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1 375 Early Prognostic Indicators in Primary Perinatal Human Immunodeficiency Virus Type 1 Infection: Importance of Viral RNA and the Timing of Transmission on Long-Term Outcome Ruth E. Dickover, Maryanne Dillon, Kwan-Moon Leung, Paul Krogstad, Susan Plaeger, Shirley Kwok, Cindy Christopherson, Audra Deveikis, Margaret Keller, E. Richard Stiehm, and Yvonne J. Bryson Department of Pediatrics, UCLA School of Medicine, Department of Biostatistics, UCLA School of Public Health, Department of Pediatrics, Long Beach Memorial Medical Center, and Department of Pediatrics, Harbor-UCLA Medical Center, Los Angeles, and Roche Molecular Systems, Alameda California The time of perinatal human immunodeficiency virus type 1 (HIV-1) transmission and the pattern of early plasma viremia as predictors of disease progression were evaluated in infected infants followed from birth. Cox proportional hazards modeling demonstrated that a 1-log higher HIV-1 RNA copy number at birth was associated with a 40% increase in the relative hazard (RH) of developing CDC class A or B symptoms (P Å.004), a 60% increase in developing AIDS (P Å.01), and an 80% increase in the of risk death (P Å.023) over the follow-up period of up to 8 years. the peak HIV-1 RNA copy number for infants during primary viremia was also predictive of progression to AIDS (RH, 9.9; 95% confidence interval [95% CI], ; P Å.008) and death (RH, 6.9; 95% CI, ; P Å.04). The results indicate that high levels of HIV-1 RNA at birth and during primary viremia are associated with early onset of symptoms and rapid disease progression to AIDS and death in perinatally infected children. About 15% 25% of infants infected perinatally with human to be an indicator of risk of disease progression [9, 11]. immunodeficiency virus type 1 (HIV-1) show onset of symptoms HIV-1 RNA levels provide a direct measure of viral replication within the first few months of life, followed by rapid in vivo as well as an indirect indicator of immune clearance; progression to AIDS and death within 1 5 years [1 2]. In the therefore, they provide a valuable tool for assessing disease remainder of infected children, HIV-1 disease progresses at a status in infected persons. Plasma HIV-1 RNA levels are an variable rate over several years and is associated with survival excellent prognostic indicator in both primary and chronic adult times of 9 years in a minority of these children [3 5]. Accu- HIV-1 infection [12 16]. Recent studies have also shown that rate prognostic indicators are needed to help guide clinicians baseline HIV-1 RNA levels in perinatally infected infants in the clinical management of HIV-1 infected children and in are associated with disease progression and mortality risk [10, the evaluation of clinical trials. In addition, a better understanding 17]. However, little data are available on HIV-1 RNA levels of the pathogenesis of primary perinatal HIV-1 infection in perinatally infected infants at birth or during long-term and risk factors associated with disease progression is essential follow-up. for the development of effective intervention strategies. To study the relationship between the early pattern of HIV- Factors potentially affecting the rate of disease progression 1 replication, the estimated time of transmission, and the rate in perinatally infected infants include the time of transmission, of disease progression, we instituted a detailed prospective cell- and humoral-mediated immunity, viral phenotype, and study of perinatally HIV-1 exposed and infected infants fol- virus load [6 10]. After birth, the pattern of HIV-1 accumula- lowed from birth up to 8 years of age. We evaluated quantitative tion in CD4 / cells as measured by quantitative DNA polymerase HIV-1 RNA PCR as a sensitive tool to determine the time chain reaction (PCR) and coculture has also been shown of transmission (i.e., in utero or intrapartum) [18] in order to assess the pattern of primary viremia as a prognostic indicator of long-term outcome. Results were correlated with other measures of virus load, including quantitative DNA PCR, immune Received 9 September 1997; revised 19 February Presented in part: 3rd National