Search tips
Search criteria 


Logo of aacPermissionsJournals.ASM.orgJournalAAC ArticleJournal InfoAuthorsReviewers
Antimicrob Agents Chemother. 1993 May; 37(5): 1095–1101.
PMCID: PMC187907

Standardized peripheral blood mononuclear cell culture assay for determination of drug susceptibilities of clinical human immunodeficiency virus type 1 isolates. The RV-43 Study Group, the AIDS Clinical Trials Group Virology Committee Resistance Working Group.


A standardized antiviral drug susceptibility assay for clinical human immunodeficiency virus type 1 (HIV-1) isolates has been developed for use in clinical trials. The protocol is a two-step procedure that first involves cocultivation of patient infected peripheral blood mononuclear cells (PBMC) with seronegative phytohemagglutinin-stimulated donor PBMC to obtain an HIV-1 stock. The virus stock is titrated for viral infectivity (50% tissue culture infective dose) by use of serial fourfold virus dilutions in donor PBMC. A standardized inoculum of 1,000 50% tissue culture infective doses per 10(6) cells is used in the second step of the procedure to acutely infect seronegative donor PBMC in a 7-day microtiter plate assay with triplicate wells containing zidovudine (ZDV) concentrations ranging from 0 to 5.0 microM. The ZDV 50% inhibitory concentrations (IC50) for reference ZDV-susceptible and ZDV-resistant HIV-1 isolates ranged from 0.002 to 0.113 microM and from 0.15 to > 5.0 microM, respectively. Use of this consensus protocol reduced interlaboratory variability for ZDV IC50 determinations with reference HIV-1 isolates. Among eight laboratories, the coefficient of variation ranged from 0.85 to 1.25 with different PBMC protocols and was reduced to 0.39 to 0.98 with the standardized assay. Among the clinical HIV-1 isolates assayed by the standardized drug susceptibility assay, the median ZDV IC50 increased gradually with more ZDV therapy. This protocol provides an efficient and reproducible means to assess the in vitro susceptibility to antiretroviral agents of virtually all clinical HIV-1 isolates.

Full text

Full text is available as a scanned copy of the original print version. Get a printable copy (PDF file) of the complete article (1.4M), or click on a page image below to browse page by page. Links to PubMed are also available for Selected References.

