Concise Communications
1146
References
10.
II.
12.
13.
14.
15.
inhibition of replication of human immunodeficiency virus type I, including that of a zidovudine-resistant isolate, by zidovudine and 2'.3'-dideoxycytidine in vitro. Antimicrob Agents Chemother 1992;36: 1559-62. Johnson VA, Byington RE. Modified HIV-I p24 antigen ELISA. Quantitative assays for virus detection. In: Aldovini A, Walker BD, eds. Techniques in HIV research. New York: Stockton Press, 1990:95-7. Chou J, Chou TC. Dose-effect analysis with microcomputers: quantitation of ED so. LD so • synergism. antagonism, low-dose risk, receptorligand binding and enzyme kinetics: a computer software for IBM-PC and manual. Cambridge, UK: Biosoft, 1989. 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. Chou TC. The median-effect principle and the combination index for quantitation of synergism and antagonism. In: Chou TC, Rideout DC, eds. Synergism and antagonism in chemotherapy. New York: Academic Press. 1991:61-102. Johnson VA, Barlow MA. Merrill DP, Chou TC, Hirsch MS. Threedrug synergistic inhibition of HIV -I replication in vitro by zidovudine. recombinant soluble CD4, and recombinant interferon-alpha A. J Infect Dis 1990;161:1059-67. Smith MS, Brian EL, Pagano JS. Resumption of virus production after human immunodeficiency virus infection of T lymphocytes in the presence of azidothymidine. J ViroI1987;61:3769-73.
Diagnosis of Vertical Human Immunodeficiency Virus Type 1 Infection by Whole Blood Culture Ariane Alimenti, Mary O'Neill, John L. Sullivan, and Katherine Luzuriaga
Department of Pediatrics and Program in Molecular Medicine, University ofMassachusetts Medical Center, Worcester
The qualitative and quantitative performance of a human immunodeficiency virus type 1 (HIV-l) whole blood culture method was assessed for use in the diagnosis of vertical HIV-I infection. This method requires
::t
MEAN
P1;P2A,C
P2B,D,F
179
2450
Figure 1. HIV-I titers in whole blood samples from 21 infected patients (untreated or on zidovudine therapy). When cultures were repeated for a single patient at different dates, the HIV-I titer from the earliest blood sample was chosen.
This method eliminates the need for lymphocyte isolation and can be done on very small volumes of blood (< I mL). Forty-three children born to HIV-seropositive mothers were studied; age at time of study ranged from 2 months to 8 years. Twenty-seven children were previously HIV-l isolation-positive more than once. At the time of first testing, 2 (7%) of these children were 18 months. Eight HIV-I-infected children were receiving zidovudine therapy at the time of testing. Sixteen children were > 15 months old, HIV-I antibody negative, and lacked clinical or laboratory evidence of HIV-1 infection. . Heparinized whole blood was cultured in duplicate by end-point dilution in 24-well plates (250,50, 10,2,0.4,0.08 JLL/well) with 106 PBMC from HIV-l-seronegative donors. Donor PBMC were stimulated with phytohemagglutinin (PHA) for 48-72 h prior to use. RPMI 1640 with 10% fetal calf serum (FCS; GIBCO Laboratories, Grand Island, NY), 5% interleukin-2 (IL-2; Pharmacia Diagnostics, Columbia, MD), and gentamicin (50 JLg/mL, GIBCO) was used to bring the total volume to 1.5 mL/well. Twice a week, 0.75 mL of supernatant was removed from each well and replaced with 0.75 mL of media (RPMI 1640 with 10% FCS, 5% IL-2, and gentamicin). The HIV-l p24 antigen content of the superna-
tant was determined once weekly by commercial antigen immunoassay (Du Pont, Wilmington, DE, or Coulter, Hialeah, FL). Cultures were maintained for 4 weeks; fresh donor cells were not added. A culture well was considered positive when the supernatant p24 antigen value was> 100 pg/mL. The lowest dilution to give a positive result was considered the end point, and HIV-1 titers were expressed as 4, 20, 100, 500, 2500, and 12,500 TCID/mL ofwhole blood. When results in duplicate wells were discordant, the last dilution to give positive results in both wells was taken as the end point. HIV-l was isolated from 36 of 38 cultures of whole blood from 27 children >2 months old. The two specimens from HIV-I-infected children that yielded negative whole blood culture results were negative by concurrent PBMC microculture (AIDS Clinical Trials Group consensus protocol) as well; other cultures of whole blood from these 2 children were positive. None of the whole blood specimens from 16 uninfected children were culture positive. Most (90%) of the positive cultures were positive by day 15; 100%were positive by day 23. HIV-I titers of21 infected patients ranged from 0 to 12,500 TCID/mL of whole blood (mean, 1044). The mean HIV-I titer for 13 asymptomatic or mildly symptomatic patients (l P-l B; 10 P-2A; 2 P-2A, C [4]) was 179 TCID/mL (figure 1), whereas it was 2450 TCID/mL for 8 more severely symptomatic patients (3 P-2D; I P-2A, C, D; I P-2B, C; 3 P-2F) (P < .004 by Student's t test and by Mann-Whitney U test). To evaluate the reproducibility of the HIV-I quantification, cultures were repeated for 6 infected patients. The HIV-I titers were the same or differed by just one fivefold dilution (data not shown). The early identification of the HIV-I-infection status of an infant born to a seropositive mother is important for the anticipated management of that infant. We have begun to evaluate the performance of this assay in infants
1146
References
10.