Conference on Human Retroviruses and complex dissociated (ICD) p24 antigen assay, CD4 / cell Related Diseases, Washington, DC, January February 1996 (abstract 217). count, use of antiretroviral therapy, disease progression, and Informed consent was obtained under the guidelines of the UCLA Human Subjects Protection Committee. survival. Grant support: NIH (HD-30629, HD-26621, and ACTG AI-27550, AI , AI-32440, and Clinical Research Center RR ); Universitywide AIDS Research Program (R91-LA-152); Pediatric AIDS Foundation, Santa Materials and Methods Monica, California. Reprints or correspondence: Dr. Yvonne J. Bryson, Dept. of Pediatrics, Study population. As part of a prospective study of maternal- UCLA Medical Center, LeConte Ave., Los Angeles, CA fetal transmission of HIV-1, 204 infants were studied in detail for (Ybryson@pediatrics.medsch.ucla.edu). virologic and immunologic parameters. The infants were born to The Journal of Infectious Diseases 1998;178: by the Infectious Diseases Society of America. All rights reserved. HIV-1 seropositive mothers in the Los Angeles Pediatric AIDS /98/ $02.00 Consortium between June 1988 and May 1995 and were followed

2 376 Dickover et al. JID 1998;178 (August) longitudinally through June To establish an early diagnosis, roviral therapy treatment throughout their follow-up period. Infants we obtained infant blood specimens at or within 48 h (or both) of who clinically progressed on zidovudine were switched to dideoxybirth; biweekly until 12 weeks of age; and at 6, 9, and 12 months inosine monotherapy (n Å 12). The remaining 9 infants did not of age for testing by DNA PCR, HIV-1 coculture, and ICD p24 receive any antiretroviral therapy during follow-up (median, 254 antigen assay. Children were followed monthly until 1 year of age weeks; 25th and 75th percentiles, ). for evidence of symptoms related to HIV-1, according to criteria A total of 197 HIV-infected pregnant women were followed established by the Centers for Disease Control and Prevention over 202 consecutive deliveries giving birth to 204 infants. Ninety- [19]. Thereafter, infected infants were followed every 1 3 months seven mothers (98 pregnancies) in our study received zidovudine for evaluation of symptoms and collection of blood samples. No therapy during pregnancy or delivery (or during both). Before infants were breast-fed, and children were defined as uninfected February 1994, 17 women (18 pregnancies), including 4 diagnosed by seroreversion on ELISA or Western blot. Infants were defined with AIDS, received open-label oral zidovudine treatment (500 as infected following two positive HIV-1 cocultures from periph- mg/day) during pregnancy on the basis of their HIV-1 disease eral blood at two separate time points or if they died of an AIDS- status (median CD4 cell count, 315/mm 3 ; 25th and 27th percentiles, defining illness. 211 and 402). Five women were enrolled in a phase I trial for According to the proposed working definition, infants were de- safety and pharmacokinetics of zidovudine during pregnancy fined as infected in utero if they had a positive HIV-1 coculture (ACTG protocols 082 and 082 addendum). Three of 6 women or DNA PCR result within 48 h of birth; infants were defined as enrolled in ACTG 076 were randomized to receive zidovudine infected intrapartum if they had a negative coculture and DNA during gestation and delivery. Five women were enrolled in a PCR within 48 h of birth and subsequent positive HIV-1 diagnostic randomized, double-blind, placebo-controlled trial of zidovudine studies within 90 days of birth [18]. in combination with HIVIG (ACTG 185). Following the publica- All HIV-1 infected infants were categorized as rapid, interme- tion of the ACTG 076 study treatment guidelines [20], 67 women diate, or slow progressors on the basis of clinical symptoms and received open-label oral zidovudine (500 mg/day) beginning in laboratory results obtained during the first 2 years of life. Rapid the second or third trimester and/or as a constant infusion (2 progressors were defined as children with the following three critemg/kg loading dose followed by 1 mg/kg per hour for a mean of ria: onset of at least two HIV-1 related symptoms (CDC class A, 11.8 