Techniques and interpretation of HIV-1 RNA quantitation
- Angela M Caliendo, MD, PhD
Angela M Caliendo, MD, PhD
- Executive Vice Chair of Medicine
- Brown University
Human immunodeficiency virus type 1 (HIV-1) RNA can be measured using qualitative or quantitative techniques. Qualitative testing (commonly referred to as nucleic acid testing or NAT) is used as a screening test to identify HIV infected individuals, such as screening possible blood donors. Quantification of HIV-RNA (viral load measurements) can be used as a diagnostic test in certain situations; however, the HIV viral load is primarily used for management/monitoring of HIV-1 infected individuals.
This topic will address the laboratory methods for quantitation of HIV-1 RNA and the use of viral load for clinical management. Information on nucleic acid, HIV-2 RNA, and CD-4 cell count testing is found elsewhere. (See "Blood donor screening: Laboratory testing", section on 'HIV-1 and HIV-2' and "Clinical manifestations and diagnosis of HIV-2 infection", section on 'Testing for HIV-2 infection' and "Techniques and interpretation of measurement of the CD4 cell count in HIV-infected patients".)
IMPORTANCE OF HIV VIRAL LOAD MEASUREMENTS
Studies have shown HIV-1 RNA levels to be a predictor of the time to progression to acquired immunodeficiency syndrome (AIDS) and death that is independent of CD4 cell counts [1-6]. Viral load measurements are also useful in determining when to initiate antiretroviral therapy, and in monitoring the response to such therapy [7-11]. (See "Selecting antiretroviral regimens for the treatment-naïve HIV-infected patient" and "Patient monitoring during HIV antiretroviral therapy".)
In specific situations (neonatal infection and acute infection), HIV-1 RNA levels also may be useful in establishing the diagnosis of HIV infection, but HIV antibody tests are primarily used for this purpose.
LABORATORY METHODS FOR QUANTITATION OF HIV-1 RNA
There are four commercial assays that have been approved by the United States Food and Drug Administration (FDA) to quantify HIV-1 RNA from plasma samples:
- Mellors JW, Rinaldo CR Jr, Gupta P, et al. Prognosis in HIV-1 infection predicted by the quantity of virus in plasma. Science 1996; 272:1167.
- O'Brien TR, Blattner WA, Waters D, et al. Serum HIV-1 RNA levels and time to development of AIDS in the Multicenter Hemophilia Cohort Study. JAMA 1996; 276:105.
- O'Brien WA, Hartigan PM, Martin D, et al. Changes in plasma HIV-1 RNA and CD4+ lymphocyte counts and the risk of progression to AIDS. Veterans Affairs Cooperative Study Group on AIDS. N Engl J Med 1996; 334:426.
- Katzenstein DA, Hammer SM, Hughes MD, et al. The relation of virologic and immunologic markers to clinical outcomes after nucleoside therapy in HIV-infected adults with 200 to 500 CD4 cells per cubic millimeter. AIDS Clinical Trials Group Study 175 Virology Study Team. N Engl J Med 1996; 335:1091.
- Shearer WT, Quinn TC, LaRussa P, et al. Viral load and disease progression in infants infected with human immunodeficiency virus type 1. Women and Infants Transmission Study Group. N Engl J Med 1997; 336:1337.
- Mellors JW, Muñoz A, Giorgi JV, et al. Plasma viral load and CD4+ lymphocytes as prognostic markers of HIV-1 infection. Ann Intern Med 1997; 126:946.
- Saag MS, Holodniy M, Kuritzkes DR, et al. HIV viral load markers in clinical practice. Nat Med 1996; 2:625.
- Hughes MD, Johnson VA, Hirsch MS, et al. Monitoring plasma HIV-1 RNA levels in addition to CD4+ lymphocyte count improves assessment of antiretroviral therapeutic response. ACTG 241 Protocol Virology Substudy Team. Ann Intern Med 1997; 126:929.
- O'Brien WA, Hartigan PM, Daar ES, et al. Changes in plasma HIV RNA levels and CD4+ lymphocyte counts predict both response to antiretroviral therapy and therapeutic failure. VA Cooperative Study Group on AIDS. Ann Intern Med 1997; 126:939.
- Yeni PG, Hammer SM, Hirsch MS, et al. Treatment for adult HIV infection: 2004 recommendations of the International AIDS Society-USA Panel. JAMA 2004; 292:251.
