A multiplex real-time PCR for quantification of HIV-1 DNA and the human albumin gene in CD4+ cells
LARS E. ERIKSSON
Department of Virology, Swedish Institute for Infectious Disease Control, Solna,
Microbiology and Tumor Biology Center, Karolinska Institutet, Stockholm, and
Department of Nursing, Karolinska Institutet, Huddinge, Sweden
Search for more papers by this authorTHOMAS LEITNER
Department of Virology, Swedish Institute for Infectious Disease Control, Solna,
Search for more papers by this authorBRITTA WAHREN
Department of Virology, Swedish Institute for Infectious Disease Control, Solna,
Microbiology and Tumor Biology Center, Karolinska Institutet, Stockholm, and
Search for more papers by this authorANN-CHARLOTTE BOSTRÖM
Department of Virology, Swedish Institute for Infectious Disease Control, Solna,
Search for more papers by this authorKERSTIN I. FALK
Department of Virology, Swedish Institute for Infectious Disease Control, Solna,
Microbiology and Tumor Biology Center, Karolinska Institutet, Stockholm, and
Search for more papers by this authorLARS E. ERIKSSON
Department of Virology, Swedish Institute for Infectious Disease Control, Solna,
Microbiology and Tumor Biology Center, Karolinska Institutet, Stockholm, and
Department of Nursing, Karolinska Institutet, Huddinge, Sweden
Search for more papers by this authorTHOMAS LEITNER
Department of Virology, Swedish Institute for Infectious Disease Control, Solna,
Search for more papers by this authorBRITTA WAHREN
Department of Virology, Swedish Institute for Infectious Disease Control, Solna,
Microbiology and Tumor Biology Center, Karolinska Institutet, Stockholm, and
Search for more papers by this authorANN-CHARLOTTE BOSTRÖM
Department of Virology, Swedish Institute for Infectious Disease Control, Solna,
Search for more papers by this authorKERSTIN I. FALK
Department of Virology, Swedish Institute for Infectious Disease Control, Solna,
Microbiology and Tumor Biology Center, Karolinska Institutet, Stockholm, and
Search for more papers by this authorAbstract
We have established a simple system for measuring HIV-1 DNA load in CD4+ cells. In a multiplex configuration, a conserved region in the HIV-1 pol gene and a section of the human albumin gene were simultaneously amplified to estimate the number of HIV-1 DNA copies per cellular genome. An established Epstein-Barr virus (EBV) standard system was used to calibrate the HIV-1 quantification. Our multiplex PCR system was tested on different in vitro developed HIV-1 strains and on longitudinal samples from eight patients. The system was able to amplify both in vitro and in vivo samples of various genetic compositions. In all eight patients, HIV-1 DNA was detected and ranged between 0.17 and 51×10−3 copies per CD4+ cell and could be monitored longitudinally, including long-term PI-ART and STI. The measured HIV-1 DNA load may be used to select the best time for the institution or re-institution of therapy.
REFERENCES
- 1 Hirsch MS, Curran J. Human immunodeficiency viruses. In: BN Fields, DM Knipe, PM Howley, editors. Fields Virology. Philadelphia, PA: Lippincott-Raven Publishers, 1996: 1953–75.
- 2 Mansky LM, Temin HM. Lower in vivo mutation rate of human immunodeficiency virus type 1 than that predicted from the fidelity of purified reverse transcriptase. J Virol 1995; 69: 5087–94.
- 3 Geijtenbeek TBH, Kwon DS, Torensma R, Van Vliet SJ, Van Duijnhoven GCF, Middel J, et al. DC-SIGN, a dendritic cell-specific HIV-1-binding protein that enhances trans-infection of T cells. Cell 2000; 100: 587–97.
- 4 Simmons G, Reeves JD, Hibbitts S, Stine JT, Gray PW, Proudfoot AEI, et al. Co-receptor use by HIV and inhibition of HIV infection by chemokine receptor ligands. Immunol Rev 2000; 177: 112–26.
- 5 Saha K, Zhang J, Gupta A, Dave R, Yimen M, Zerhouni B. Isolation of primary HIV-1 that target CD8+ T lymphocytes using CD8 as a receptor. Nat Med 2001; 7: 65–72.
- 6 Catalan J. Psychiatric manifestations of HIV disease. Ballieres Clin Gastroenterol 1990; 4: 547–62.
- 7 Saag MS. Natural history of HIV-1 disease. In: S Broder, C Thomas, J Merigan, D Bolognesi, editors. Textbook of AIDS Medicine. Baltimore: Williams & Wilkins, 1994: 45–53.