h) during labor and delivery. B, or C) [19] within 6 months of birth; significant and sustained As part of our study on maternal-fetal transmission of HIV-1, fall in CD4 cell count (õ1500/mm 3 or õ25%) within 1 year of blood samples were available at delivery from 20 of the 31 mothers birth; and a diagnosis of AIDS (CDC class C) within 2 years of of HIV-1 infected infants. Samples were not available at delivery birth. Slow progressors were defined as children with the following for 11 mothers who delivered prior to the initiation of the maternalcriteria: no HIV-1 related symptoms until ú6 months after birth; fetal transmission study (May 1989) or who delivered at sites stable CD4 cell count ( 1500/mm 3 or 25%) within 1 year of outside of the consortium; however, samples were available from birth; and lack of progression to AIDS (CDC class C) within at their infants within 48 h of birth. least 2 years of birth. Intermediate progressors were defined as Laboratory methods. Ficoll-hypaque density gradient centrifuchildren with onset of CDC class A or B symptoms within 6 gation was used to prepare peripheral blood mononuclear cells months of birth; apparent response coincident with antiretroviral (PBMC) from heparinized (HIV-1 coculture) or EDTA-treated therapy, with a resolution of the initial symptoms and an increase (PCR) samples. Mononuclear cells were washed twice with normal in CD4 cell count to ú1500/mm 3 in the first year of life; and lack saline and enumerated; cells not used immediately were stored of disease progression (no change in CDC classification status) within the first 2 years of birth. under liquid nitrogen. A total of 75 infants (4 infected, 71 uninfected) received oral HIV-1 antibodies were detected by ELISA and confirmed by zidovudine (2 mg/kg four times daily) in the first 6 weeks of life. Western blot assay (Abbott Laboratories, Abbott Park, IL). The Three of 6 infants born to women enrolled in a randomized, double- ICD p24 antigen assay was done on batched samples, using the blind, placebo-controlled trial of zidovudine for the prevention of Coulter assay (Coulter Immunology, Hialeah, FL) [21]. Samples perinatal HIV transmission (AIDS Clinical Trials Group [ACTG] with õ10 pg/ml ICD p24 antigen were assigned a value of 5 protocol 076 [20]) were randomized to receive zidovudine during pg/ml in the statistical analysis. the first 6 weeks of life; all 3 infants were uninfected. One infected Quantitative HIV-1 RNA PCR was done on batched plasma and 4 uninfected children born to mothers enrolled in a randomlecular Systems, Somerville NJ) according to the manufacturer s samples, using an assay (AMPLICOR HIV-1 Monitor; Roche Mo- ized, double-blind, placebo-controlled trial of hyperimmune HIVinstructions. 1 gamma globulin (HIVIG) for the prevention of perinatal transplasma EDTA-, acid-citrate-dextrose, or heparin-treated mission (ACTG 185) also received oral zidovudine for the first 6 was processed within 4 h after collection and stored at weeks after birth. Following the guidelines established by the 070 C until use. RNA was prepared from EDTA- and ACD- ACTG 076 study [20], 3 infected and 64 uninfected infants born treated plasma (n Å 300) according to the assay protocol and from after February 1994 were treated with oral zidovudine for the first heparinized samples (n Å 15), using a modification of a previously 6 weeks following birth. described method [22, 23]. Fifty microliters of each prepared RNA For infected infants who received antiretroviral therapy after 6 sample was used for PCR. After amplification and detection of weeks of age, treatment was initiated according to the standard of the PCR product, the starting HIV-1 RNA copy number in each care at the time and after the onset of HIV-1 related symptoms. sample was calculated by comparison with the internal quantitation Treatment consisted of zidovudine monotherapy for most children standard, and results were expressed as HIV-1 RNA copies/ml (n Å 20); 2 infants in ACTG study 180 received nevirapine followed plasma. Samples in which HIV-1 RNA was not detected were by zidovudine. Infected infants continued to receive antiret- assigned a value of 50 RNA copies/ml in the statistical analysis.