- Panel on Antiretroviral Guidelines for Adults and Adolescents. Guidelines for the use of antiretroviral agents in HIV-1-infected adults and adolescents. Department of Health and Human Services. Available at http://www.aidsinfo.nih.gov/contentfiles/lvguidelines/adultandadolescentgl.pdf (Accessed on October 31, 2013).
- Mulder J, McKinney N, Christopherson C, et al. Rapid and simple PCR assay for quantitation of human immunodeficiency virus type 1 RNA in plasma: application to acute retroviral infection. J Clin Microbiol 1994; 32:292.
- Pachl C, Todd JA, Kern DG, et al. Rapid and precise quantification of HIV-1 RNA in plasma using a branched DNA signal amplification assay. J Acquir Immune Defic Syndr Hum Retrovirol 1995; 8:446.
- Schumacher W, Frick E, Kauselmann M, et al. Fully automated quantification of human immunodeficiency virus (HIV) type 1 RNA in human plasma by the COBAS AmpliPrep/COBAS TaqMan system. J Clin Virol 2007; 38:304.
- Gueudin M, Plantier JC, Lemée V, et al. Evaluation of the Roche Cobas TaqMan and Abbott RealTime extraction-quantification systems for HIV-1 subtypes. J Acquir Immune Defic Syndr 2007; 44:500.
- Foulongne V, Montes B, Didelot-Rousseau MN, Segondy M. Comparison of the LCx human immunodeficiency virus (HIV) RNA quantitative, RealTime HIV, and COBAS AmpliPrep-COBAS TaqMan assays for quantitation of HIV type 1 RNA in plasma. J Clin Microbiol 2006; 44:2963.
- Berger A, Scherzed L, Stürmer M, et al. Comparative evaluation of the Cobas Amplicor HIV-1 Monitor Ultrasensitive Test, the new Cobas AmpliPrep/Cobas Amplicor HIV-1 Monitor Ultrasensitive Test and the Versant HIV RNA 3.0 assays for quantitation of HIV-1 RNA in plasma samples. J Clin Virol 2005; 33:43.
- Damond F, Roquebert B, Bénard A, et al. Human immunodeficiency virus type 1 (HIV-1) plasma load discrepancies between the Roche COBAS AMPLICOR HIV-1 MONITOR Version 1.5 and the Roche COBAS AmpliPrep/COBAS TaqMan HIV-1 assays. J Clin Microbiol 2007; 45:3436.
- Cao Y, Ho DD, Todd J, et al. Clinical evaluation of branched DNA signal amplification for quantifying HIV type 1 in human plasma. AIDS Res Hum Retroviruses 1995; 11:353.
- Lin HJ, Myers LE, Yen-Lieberman B, et al. Multicenter evaluation of quantification methods for plasma human immunodeficiency virus type 1 RNA. J Infect Dis 1994; 170:553.
- Elbeik T, Charlebois E, Nassos P, et al. Quantitative and cost comparison of ultrasensitive human immunodeficiency virus type 1 RNA viral load assays: Bayer bDNA quantiplex versions 3.0 and 2.0 and Roche PCR Amplicor monitor version 1.5. J Clin Microbiol 2000; 38:1113.
- Farzadegan H, Hoover DR, Astemborski J, et al. Sex differences in HIV-1 viral load and progression to AIDS. Lancet 1998; 352:1510.
- Jain V, Liegler T, Kabami J, et al. Assessment of population-based HIV RNA levels in a rural east African setting using a fingerprick-based blood collection method. Clin Infect Dis 2013; 56:598.
- Dickover RE, Herman SA, Saddiq K, et al. Optimization of specimen-handling procedures for accurate quantitation of levels of human immunodeficiency virus RNA in plasma by reverse transcriptase PCR. J Clin Microbiol 1998; 36:1070.
- Holodniy M, Mole L, Yen-Lieberman B, et al. Comparative stabilities of quantitative human immunodeficiency virus RNA in plasma from samples collected in VACUTAINER CPT, VACUTAINER PPT, and standard VACUTAINER tubes. J Clin Microbiol 1995; 33:1562.
- Holodniy M, Rainen L, Herman S, Yen-Lieberman B. Stability of plasma human immunodeficiency virus load in VACUTAINER PPT plasma preparation tubes during overnight shipment. J Clin Microbiol 2000; 38:323.
- García-Bujalance S, Ladrón de Guevara C, González-García J, et al. Elevation of viral load by PCR and use of plasma preparation tubes for quantification of human immunodeficiency virus type 1. J Microbiol Methods 2007; 69:384.
