Molecular Diagnosis of HIV-1 by PCR

  • Susan A. Fiscus
Part of the Methods in Molecular Medicine™ book series (MIMM, volume 20)


Most individuals can be diagnosed as being infected with HIV-1 with an enzyme immunoassay (EIA), which detects antibodies to the virus. Repeatedly positive results must be confirmed, usually by Western blot. These assays have been improved since their introduction in 1985. When used together, the EIA and Western blot are now extremely reliable, with sensitivities of 99.5% and specificities of 99.8% for the diagnosis of HIV-1 (1). These tests are fairly rapid, reproducible across laboratories, well quality-controlled, relatively inexpensive, and form the basis of the protection of the blood supply.


Roche Molecular System NASBA Assay Infected Peripheral Blood Mononuclear Cell Copy Number Input Hybridization Protection Assay 
These keywords were added by machine and not by the authors. This process is experimental and the keywords may be updated as the learning algorithm improves.


  1. 1.
    Davey, R. and Lane, C. (1990) Laboratory methods in the diagnosis and prognostic staging of infection with human immunodeficiency virus type 1. Rev. Infect. Dis. 12, 912–930.PubMedGoogle Scholar
  2. 2.
    Niu, M. T., Stein, D. S., and Schnittman, S. M. (1993) Primary human immunodeficiency virus type 1 infection: review of pathogenesis and early treatment intervention in humans and animal retrovirus infections. J. Infect. Dis. 168, 1490–1501.PubMedGoogle Scholar
  3. 3.
    Schacker T., Collier, A. C., Hughes, J., Shea, T., and Corey, L. (1996) Clinical and epidemiologic features of primary HIV infection. Ann. Intern. Med. 125, 257–264.PubMedGoogle Scholar
  4. 4.
    Farzedegan H., Vlahov, D., Solomon, L., Munoz, A., Astemborski, J., Taylor, E., et al. (1993) Detection of human immunodeficiency virus type 1 infection by polymerase chain reaction in a cohort of seronegative intravenous drug users. J. Infect. Dis. 168, 327–331.Google Scholar
  5. 5.
    Bruisten, S., Koppleman, M., Dekker, J., Bakker, M., de Goede, R. E., Roos, M. T., et al. (1992) Concordance of human immunodeficiency virus detection by polymerase chain reaction and by serologic assays in a Dutch cohort of seronegative homosexual men. J. Infect. Dis. 166, 620–622.PubMedGoogle Scholar
  6. 6.
    Centers for Disease Control and Protection (1996) US Public Health Service guidelines for testing and counseling blood and plasma donors for HIV type 1 antigen. M. M. W. R. 45 (no. RR-2).Google Scholar
  7. 7.
    Nishanian, P., Huskins, K. R., Stehn, S., Detels, R., and Fahey, J. L. (1990) A simple method for improved assay demonstrates that HIV p24 antigen is present as immune complexes in most sera from HIV infected individuals. J. Infect. Dis. 162, 21–28.PubMedGoogle Scholar
  8. 8.
    Clark S. J., Saag, M. S., Decker, W. D., Campbell-Hill, S., Roberson, J. L., Veldkamp, P. J., et al. (1991) High titers of cytopathic virus in plasma of patients with symptomatic primary HIV-1 infection. N. Engl. J. Med. 324, 954–960.PubMedCrossRefGoogle Scholar
  9. 9.
    Daar, E. S., Mougdil, T.; Meyer, R. D., and Ho, D. D. (1991) Transient high levels of viremia in patients with primary human immunodeficiency virus type 1 infection. N. Engl. J. Med. 324, 961–964.PubMedCrossRefGoogle Scholar
  10. 10.
    Busch, M. P., Lee, L. L. L., Satten, G. A., Henrard, D. R., Farzadegar, H., Nelson, K. E., et al. (1995) Time course of detection of viral and serologic markers preceding human immunodeficiency virus type 1 seroconversion: implications for screening of blood and tissue donors. Transfusion 35, 91–97.PubMedCrossRefGoogle Scholar
  11. 11.
    Schrieber, G. B., Busch, M. P., Kleinman, S. H., Korelitz, J. J., and the Retrovirus Epidemiology Donor Study (1996) The risk of transfusion-transmitted viral infections. N. Engl. J. Med. 334, 1685–1690.CrossRefGoogle Scholar
  12. 12.
    Ho, D. D. (1995) Time to hit HIV, early and hard. N. Engl. J. Med. 333 450,451.PubMedCrossRefGoogle Scholar
  13. 13.
    Henrard, D., Fauvel, M., Samson J., Delage, G., Boucher, M., Hankins, C, et al. (1993) Ontogeny of the humoral immune response to human immunodeficiency virus type 1 in infants. J. Infect. Dis. 168, 288–291.PubMedGoogle Scholar
  14. 14.
