New York at Stony Brook, Stony Brook, New York 11794; Abbott Laboratories, North Chicago, Illinois 600642; and. Division ofHematology Research, Long ...
Vol. 26, No. 9
JOURNAL OF CLINICAL MICROBIOLOGY, Sept. 1988, p. 1890-1892 0095-1137/88/091890-03$02.00/0 Copyright © 1988, American Society for Microbiology
Comparison of Antigen Immunoassay and Reverse Transcriptase Assay for Monitoring Human Immunodeficiency Virus Infection in an Antiviral Trial HAROLD BURGER,1* DEBORAH PAUL,2 FREDERICK P. SIEGAL,3 INGRID WENDEL,' SHERRY NEFF,1 DAVID EILBOTT,' KEVIN GEHAN,3 ROGER GRIMSON,4 AND BARBARA WEISER' Division of Infectious Diseases, Department of Medicine,' and Department of Community Medicine,4 State University of New York at Stony Brook, Stony Brook, New York 11794; Abbott Laboratories, North Chicago, Illinois 600642; and Division of Hematology Research, Long Island Jewish Medical Center, New Hyde Park, New York 110423 Received 25 February 1988/Accepted 7 June 1988
We compared the Abbott enzyme immunoassay for human immunodeficiency virus (HIV) antigen with the of monitoring HIV infection during an antiviral trial. The Abbott than RTA whether or not the patients were antigenemic and HIV in cultures used for monitoring clinical trials.
reverse transcriptase assay (RTA) as a means enzyme immunoassay detected HIV earlier appears to be superior to RTA for detecting
The human immunodeficiency virus (HIV), a human retrovirus, is the etiologic agent of acquired immunodeficiency syndrome. Clinical trials of antiretroviral agents are now in progress to develop effective therapies for acquired immunodeficiency syndrome and HIV infection. An important component in the evaluation of drug therapy for HIV infection is virologic monitoring. HIV cultivation from patient blood, however, is costly in terms of time, effort, and supplies. An improvement in ease and time of detection of the virus is desirable. HIV is most sensitively detected in patients by cocultivating peripheral blood mononuclear cells (PBMCs) from the patient with PBMCs from normal donors. HIV production in culture was first monitored by assaying culture supernatants for reverse transcriptase activity (1, 3, 6). This method, however, is time consuming, uses radioactive isotopes, and requires significant time in culture before the reverse transcriptase activity is detectable. The Abbott enzyme immunoassay (EIA) for HIV antigen detects mainly the viral core protein p24 in culture supernatants as well as in blood. This study aims to determine whether the Abbott EIA is superior to the reverse transcriptase assay (RTA) for detecting HIV in culture from patients in a clinical antiviral trial by addressing the following questions. (i) Does EIA detect all cultures positive by RTA? (ii) Does EIA detect positive cultures significantly before RTA? (iii) Is EIA useful for detecting positive cultures from patients who are not antigenemic as well as those from patients who have detectable HIV antigen in their plasma? HIV was isolated from the blood of patients by cocultivation with normal PBMCs using standard procedures (1, 3, 6) to maximize detection of the virus. HIV was detected by using the RTA (1, 3, 6) and the Abbott EIA (7). The RTA was performed on culture supernatants as follows. One milliliter of coculture, cleared of cells by low-speed centrifugation for 2 min at 2,000 x g, was concentrated by centrifugation in a Tomy high-speed microcentrifuge (15,860 x g) for 40 min at 4°C. The resulting virus pellet was suspended in 50 ,ul of reaction mixture (1, 3, 6), and the reaction was carried out and assayed by standard methods (1, 3, 6). A reverse transcriptase activity of at least six times above background was considered positive. *
HIV CULTURE SUPERNATANT ASSAYS
SAMPLE PLASMA #
p24 Ag ASSAY
25 248B 24A 23B 23A 228 22A 21B 21A 20B 20A 198 19A 18 17B 1
7A
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15B 15A 14B 14A 138 13A 128 12A 118
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DAYS IN CULTURE plasma and culture supernatants. Sym-
FIG. 1. HIV detection in bols: *, positive reverse transcriptase activity; O, negative reverse transcriptase activity; *, positive HIV p24 antigen; El, negative HIV p24 antigen.