II.
12.
13.
14.
15.
inhibition of replication of human immunodeficiency virus type I, including that of a zidovudine-resistant isolate, by zidovudine and 2'.3'-dideoxycytidine in vitro. Antimicrob Agents Chemother 1992;36: 1559-62. Johnson VA, Byington RE. Modified HIV-I p24 antigen ELISA. Quantitative assays for virus detection. In: Aldovini A, Walker BD, eds. Techniques in HIV research. New York: Stockton Press, 1990:95-7. Chou J, Chou TC. Dose-effect analysis with microcomputers: quantitation of ED so. LD so • synergism. antagonism, low-dose risk, receptorligand binding and enzyme kinetics: a computer software for IBM-PC and manual. Cambridge, UK: Biosoft, 1989. 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. Chou TC. The median-effect principle and the combination index for quantitation of synergism and antagonism. In: Chou TC, Rideout DC, eds. Synergism and antagonism in chemotherapy. New York: Academic Press. 1991:61-102. Johnson VA, Barlow MA. Merrill DP, Chou TC, Hirsch MS. Threedrug synergistic inhibition of HIV -I replication in vitro by zidovudine. recombinant soluble CD4, and recombinant interferon-alpha A. J Infect Dis 1990;161:1059-67. Smith MS, Brian EL, Pagano JS. Resumption of virus production after human immunodeficiency virus infection of T lymphocytes in the presence of azidothymidine. J ViroI1987;61:3769-73.
Diagnosis of Vertical Human Immunodeficiency Virus Type 1 Infection by Whole Blood Culture Ariane Alimenti, Mary O'Neill, John L. Sullivan, and Katherine Luzuriaga
Department of Pediatrics and Program in Molecular Medicine, University ofMassachusetts Medical Center, Worcester
The qualitative and quantitative performance of a human immunodeficiency virus type 1 (HIV-l) whole blood culture method was assessed for use in the diagnosis of vertical HIV-I infection. This method requires
::t
MEAN
P1;P2A,C
P2B,D,F
179
2450
Figure 1. HIV-I titers in whole blood samples from 21 infected patients (untreated or on zidovudine therapy). When cultures were repeated for a single patient at different dates, the HIV-I titer from the earliest blood sample was chosen.
This method eliminates the need for lymphocyte isolation and can be done on very small volumes of blood (< I mL). Forty-three children born to HIV-seropositive mothers were studied; age at time of study ranged from 2 months to 8 years. Twenty-seven children were previously HIV-l isolation-positive more than once. At the time of first testing, 2 (7%) of these children were 18 months. Eight HIV-I-infected children were receiving zidovudine therapy at the time of testing. Sixteen children were > 15 months old, HIV-I antibody negative, and lacked clinical or laboratory evidence of HIV-1 infection. . Heparinized whole blood was cultured in duplicate by end-point dilution in 24-well plates (250,50, 10,2,0.4,0.08 JLL/well) with 106 PBMC from HIV-l-seronegative donors. Donor PBMC were stimulated with phytohemagglutinin (PHA) for 48-72 h prior to use. RPMI 1640 with 10% fetal calf serum (FCS; GIBCO Laboratories, Grand Island, NY), 5% interleukin-2 (IL-2; Pharmacia Diagnostics, Columbia, MD), and gentamicin (50 JLg/mL, GIBCO) was used to bring the total volume to 1.5 mL/well. Twice a week, 0.75 mL of supernatant was removed from each well and replaced with 0.75 mL of media (RPMI 1640 with 10% FCS, 5% IL-2, and gentamicin). The HIV-l p24 antigen content of the superna-
tant was determined once weekly by commercial antigen immunoassay (Du Pont, Wilmington, DE, or Coulter, Hialeah, FL). Cultures were maintained for 4 weeks; fresh donor cells were not added. A culture well was considered positive when the supernatant p24 antigen value was> 100 pg/mL. The lowest dilution to give a positive result was considered the end point, and HIV-1 titers were expressed as 4, 20, 100, 500, 2500, and 12,500 TCID/mL ofwhole blood. When results in duplicate wells were discordant, the last dilution to give positive results in both wells was taken as the end point. HIV-l was isolated from 36 of 38 cultures of whole blood from 27 children >2 months old. The two specimens from HIV-I-infected children that yielded negative whole blood culture results were negative by concurrent PBMC microculture (AIDS Clinical Trials Group consensus protocol) as well; other cultures of whole blood from these 2 children were positive. None of the whole blood specimens from 16 uninfected children were culture positive. Most (90%) of the positive cultures were positive by day 15; 100%were positive by day 23. HIV-I titers of21 infected patients ranged from 0 to 12,500 TCID/mL of whole blood (mean, 1044). The mean HIV-I titer for 13 asymptomatic or mildly symptomatic patients (l P-l B; 10 P-2A; 2 P-2A, C [4]) was 179 TCID/mL (figure 1), whereas it was 2450 TCID/mL for 8 more severely symptomatic patients (3 P-2D; I P-2A, C, D; I P-2B, C; 3 P-2F) (P < .004 by Student's t test and by Mann-Whitney U test). To evaluate the reproducibility of the HIV-I quantification, cultures were repeated for 6 infected patients. The HIV-I titers were the same or differed by just one fivefold dilution (data not shown). The early identification of the HIV-I-infection status of an infant born to a seropositive mother is important for the anticipated management of that infant. We have begun to evaluate the performance of this assay in infants