- Griffith BP, Mayo DR. Increased levels of HIV RNA detected in samples with viral loads close to the detection limit collected in Plasma Preparation Tubes (PPT). J Clin Virol 2006; 35:197.
- Erice A, Brambilla D, Bremer J, et al. Performance characteristics of the QUANTIPLEX HIV-1 RNA 3.0 assay for detection and quantitation of human immunodeficiency virus type 1 RNA in plasma. J Clin Microbiol 2000; 38:2837.
- Sun R, Ku J, Jayakar H, et al. Ultrasensitive reverse transcription-PCR assay for quantitation of human immunodeficiency virus type 1 RNA in plasma. J Clin Microbiol 1998; 36:2964.
- Holodniy M, Mole L, Winters M, Merigan TC. Diurnal and short-term stability of HIV virus load as measured by gene amplification. J Acquir Immune Defic Syndr 1994; 7:363.
- Brambilla D, Reichelderfer PS, Bremer JW, et al. The contribution of assay variation and biological variation to the total variability of plasma HIV-1 RNA measurements. The Women Infant Transmission Study Clinics. Virology Quality Assurance Program. AIDS 1999; 13:2269.
- Donovan RM, Bush CE, Markowitz NP, et al. Changes in virus load markers during AIDS-associated opportunistic diseases in human immunodeficiency virus-infected persons. J Infect Dis 1996; 174:401.
- Mole L, Ripich S, Margolis D, Holodniy M. The impact of active herpes simplex virus infection on human immunodeficiency virus load. J Infect Dis 1997; 176:766.
- O'Brien WA, Grovit-Ferbas K, Namazi A, et al. Human immunodeficiency virus-type 1 replication can be increased in peripheral blood of seropositive patients after influenza vaccination. Blood 1995; 86:1082.
- Staprans SI, Hamilton BL, Follansbee SE, et al. Activation of virus replication after vaccination of HIV-1-infected individuals. J Exp Med 1995; 182:1727.
- Hu DJ, Dondero TJ, Rayfield MA, et al. The emerging genetic diversity of HIV. The importance of global surveillance for diagnostics, research, and prevention. JAMA 1996; 275:210.
- Cartwright CP. The changing epidemiology of HIV/AIDS at a Minnesota hospital: impact of demographic change and viral diversity. J Med Virol 2006; 78 Suppl 1:S19.
- Lin HH, Gaschen BK, Collie M, et al. Genetic characterization of diverse HIV-1 strains in an immigrant population living in New York City. J Acquir Immune Defic Syndr 2006; 41:399.
- Parekh B, Phillips S, Granade TC, et al. Impact of HIV type 1 subtype variation on viral RNA quantitation. AIDS Res Hum Retroviruses 1999; 15:133.
- Jagodzinski LL, Wiggins DL, McManis JL, et al. Use of calibrated viral load standards for group M subtypes of human immunodeficiency virus type 1 to assess the performance of viral RNA quantitation tests. J Clin Microbiol 2000; 38:1247.
- Swanson P, Huang S, Abravaya K, et al. Evaluation of performance across the dynamic range of the Abbott RealTime HIV-1 assay as compared to VERSANT HIV-1 RNA 3.0 and AMPLICOR HIV-1 MONITOR v1.5 using serial dilutions of 39 group M and O viruses. J Virol Methods 2007; 141:49.
- Rouet F, Chaix ML, Nerrienet E, et al. Impact of HIV-1 genetic diversity on plasma HIV-1 RNA Quantification: usefulness of the Agence Nationale de Recherches sur le SIDA second-generation long terminal repeat-based real-time reverse transcriptase polymerase chain reaction test. J Acquir Immune Defic Syndr 2007; 45:380.
- IMPORTANCE OF HIV VIRAL LOAD MEASUREMENTS
- LABORATORY METHODS FOR QUANTITATION OF HIV-1 RNA
- Specimen collection
- Quantification and linear range
- Clinically significant changes in viral load
- Exogenous factors that may affect HIV RNA levels
- Relationship of HIV-1 RNA to CD4 cell count
- HIV-1 subtypes
- DIAGNOSTIC TESTING
- GUIDELINES FOR THE USE OF HIV-1 RNA LEVELS IN CLINICAL PRACTICE
- AVAILABILITY AND COST OF VIRAL LOAD TESTS
- SUMMARY AND RECOMMENDATIONS