Corresponding author. 1890
VOL. 26, 1988
NOTES
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TABLE 1. Comparison of HIV culture supernatant assays in patients seropositive and seronegative for HIV p24 antigen by EIA No. of cultures
Patients
p24 Antigen Seropositive Seronegative Total
No. of patients
8 8 16
Total
RTA positive
23 18 41
23 18 41
Mean no. of days (range) p24 positive bT EIA positive before RTA positive To positive RTA EIA
23 18 41
12.5 (8-17) 15.2 (8-23) 13.7 (8-23)
6.2 (2-12)c 5.2 (3_10)c 5.9 (2-12)d
a The plasma EIA result did not vary on individual patients tested at different times. bThe detection of a positive EIA earlier than the detection of a positive RTA is statistically significant by the sign test. c p = 0.004.
dp=
0.00003.
The Abbott HIV antigen EIA is a sandwich solid-phase EIA. It is most sensitive for detection of HIV p24 core antigen and only faintly detects HIV envelope antigen (7). The assay was performed according to the directions of the manufacturer. The assay was read at A492 in a Quantum Dual Wavelength spectrophotometer (Abbott Laboratories). A sample was considered positive if the optical density was .0.050 plus the mean of three negative human plasma samples or negative culture supernatants. In order to analyze the data statistically, the sign test was used to test for differences between the time to a positive EIA and RTA in HIV cultures. The units of observation were the 16 patients. Those patients cultured on more than one occasion were counted once, using the culture that gave the most conservative result for statistical testing purposes. Nonparametric methods were used because the distribution of days between times of response types was irregular. We compared the Abbott EIA for HIV antigen with the RTA as a means of monitoring HIV cultures of patients during an antiviral trial. Patients with acquired immunodeficiency syndrome-related complex (ARC) who had antibody to HIV and were culture positive for HIV by the RTA were enrolled in a phase I to Il study of rifabutin (Adria Labs) as therapy for HIV infection. We tested 25 specimens of peripheral blood from 16 ARC patients sampled at various times during the rifabutin trial to detect HIV. Of the 25 plasma samples, 13 (52%) were positive for HIV antigen by EIA. Since some of these patients were tested multiple times, a total of 16 patients were tested for antigenemia, 8 of whom (50%) were positive on one or more occasions. The plasma of each patient remained positive or negative by EIA when serial samples were tested. PBMCs obtained at the same time as the plasma were cultured in duplicate to detect HIV. PBMCs from all 16 ARC patients were found to be culture positive by both RTA and EIA. Some patients had their PBMCs cultured on more than one occasion: the PBMCs were cultured four times for 1 patient, three times for another patient, twice for 4 patients, and once for 10 patients. In order to determine whether EIA detected all cultures positive by RTA and whether EIA detected positive cultures significantly earlier, we assayed the same tissue culture supernatants by both methods (Fig. 1). For a culture to be evaluable in this comparison, a negative RTA result was required prior to a positive result. Forty-one cultures from the 25 peripheral blood samples were evaluable. By using RTA performed approximately every 3 days, HIV was detected in all 41 cultures of PBMCs from the 16 ARC patients. Comparison of RTA and EIA on the 41 culture supernatants showed that all cultures that tested positive by RTA were also positive by EIA. Viral antigen, however, was detected 2 to 12 days earlier than reverse transcriptase