- 8 Eriksson LE, Nordström G, Berglund T, Sandström E. The health-related quality of life in a Swedish sample of HIV-infected persons. J Adv Nurs 2000; 32: 1213–23.
- 9 British HIV Association. British HIV Association (BHIVA) guidelines for the treatment of HIV-infected adults with antiretroviral therapy. HIV Med 2000; 1: 76–101.
- 10 Carpenter CC, Cooper DA, Fischl MA, Gatell JM, Gazzard BG, Hammer SM, et al. Antiretroviral therapy in adults: updated recommendations of the International AIDS Society-USA Panel. JAMA 2000; 283: 381–90.
- 11 Bratt G, Karlsson A, Leandersson A-C, Albert J, Wahren B, Sandström E. Treatment history and baseline viral load, but not viral tropism or CCR-5 genotype, influence prolonged antiviral efficacy of highly active antiretroviral treatment.. AIDS 1998; 12: 2193–202.
- 12 Palella FJJ, Delaney KM, Moorman AC, Loveless MO, Fuhrer J, Satten GA, et al. Declining morbidity and mortality among patients with advanced human immunodeficiency virus infection. HIV Outpatient Study Investigators. N Engl J Med 1998; 338: 853–60.
- 13 Mocroft A, Katlama C, Johnson AM, Pradier C, Antunes F, Mulcahy F, et al. AIDS across Europe, 1994–98: the EuroSIDA study. Lancet 2000; 356: 291–6.
- 14 Hejdeman B, Lenkei R, Leandersson AC, Hultström AL, Wahren B, Sandström E, et al. Clinical and immunological benefits from highly active antiretroviral therapy in spite of limited viral load reduction in HIV type 1 infection. AIDS Res Hum Retroviruses 2001; 17: 227–86.
- 15 Safrin S, Grunfeld C. Fat distribution and metabolic changes in patients with HIV infection.. AIDS 1999; 13: 2493–505.
- 16 Koppel K, Bratt G, Eriksson M, Sandström E. Serum lipid levels associated with increased risk for cardiovascular disease is associated with highly active antiretroviral therapy (HAART) in HIV-1 infection. Int J STD AIDS 2000; 11: 451–5.
- 17 Garcia F, Plana M, Vidal C, Cruceta A, O'Brien WA, Pantaleo G, et al. Dynamics of viral load rebound and immunological changes after stopping effective antiretroviral therapy.. AIDS 1999; 13: F79–86.
- 18 Ruiz L, Martinez-Picado J, Romeu J, Paredes R, Zayat MK, Marfil S, et al. Structured treatment interruption in chronically HIV-1 infected patients after long-term viral suppression. AIDS 2000; 14: 397–403.
- 19 Harrigan PR, Whaley M, Montaner JSG. Rate of HIV-1 RNA rebound upon stopping antiretroviral therapy.. AIDS 1999; 13: F59–62.
- 20 Neumann AU, Tubiana R, Calvez V, Robert C, Li TS, Agut H, et al. HIV-1 rebound during interruption of highly active antiretroviral therapy has no deleterious effect on reinitiated treatment. AIDS 1999; 13: 677–83.
- 21 Phillips AN, McLean AR, Loveday C, Tyrer M, Bofill M, Devereux H, et al. In vivo HIV-1 replicative capacity in early and advanced infection. AIDS 1999; 13: 67–73.
- 22 Hockett RD, Saag MS, Kilby JM, Sfakianos G, Wakefield TB, Bucy RP. Stability in the HIV vDNA pool in peripheral CD4+ T cells of untreated patients by single tube quantitative PCR. J Virol Methods 2000; 87: 1–12.
- 23 Kostrikis LG, Touloumi G, Karanicolas R, Pantazis N, Anastassopoulou C, Karafoulidou A, et al. Quantitation of human immunodeficiency virus type 1 DNA forms with the second template switch in peripheral blood cells predicts disease progression independently of plasma RNA load. J Virol 2002; 76: 10099–108.
- 24 Aleman S, Visco-Comandini U, Loré K, Sönnerborg A. Long-term effects of antiretroviral combination therapy on HIV type 1 DNA levels. AIDS Res Hum Retroviruses 1999; 15: 1249–54.
- 25 Désiré N, Dehée A, Schneider V, Jacomet C, Goujon C, Girard PM, et al. Quantification of human immunodeficiency virus type 1 proviral load by a TaqMan real-time PCR assay. J Clin Microbiol 2001; 39: 1303–10.