3 JID 1998;178 (August) RNA Load in Perinatal HIV Disease Progression 377 HIV-1 DNA in PBMC were quantitated by use of previously so the overall transmission rate during the entire study period published PCR methods [24]. Samples with copy numbers õ10 was 16.7%. Three of 34 HIV-1 infected infants who were were assigned a value of 5 HIV-1 DNA copies/mg PBMC DNA followed from within 1 month of birth but who did not have in the statistical analysis. On the basis of the assumptions that available birth samples defining the time of transmission were infected cells are CD4 positive and that each cell carries 1 copy excluded from this study. Twenty-eight of 31 infected infants of HIV-1 proviral DNA [25], we calculated the percentage of followed from within 48 h of birth had multiple samples CD4 / lymphocytes infected with HIV-1 in the circulation from the HIV-1 DNA copy number/mg PBMC DNA and the percentage available for virologic and immunologic analysis. Blood sam- of PBMC that were CD4 positive as determined by flow cytometry ples after the samples obtained within 48 h of birth were not on samples from the same date. available for 2 rapid progressors who died soon after birth Quantitative cocultures were done according to the National (10 and 12 weeks) and from 1 slowly progressing infant who Institutes of Health/National Institute of Allergy and Infectious moved to a location outside of the consortium. Disease Clinical (NIH/NIAID) Trials Group consensus protocol The 31 HIV-1 infected infants who were followed from in 24-well plates [26]. Negative HIV-1 cocultures were assigned within 48 h of birth showed 3 different patterns of clinical a value of 0.1 HIV-1 infected PBMC/10 6 in the statistical analysis. progression in the first 2 years of life. Of the 31 infants, 10 HIV-1 isolates from the zidovudine-treated transmitters at delivmet our criteria for rapid progressors by having early onset of ery and the first isolates from their infants were tested for zidovu- HIV-1 related symptoms (median, 6 weeks; 25th and 75th dine sensitivity using the NIH/NIAID Clinical Trials Group phenopercentiles, 5 and 12) and subsequent clinical progression to typic assay [26]. Flow cytometry of PBMC was done as previously published AIDS within 2 years despite antiviral therapy in 8 of these 10 [27]. Age-adjusted CD4 cell counts were determined by compariprogressors; they showed early onset of symptoms (median, 9 infants. Five of the 31 infants were considered intermediate son with age-related standards established for uninfected children born to HIV-1 infected mothers and are expressed as the CD4 weeks; 25th and 75th percentiles, 5 and 19) with a lack of cell percentile [28]. further disease progression until at least 2 years of age (coinci- Statistics. Descriptive statistics are provided as median (25th dent with the initiation of antiretroviral treatment at a median and 75th percentiles). Correlations between individual parameters of 15 weeks of age [25th and 75th percentiles, 12 and 18]). Of were determined using Spearman s rank correlation coefficient. the 5 intermediate progressors, 4 had a transient decrease in Virologic and immunologic parameters between groups were com- CD4 cells to õ1500/mm 3, and the fifth child showed a gradual pared using the Mann-Whitney U and Kruskal-Wallis tests. Median decrease in CD4 cells during the first year. All 5 intermediate time to onset of symptoms, AIDS, death, and treatment between progressors showed a resolution of initial symptoms and an groups were compared using the Mann-Whitney U and Kruskalincrease in CD4 cells ( 1500/mm 3 ) by at least 1 year after Wallis tests. The area under the curve for plasma HIV-1 RNA levels over the first 12 weeks of life was calculated for each child, birth coincident with the initiation of antiretroviral therapy. using the tangential method. The distribution of times to events Thirteen of the 31 infants were considered slow progressors; was estimated by Kaplan-Meier product limit analysis, with P they had late onset of symptoms (median, 42 weeks; 25th and values calculated by the log-rank test. The relative hazard (RH) 75th