/$08.00+0     DOI: 10.1128/JCM.41.10.4531-4536.2003
Copyright © 2003, American Society for Microbiology. All Rights Reserved.



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