activity in the 41 cultures (Table 1), with the difference being statistically significant (P = 0.00003). Twenty-three of the 41 cultures were from antigenemic patients, and 18 were from patients without detectable antigenemia. There was no significant difference in the mean number of days to detection of p24 antigen in supernatants of cultures from patients with or without antigenemia (Table 1). The culture supernatant EIA was positive significantly before the RTA whether or not the patient was antigenemic. Previous studies have shown that EIA is capable of detecting HIV in culture (2, 4, 5). Gupta et al. (4) found that EIA and RTA displayed comparable sensitivities on random samples, with a 1.5% disagreement between the two tests. In addition, these two tests showed nearly identical kinetic patterns of HIV appearance in culture supernatants (4). Studies by Feorino et al. (2) and Paul et al. (7) compared EIA and RTA for sensitivity and specificity of detection of purified HIV. They found that EIA was 100-fold more sensitive than RTA and highly specific for HIV, without cross-reactivity to human T-cell leukemia virus types I and II, several DNA viruses, or uninfected cells (2, 7). In addition, they summarized data from 34 positive cultures which indicated that for most cultures, EIA detected HIV before RTA did (2). The question of whether EIA should replace RTA, the original standard for HIV detection, in monitoring HIV cultures in clinical antiviral trials has not been addressed in a systematic way. In this study of ARC patients enrolled in a phase I to II study of rifabutin as an antiviral agent, EIA was compared with RTA for culture monitoring. EIA detected HIV in every culture that RTA did; EIA detected HIV significantly earlier than RTA whether or not the patients were antigenemic. Because of the earlier detection of HIV in culture as well as the high specificity and increased efficiency for handling large numbers of specimens, the EIA appears to be superior to RTA for monitoring HIV cultures in clinical antiviral trials. This work was supported by a grant from the New York State AIDS Institute and by a contract from Adria Laboratories. We thank David Mack for technical assistance and Barbara Baker for help in preparation of the manuscript. LITERATURE CITED 1. Barre-Sinoussi, F., J. C. Chermann, F. Rey, M. T. Nugeyre, S. Charmaret, J. Gruest, C. Dauguet, C. Axler-Blin, F. BrunVezinet, C. Rouzioux, W. Rozenbaum, and L. Montagnier. 1983. Isolation of a T-lymphotropic retrovirus from a patient at risk for acquired immune deficiency syndrome (AIDS). Science 220:868870. 2. Feorino, P., B. Forrester, C. Schable, D. Warfield, and G. Schochetman. 1987. Comparison of antigen assay and reverse transcriptase assay for detecting human immunodeficiency virus in culture. J. Clin. Microbiol. 25:2344-2346.
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3. Gallo, R. C., S. Z. Salahuddin, M. Popovic, G. M. Shearer, M. Kaplan, B. F. Haynes, T. J. Palker, R. Redfield, J. Oleske, B. Safai, G. White, P. Foster, and P. D. Markham. 1984. Frequent detection and isolation of cytopathic retrovirus (HTLV llI) from patients with AIDS and at risk for AIDS. Science 224:500502. 4. Gupta, P., R. Balachandran, K. Grovit, D. Webster, and C. Rinaldo, Jr. 1987. Detection of human immunodeficiency virus by reverse transcriptase assay, antigen capture assay, and radioimmunoassay. J. Clin. Microbiol. 25:1122-1125. 5. Jackson, B., K. Sannerud, F. Rhame, R. Tsang, and H. H.
J. CLIN. MICROBIOL.
Balfour, Jr. 1987. Comparison of reverse transcriptase assay with the Retro-Tek viral capture assay for detection of human immunodeficiency virus. Diagn. Microbiol. Infect. Dis. 7:185-192. 6. Levy, J., D. Hoffman, S. M. Kramer, J. A. Landis, J. M. Shimabukuro, and L. S. Oshiro. 1984. Isolation of lymphocytopathic retroviruses from San Francisco patients with AIDS. Science 225:840-842. 7. Paul, D. A., L. A. Falk, H. A. Kessier, R. M. Chase, B. Blaauw, D. S. Chudwin, and A. L. Landay. 1987. Correlation of serum HIV antigen and antibody with clinical status in HIV-infected patients. J. Med. Virol. 22:357-363.