percentiles, 28 and 130) followed by a lack of further of HIV-1 related symptoms, AIDS, and death were calculated by disease progression until at least 2 years of age, regardless of univariate Cox proportional hazards modeling, with time-depen- treatment status. The 3 remaining slow progressors were free dent variables if needed. The corresponding 95% confidence inter- of HIV-1 related symptoms as of their last follow-up visit vals (CIs) constructed around each estimate and P values deter- ( weeks of age). mined by x 2 likelihood ratio analysis were also provided. P Eighteen infected infants were defined as infected in utero.05 was considered statistically significant. P.10 represents on the basis of positive results for HIV-1 coculture or DNA nonsignificant results. PCR within 48 h following birth (table 1). The remaining 13 infected children had negative results for HIV-1 cultures and Results DNA PCRs at birth but developed evidence of infection on subsequent studies (2 8 weeks after birth) and were thus defined Patient characteristics. Samples were available for analysis as infected intrapartum. from 204 infants with known infection outcomes; the No significant difference was seen between in utero and infants were followed prospectively in the Los Angeles Pediatric intrapartum-infected infants with regard to gestational age AIDS Consortium between June 1988 and May (median, 38 weeks; 25th and 75th percentiles, 38 and 40, vs. Of 34 perinatally infected infants born during this period, 31 median, 38 weeks; 25th and 75th percentiles, 35 and 39) or who had samples within 48 h of birth were followed clinically birth weight (median, 3.1 kg; 25th and 75th percentiles, 2.7 for a median of 186 weeks (25th and 75th percentiles, 96 and 3.4, vs. median, 2.4 kg; 25th and 75th percentiles, 2.1 and and 311). Before zidovudine treatment was begun in the 3.3). Nine of 18 in utero infected infants showed rapid disease mothers alone or in both mothers and infants (according to progression, while 5 (28%) were intermediate progressors, and the ACTG 076 regimen), the transmission rate within our 4 (22%) were slow progressors (table 1). Intrapartum-infected cohort was 27.9%. After the initiation of the 076 treatment infants were significantly less likely to show rapid (1/13) or regimen, the transmission rate in our cohort dropped to 9.1%, intermediate (0/13) disease progression, and 12 of the 13 were

4 378 Dickover et al. JID 1998;178 (August) Table 1. Infection status and disease progression in infants at risk At birth, the children who had rapid disease progression had for perinatal HIV-1 infection. significantly higher median HIV-1 RNA and DNA levels than those who demonstrated intermediate or slow disease progres- In utero, Intrapartum, No. total no. (%) no. (%) (%) sion (table 2). A statistically significant difference between these 3 groups on the basis of other measures, including quanti- Outcome (n Å 204) tative cocultures, ICD p24 antigen levels, and CD4 cell count Infected (total) 34 (17) at birth, was not seen (table 2). In utero 18 (53) Infant and maternal HIV-1 RNA levels at birth correlated Intrapartum 13 (34) Indeterminant* 3 (13) strongly (r Å 0.63, P Å.008; n Å 20). Maternal CD4 cell Uninfected 170 (83) count at delivery correlated with infant CD4 cell count within Disease progression (n Å 31) 1 month of birth (r Å 0.59, P Å.002; n Å 18) and showed Rapid 9 (50) 1 (8) 10 (32) an inverse correlation with infant HIV-1 RNA levels at birth Intermediate 5 (28) 0 (0) 5 (16) (r Å00.56, P Å.02; n Å 18). Slow 4 (22) 12 (92) 16 (52) Total 18 (58) 13 (42) 31 (100) Virologic and immunologic parameters over time. The 28 HIV-1 infected children who were followed virologically and * No samples available within 48 h of birth. immunologically over time were tested with an average of 10 quantitative cocultures, 10 quantitative DNA PCRs, 10 quantitative RNA PCRs, 6 ICD p24 antigen assays, and 12 lymphocyte subset determinations. One time point during the first 6 slow progressors (P õ.001). The Kaplan Meier plot of time months of follow-up was randomly selected for determination to onset of HIV-1 related