    Consensus Workshop (1992) Early diagnosis of HIV infection in infants. J. AIDS 5, 1169–1178.Google Scholar
  15. 15.
    Fiscus, S. A., Welles, S. L., Spector, S. A., and Lathey, J. L. (1995) Length of incubation time for human immunodeficiency virus culture. J. Clin. Microbiol. 33, 246,247.PubMedGoogle Scholar
  16. 16.
    Paul, M. O., Tetali, S., Lesser, M. L., Abrams, E. J., Wang, X. P., Kowalski, R., et al. (1996) Laboratory diagnosis of infection status in infants perinatally exposed to human immunodeficiency virus type 1. J. Infect. Dis. 173, 68–76.PubMedGoogle Scholar
  17. 17.
    Cassol, S, Lapointe N., Salas, T., et al. (1992) Diagnosis of vertical HIV-1 transmission using the polymerase chain reaction and dried blood spot specimens. J. AIDS 5, 3–119.Google Scholar
  18. 18.
    Palumbo P. E., Kwok, S., Waters, S., et al. (1995) Viral measurement by polymerase chain reaction-based assays in human immunodeficiency virus-infected infants. J. Pediatr. 126, 592–595.PubMedCrossRefGoogle Scholar
  19. 19.
    Holodniy, M., Katzenstein, D. A., Sengupta, S., et al. (1991) Detection and quantification of human immunodeficiency virus RNA in patient serum by use of the polymerase chain reaction. J. Infect. Dis. 163, 862–866.PubMedGoogle Scholar
  20. 20.
    Semple, M., Loveday, C, Weller, I., and Tedder, R. (1991) Direct measurement of viremia in patients infected with HIV-1 and its relationship to disease progression and zidovudine therapy. J. Med. Virol. 35, 38–45.PubMedCrossRefGoogle Scholar
  21. 21.
    Bruisten, S. M., Koppelman, M. H., Roos, M. T., Loeliger, A. E., Reiss, P., Boucher, C. A., et al. (1993) Use of a competitive polymerase chain reaction to determine HIV-1 levels in response to antiviral treatments. AIDS 7(Suppl. 2), S15–S20.PubMedCrossRefGoogle Scholar
  22. 22.
    Vandamme, A.-M., Schmit, J.-C, Van Dooren, S., Van Laethem, K., Gobbers, E., Kok, W., et al. (1996) Quantification of HIV-1 RNA in plasma: comparable results with the NASBA HIV-1 RNA QT and the Amplicor HIV monitor test. J. AIDS 13, 127–139.Google Scholar
  23. 23.
    Revets H., Marissens, D., DeWit, S., Lacor, P., Clumeck, N., Lauwers, S., et al. (1996) Comparative evaluation of NASBA HIV-1 RNA QT, Amplicor HIV Monitor, and Quantiplex HIV RNA assay, three methods for quantification of human immunodeficiency virus type 1 RNA in plasma. J. Clin. Microbiol. 34, 1058–1064.PubMedGoogle Scholar
  24. 24.
    Lin, H. J., Myers L. E., Yen-Lieberman, B., Hollinger, F. B., Henrard, D., Hooper, C. J., et al. (1994) Multicenter evaluation of quantification methods for plasma human immunodeficiency virus type 1 RNA. J. Infect. Dis. 170, 353–362.Google Scholar
  25. 25.
    Schuurman R., Deschamps, D., Weverling, G. J., et al. (1996) Multicenter comparison of three commercial methods for quantification of human immunodeficiency virus type 1 RNA in plasma. J. Clin. Microbiol. 34, 3016–3022.PubMedGoogle Scholar
  26. 26.
    Chernoff, D. (1996). Reported at the Technology Utilization for HIV Blood Evaluation and Standardization in Pediatrics: A Special Emphasis on Plasma RNA Assays Meeting, held in Solomon’s Island, MD, June 1996.Google Scholar
  27. 27.
    Gobbers, E., Hilgers, P., Fransen, K., van de Wiel, P., and van der Groen, G. (1996) Sensitivity and amplification efficiency of the NASBA HIV-1 RNA amplification system with regard to different HIV-1 genotypes. Abstract We. B. 3168. 11th International Conference on AIDS, Vancouver, BC, Canada, July 1996.Google Scholar
  28. 28.
    Gleeson, T. and Montpetit, M. (1996) Limited detection of HIV-1 subtypes by the Roche Amplicor diagnostic polymerase chain reaction assay. Abstract Mo. A. 160. 11th International Conference on AIDS, Vancouver, BC, Canada, July 1996.Google Scholar
  29. 29.
    Christopherson, C. D. and Kwok, S. (1996) The effects of mutations on HIV-1 quantification by RT/PCR. Abstract Mo. A. 161. 11th International Conference on AIDS, Vancouver, BC, Canada, July 1996.Google Scholar
  30. 30.
    Katzenstein, D. A., Holodniy, M, Israelski, D. M., Sengupta, S., Mole, L. A., Bubp, J. L., and Merigan, T. C, et al. (1992) Plasma viremia in human immunodeficiency virus infection: relationship to stage of disease and antiviral treatment. J. AIDS 5, 7–112.Google Scholar