- 26 Riva E, Pistello M, Narciso P, D'Offizi G, Isola P, Galati V, et al. Decay of HIV type 1 DNA and development of drug-resistant mutants in patients with primary HIV type 1 infection receiving highly active antiretroviral therapy. AIDS Res Hum Retroviruses 2001; 17: 1599–604.
- 27 Douek DC, Brenchley JM, Betts MR, Ambrozak DR, Hill BJ, Okamoto Y, et al. HIV preferentially infects HIV-specific CD4+ T cells. Nature 2002; 417: 95–8.
- 28
Bièche I,
Olivi M,
Champème MH,
Vidaud D,
Lidereau R,
Vidaud M.
Novel approach to quantitative polymerase chain reaction using real-time detection: application to the detection of gene amplification in breast cancer.
Int J Cancer
1998; 78: 661–6.
10.1002/(SICI)1097-0215(19981123)78:5<661::AID-IJC22>3.0.CO;2-I CAS PubMed Web of Science® Google Scholar
- 29 Folks TM, Powell D, Lightfoote M, Koening S, Faucl AS, Benn S, et al. Biological and biochemical characterization of a cloned Leu-3-cell surviving infection with the acquired immune deficiency syndrome retrovirus. J Exp Med 1986; 164: 280–90.
- 30 Popovic M, Sarngadharan MG, Read E, Gallo RC. Detection, isolation, and continuous production of cytopathic retroviruses (HTLV-III) from patients with AIDS and pre-AIDS. Science 1984; 224: 497–500.
- 31 Cargano S, Caporossi D, Gualandi G, Calef E. Different localization of Epstein-Barr virus genome in two subclones of the Burkitt lymphoma cell line Namalwa. Genes Chromosomes Cancer 1992; 4: 205–10.
- 32 Lenoir GM, Preud'homme JL, Bernheim A, Berger R. Correlation between immunoglobulin light chain expression and variant translocation in Burkitt's lymphoma. Nature 1982; 298: 474–6.
- 33 Heid CA, Stevens J, Livak KJ, Williams PM. Real time quantitative PCR. Genome Res 1996; 6: 986–94.
- 34 Gibson UE, Heid CA, Williams PM. A novel method for real time quantitative RT-PCR. Genome Res 1996; 6: 995–1001.
- 35 C Kuiken, B Foley, B Hahn, P Marx, F McCutchan, J. Mellors, et al., editors. HIV Sequence Compendium 2001. Los Alamos, New Mexico: Theoretical Biology and Biophysics Group, Los Alamos National Laboratory, 2001.
- 36 Enbom M, Strand A, Falk KI, Linde A. Detection of Epstein-Barr virus, but not human herpesvirus 8, DNA in cervical secretions from Swedish women by real-time polymerase chain reaction. Sex Transm Dis 2001; 28: 300–6.
- 37 Blom N. Nucleotide data, www.cbs.dtu.dk/kikob/sciencenuc_table.htm. The Technical University of Denmark 1996.
- 38 Lewin B. Genes VII. Seventh ed. New York: Oxford University Press, 2000.
- 39 Cone RW, Gowland P, Opravil M, Grob P, Ledergerber B. Swiss HIV Cohort Study. Levels of HIV-infected peripheral blood cells remain stable throughout the natural history of HIV-1 infection.. AIDS 1998; 12: 2253–60.
- 40 Ibáñez A, Puig T, Elias J, Clotet B, Ruiz L, Martınez MA. Quantification of integrated and total HIV-1 DNA after long-term highly active antiretroviral therapy in HIV-1–infected patients.. AIDS 1999; 13: 1045–9.
- 41 Russell RR, Bowmer MI, Nguyen C, Grant MD. HIV-1 DNA burden in peripheral blood CD4+ cells influences disease progression, antiretroviral efficacy, and CD4+ T-cell restoration. Viral Immunol 2001; 14: 379–89.
- 42 Chun TW, Stuyver L, Mizell SB, Ehler LA, Mican JAM, Baseler M, et al. Presence of an inducible HIV-1 latent reservoir during highly active antiretroviral therapy. Proc Natl Acad Sci USA 1997; 94: 13193–7.
- 43 Altfeld M, Rosenberg ES, Shankarappa R, Mukherjee JS, Hecht F, Eldridge RL, et al. Immune control of HIV infection. Abstract no. 004. In: Keystone Symposia, HIV Pathogenesis. Keystone, Colorado: Keystone Symposia, 2001: 236.