symptoms (CDC class A or B or of the correlations between virologic and immunologic assays C) in infants according to the timing of transmission is shown in the 28 infants. Among the assays that were done on the in figure 1. In utero infected infants developed symptoms same days, results for the quantitative RNA PCR correlated significantly earlier than did intrapartum-infected infants well with results for the quantitative DNA PCR (r Å 0.51, P (median, 9 weeks; 25th and 75th percentiles, 6 and 18, vs. õ.001), the quantitative coculture (r Å 0.49, P õ.001), and median, 28 weeks; 25th and 75th percentiles, 19 and 43; the ICD p24 antigen assay (r Å 0.44, P õ.001) and with the P Å.003). percentage of infected CD4-positive cells (r Å.47, P õ.001). Virologic and immunologic parameters at birth. HIV-1 co- Plasma HIV-1 RNA levels showed an inverse correlation with culture was done for all 201 infants who were followed from the absolute CD4 cell count (r Å00.29, P Å.009) and the within 48 h of birth. We also performed HIV-1 DNA PCR for age-adjusted CD4 cell count (r Å00.36, P Å.001). 160 of the infants and RNA PCR for 46. Of the 18 in utero HIV-1 RNA levels during primary infection. After birth, infected infants, 16 had positive HIV-1 cocultures (median, 1 infected infants showed rapid accumulation of HIV-1 RNA in infected PBMC/10 6 ; 25th and 75th percentiles, 1 and 1) and positive DNA PCRs (median, 11 HIV-1 DNA copies/mg PBMC DNA; 25th and 75th percentiles, 10 and 18) within 48 h of birth. Two infected infants were considered as in utero infected on the basis of positive results for HIV-1 coculture (n Å 1, 1 infected PBMC/10 6 ) or HIV-1 DNA PCR (n Å 1, 10 DNA copies/mg PBMC DNA) at birth and with subsequent positive follow-up tests. Thirteen intrapartum-infected infants had negative DNA PCRs and cocultures within 48 h of birth with subsequent positive follow-up tests. Figure 2 shows the quantitative RNA PCR results at birth for 46 infants born to HIV-1 infected mothers in our cohort, including 15 randomly selected seroreverters. All 18 infants defined as in utero infected by DNA PCR or coculture (or both) were also positive with a wide range of plasma HIV- 1 RNA values within 48 h of birth and a median level of 26,940 HIV-1 RNA copies/ml (25th and 75th percentiles, 1556 and 468,390). In contrast, none of the 13 infants pre- Figure 1. Kaplan-Meier plot of onset of HIV-1 related symptoms viously defined as intrapartum infected had detectable levels (CDC class A or B or C) in infected infants according to time of of plasma HIV-1 RNA within 48 h of birth. Similarly, 0 of transmission. Children positive for HIV-1 by coculture or DNA poly- merase chain reaction within 48 h of birth were defined as in utero 5 cord-blood and 0 of 10 day-one peripheral blood samples infected (n Å 18). Children negative for HIV-1 within 48 h of birth but from the randomly selected seroreverters were positive for with subsequent positive results (n Å 13) were defined as intrapartum HIV-1 RNA. infected.

5 JID 1998;178 (August) RNA Load in Perinatal HIV Disease Progression 379 Figure 2. Levels of plasma HIV-1 RNA in children at risk for perinatal infection at birth. Arrow indicates cutoff for RNA polymerase chain reaction assay. their plasma, reaching peak levels at an overall median of 8 vs. median, 10; 25th and 75th percentiles, 8 and 12 [n Å 5] weeks of age (25th and 75th percentiles, 5 and 11). The infected vs. median, 6; 25th and 75th percentiles, 4 and 7, [n Å 15]; infants reached their lowest HIV-1 RNA levels (trough) during P Å.02). At the time of the peak in primary viremia, levels the first year of life, at a median of 24 weeks of age (25th and of cell-free and cell-associated HIV-1 were significantly 75th percentiles, 20 and 26). We used the period from birth to higher in both rapid and intermediate progressors, and CD4 HIV-1 RNA level trough (Ç6 months of age) to define primary cell counts were significantly lower than those in slow proviremia in perinatally infected infants followed from birth. gressors (table 3). Rapid progressors reached their peak in primary plasma viremia We calculated the area under the