Selected References

These references are in PubMed. This may not be the complete list of references from this article.
  • Burke DS, Redfield RR, Bjornson DC, Fowler AK, Oster CN. Frequent isolation of HIV-1 from the blood of patients receiving zidovudine (AZT) therapy. N Engl J Med. 1989 Dec 14;321(24):1682–1682. [PubMed]
  • Chou TC, Talalay P. Quantitative analysis of dose-effect relationships: the combined effects of multiple drugs or enzyme inhibitors. Adv Enzyme Regul. 1984;22:27–55. [PubMed]
  • Coombs RW, Collier AC, Allain JP, Nikora B, Leuther M, Gjerset GF, Corey L. Plasma viremia in human immunodeficiency virus infection. N Engl J Med. 1989 Dec 14;321(24):1626–1631. [PubMed]
  • Eron JJ, Gorczyca P, Kaplan JC, D'Aquila RT. Susceptibility testing by polymerase chain reaction DNA quantitation: a method to measure drug resistance of human immunodeficiency virus type 1 isolates. Proc Natl Acad Sci U S A. 1992 Apr 15;89(8):3241–3245. [PubMed]
  • Ho DD, Moudgil T, Alam M. Quantitation of human immunodeficiency virus type 1 in the blood of infected persons. N Engl J Med. 1989 Dec 14;321(24):1621–1625. [PubMed]
  • Hollinger FB, Bremer JW, Myers LE, Gold JW, McQuay L. Standardization of sensitive human immunodeficiency virus coculture procedures and establishment of a multicenter quality assurance program for the AIDS Clinical Trials Group. The NIH/NIAID/DAIDS/ACTG Virology Laboratories. J Clin Microbiol. 1992 Jul;30(7):1787–1794. [PMC free article] [PubMed]
  • Jackson JB, Coombs RW, Sannerud K, Rhame FS, Balfour HH., Jr Rapid and sensitive viral culture method for human immunodeficiency virus type 1. J Clin Microbiol. 1988 Jul;26(7):1416–1418. [PMC free article] [PubMed]
  • Japour AJ, Chatis PA, Eigenrauch HA, Crumpacker CS. Detection of human immunodeficiency virus type 1 clinical isolates with reduced sensitivity to zidovudine and dideoxyinosine by RNA.RNA hybridization. Proc Natl Acad Sci U S A. 1991 Apr 15;88(8):3092–3096. [PubMed]
  • Johnson VA, Merrill DP, Videler JA, Chou TC, Byington RE, Eron JJ, D'Aquila RT, Hirsch MS. Two-drug combinations of zidovudine, didanosine, and recombinant interferon-alpha A inhibit replication of zidovudine-resistant human immunodeficiency virus type 1 synergistically in vitro. J Infect Dis. 1991 Oct;164(4):646–655. [PubMed]
  • Jung M, Agut H, Candotti D, Ingrand D, Katlama C, Huraux JM. Susceptibility of HIV-1 isolates to zidovudine: correlation between widely applicable culture test and PCR analysis. J Acquir Immune Defic Syndr. 1992;5(4):359–364. [PubMed]
  • Kellam P, Boucher CA, Larder BA. Fifth mutation in human immunodeficiency virus type 1 reverse transcriptase contributes to the development of high-level resistance to zidovudine. Proc Natl Acad Sci U S A. 1992 Mar 1;89(5):1934–1938. [PubMed]
  • Land S, Terloar G, McPhee D, Birch C, Doherty R, Cooper D, Gust I. Decreased in vitro susceptibility to zidovudine of HIV isolates obtained from patients with AIDS. J Infect Dis. 1990 Feb;161(2):326–329. [PubMed]
  • Larder BA, Chesebro B, Richman DD. Susceptibilities of zidovudine-susceptible and -resistant human immunodeficiency virus isolates to antiviral agents determined by using a quantitative plaque reduction assay. Antimicrob Agents Chemother. 1990 Mar;34(3):436–441. [PMC free article] [PubMed]
  • Larder BA, Darby G, Richman DD. HIV with reduced sensitivity to zidovudine (AZT) isolated during prolonged therapy. Science. 1989 Mar 31;243(4899):1731–1734. [PubMed]
  • Larder BA, Kemp SD. Multiple mutations in HIV-1 reverse transcriptase confer high-level resistance to zidovudine (AZT). Science. 1989 Dec 1;246(4934):1155–1158. [PubMed]
  • Mayers DL, McCutchan FE, Sanders-Buell EE, Merritt LI, Dilworth S, Fowler AK, Marks CA, Ruiz NM, Richman DD, Roberts CR, et al. Characterization of HIV isolates arising after prolonged zidovudine therapy. J Acquir Immune Defic Syndr. 1992;5(8):749–759. [PubMed]
  • Nunberg JH, Schleif WA, Boots EJ, O'Brien JA, Quintero JC, Hoffman JM, Emini EA, Goldman ME. Viral resistance to human immunodeficiency virus type 1-specific pyridinone reverse transcriptase inhibitors. J Virol. 1991 Sep;65(9):4887–4892. [PMC free article] [PubMed]
  • Richman D, Shih CK, Lowy I, Rose J, Prodanovich P, Goff S, Griffin J. Human immunodeficiency virus type 1 mutants resistant to nonnucleoside inhibitors of reverse transcriptase arise in tissue culture. Proc Natl Acad Sci U S A. 1991 Dec 15;88(24):11241–11245. [PubMed]
  • Richman DD. HIV drug resistance. AIDS Res Hum Retroviruses. 1992 Jun;8(6):1065–1071. [PubMed]
  • Richman DD, Grimes JM, Lagakos SW. Effect of stage of disease and drug dose on zidovudine susceptibilities of isolates of human immunodeficiency virus. J Acquir Immune Defic Syndr. 1990;3(8):743–746. [PubMed]
  • Rooke R, Tremblay M, Soudeyns H, DeStephano L, Yao XJ, Fanning M, Montaner JS, O'Shaughnessy M, Gelmon K, Tsoukas C, et al. Isolation of drug-resistant variants of HIV-1 from patients on long-term zidovudine therapy. Canadian Zidovudine Multi-Centre Study Group. AIDS. 1989 Jul;3(7):411–415. [PubMed]
  • St Clair MH, Martin JL, Tudor-Williams G, Bach MC, Vavro CL, King DM, Kellam P, Kemp SD, Larder BA. Resistance to ddI and sensitivity to AZT induced by a mutation in HIV-1 reverse transcriptase. Science. 1991 Sep 27;253(5027):1557–1559. [PubMed]
  • Tudor-Williams G, St Clair MH, McKinney RE, Maha M, Walter E, Santacroce S, Mintz M, O'Donnell K, Rudoll T, Vavro CL, et al. HIV-1 sensitivity to zidovudine and clinical outcome in children. Lancet. 1992 Jan 4;339(8784):15–19. [PubMed]
  • Wahlberg J, Albert J, Lundeberg J, Cox S, Wahren B, Uhlén M. Dynamic changes in HIV-1 quasispecies from azidothymidine (AZT)-treated patients. FASEB J. 1992 Jul;6(10):2843–2847. [PubMed]

Articles from Antimicrobial Agents and Chemotherapy are provided here courtesy of American Society for Microbiology (ASM)