  31. 31.
    Holodniy, M., Kim, S., Katzenstein, D., Konrad, M., Groves, E., and Merigan, T. (1991) Inhibition of human immunodeficiency virus gene amplification by heparin. J. Clin. Microbiol. 29, 676–679.PubMedGoogle Scholar
  32. 32.
    Yen-Lieberman, B., Brambilla, D., Jackson, J. B., Bremer, J., Coombs, R., Cronin, M., et al. (1996) Evaluation of a quality assurance program for quantitation of human immunodeficiency virus type 1 RNA in plasma by the AIDS Clinical Trials Group Laboratories. J. Clin. Microbiol. 34, 2695–2701.PubMedGoogle Scholar
  33. 33.
    Reichelderfer, P. S. and Jackson, J. B. (1994) Quality assurance and use of PCR in clinical trials. PCR Methods Appl. 4, S141–149.PubMedGoogle Scholar
  34. 34.
    National Institutes of Health (1996) ACTG Virology Manual, 3rd ed. National Institutes of Health, Bethesda, MD.Google Scholar
  35. 35.
    Whetsell A., Drew, J., Milman, G., Hoff, R., Dragon, E. A., Adler, K., et al. (1992) Comparison of three non-isotopic polymerase chain reaction-based methods for detection of human immunodeficiency virus type 1. J. Clin. Microbiol. 30, 845–853.PubMedGoogle Scholar
  36. 36.
    Jackson, J. B., Drew, J., Lin, H. J., et al. (1993) Establishment of a quality assurance program for human immunodeficiency virus type 1 DNA polymerase chain reaction assays by the AIDS Clinical Trials Group. J. Clin. Microbiol. 31, 3123–3128.PubMedGoogle Scholar
  37. 37.
    Kwok, S. and Sninsky, J. J. (1993) PCR detection of human immunodeficiency virus type 1 proviral DNA sequences, in Diagnostic Molecular Microbiology, ASM, Washington, DC, pp. 309–315.Google Scholar
  38. 38.
    Coutlee, F., Viscidi, R., Sainte-Antoine, P., Kessols, A., and Yolken, R. (1991) The polymerase chain reaction: a new tool for the understanding and diagnosis of HIV-1 infection at the molecular level. Mol. Cell Probes 5, 241–259.PubMedCrossRefGoogle Scholar
  39. 39.
    Candotti, D., Jung, M., Keroudan, D., Rosenheim, M., Gentilini, M., M’Pele, P., et al. (1991) Genetic variability affects the detection of HIV by polymerase chain reaction. AIDS 5, 1003–1007.PubMedCrossRefGoogle Scholar
  40. 40.
    Cassol, S., Salas, T., Lapointe, N., Arella, M., Rudnik, J., and O’Shaughnessy, M. (1991) Improved detection of HIV-1 envelope sequences using optimized PCR and inosine-substituted primers. Mol. Cell Probes 5, 157–160.PubMedCrossRefGoogle Scholar
  41. 41.
    Kwok, S., Kellogg, D. E., McKinney, N., Spasik, D., Goda, L., Levenson, C, et al. (1990) Effects of primer-template mismatches on the polymerase chain reaction: human immunodeficiency virus type 1 model studies. Nucleic Acids Res. 18, 999–1005.PubMedCrossRefGoogle Scholar
  42. 42.
    Sambrook, J., Frisch, E. F., and Maniatis, T. (1989) Molecular Cloning: A Laboratory Manual, 2nd ed. Cold Spring Harbor Laboratory, Cold Spring Harbor, NY.Google Scholar
  43. 43.
    Fletcher, M. A., Klimas, N., Morgan, R., and Gjerset, G. (1992) Manual of Clinical Laboratory Immunology, 4th ed. ASM, Washington, DC, pp. 213–219.Google Scholar
  44. 44.
    Farzedegan, H., Polis, M. A., Wolinsky, S. M., Rinaldo, C. R., Srinsky, J. J., Kwok, S., et al. (1988) Loss of human immunodeficiency virus type 1 (HIV-1) antibodies with evidence of viral infection in asymptomatic homosexual men. Ann. Intern. Med. 108, 785–790.Google Scholar
  45. 45.
    Clewly, J. (1989) The polymerase chain reaction, a review of the practical limitations for human immunodeficiency virus diagnosis. J. Virol. Methods 25, 179–188.CrossRefGoogle Scholar
  46. 46.
    Kwok, S. and Higuchi, R. (1989) Avoiding false positives with PCR. Nature 339, 237,238.PubMedCrossRefGoogle Scholar
  47. 47.
    McCreedy, B. J. and Callaway, T. H. (1993) Laboratory design and work flow, in Diagnostic Molecular Microbiology, ASM, Washington, DC, pp. 149–159.Google Scholar
  48. 48.
    Dragon, E. A., Spandoro, J. P., and Madej, R. (1993) Quality control of polymerase chain reaction, in Diagnostic Molecular Microbiology, ASM, Washington, DC, pp. 160–168.Google Scholar

Copyright information

© Humana Press Inc., Totowa, NJ 1999

Authors and Affiliations

  • Susan A. Fiscus
    • 1
  1. 1.Department of MedicineUniversity of North Carolina at Chapel HillChapel Hill

Personalised recommendations