curve for plasma HIV-1 significantly later than intermediate and slow progressors (me- RNA levels over the first 12 weeks after birth in 8 rapid, 5 dian, 12 weeks; 25th and 75th percentiles, 7 and 12, [n Å 8] intermediate, and 15 slow progressors. The combination of a Table 2. Biologic variables in HIV-1 infected infants at birth, according to rapid, intermediate, or slow disease progression status. Rapid progressors Intermediate progressors Slow progressors Median Median Median (25th and 75th (25th and 75th (25th and 75th percentiles) n percentiles) n percentiles) n P HIV RNA copies/ml 245, (1556 and 960,920) (895 and 42,861) (50 and 75) HIV copies/mg PBMC DNA (10 and 21) (10 and 14) (5 and 5) % CD4 cells infected (0.008 and 0.022) (0.012 and 0.016) (0.005 and 0.009) Infected PBMC/ (1 and 1) (1 and 1) (0.1 and 0.55) ICD p24 (pg/ml) NS (5 and 68) (5 and 86) (5 and 5) CD4 cell count (/mm 3 ) (1944 and 3634) (3935 and 4502) (2527 and 3472) % CD4 / cells NS (40 and 64) (52 and 55) (43 and 65) CD4/CD8 ratio NS (0.33 and 2.31) (1.56 and 2.70) (1.49 and 2.81) NOTE..10). PBMC, peripheral blood mononuclear cells; ICD, immune complex dissociated. P calculated using Kruskal-Wallis test. NS, not significant (P ú

6 380 Dickover et al. JID 1998;178 (August) Table 3. Biologic variables at the time of peak viremia in HIV-1 infected infants followed from birth, according to rapid, intermediate, or slow disease progression status. Rapid progressors Intermediate progressors Slow progressors Median Median Median (25th and 75th (25th and 75th (25th and 75th percentiles) n percentiles) n percentiles) n P HIV RNA copies/ml 3,064, ,913, , õ.001 (2,017,877 and 5,632,026) (1,547,408 and 4,724,300) (512,897 and 1,021,541) HIV copies/mg PBMC DNA õ.001 (310 and 441) (253 and 347) (137 and 204) % CD4 cells infected õ.001 (1.1 and 2.1) (0.52 and 0.68) (0.22 and.035) Infected PBMC/ (125 and 625) (81 and 625) (10 and 125) ICD p24 (pg/ml) (459 and 890) (260 and 976) (5 and 305) CD4 cell count (/mm 3 ) , , õ.001 (167 and 1,717) (1304 and 2295) (2070 and 2720) % CD4 / cells (12 and 38) (27 and 38) (34 and 47) CD4/CD8 ratio (0.35 and 1.42) (1.2 and 1.7) (1.4 and 2.2) Age adjusted* values (percentile) CD4 cell count (/mm 3 ) õ.001 (1 and 7) (4 and 24) (17 and 41) % CD4 / cells (1 and 3) (3 and 19) (10 and 46) CD4/CD8 ratio (1 and 14) (11 and 24) (10 and 42) NOTE. PBMC, peripheral blood mononuclear cells; ICD, immune complex dissociated. P calculated using Kruskal-Wallis test. * Values were compared with the distribution of normal lymphocyte subpopulations in uninfected children in the same age ranges. higher virus load and a longer duration of primary viremia in tiles, 5- and 10-fold) prior to the initiation of antiretroviral rapid and intermediate progressors resulted in a significantly therapy (figure 4E). Thirteen of the 15 slow progressors fol- higher area under the curve compared with that for slow prog- lowed longitudinally from birth had õ10 5 /ml HIV-1 RNA ressors: rapid progressors Å median, 27,572,490 HIV-1 RNA copies by 1 year of age, and the remaining 2 had levels õ10 5 / copies 1 week; 25th and 75th percentiles, 21,956,342 and ml by 2 years of age. The slow progressors also showed a 62,789,384, (n Å 10) vs. intermediate progressors Å median, more gradual, age-related decline in CD4 cells (figure 4F). 25,483,340 HIV-1 RNA copies 1 week; 25th and 75th percen- The intermediate progressors showed high rates of HIV-1 replication, tiles, 9,633,281 and 33,274,582, (n Å 5) vs. slow progressors which were similar to those for the rapid progressors Å 5,787,257 HIV-1 RNA copies 1 week; 25th and 75th percen- soon after birth, and in contrast to rapid progressors, they had a tiles, 2,334,998 and 7,767,661, (n Å 16); P õ.001 (figure 3). significant decline in HIV-1 RNA levels following the initiation Figure 4 shows individual examples of the three early pat- of antiretroviral therapy (figure 4C). Two of these children received terns of HIV-1 plasma viremia and CD4 cell decline in 12 nevirapine followed by zidovudine, and the 3 remaining infected infants, who were representative of the cohort, over children received zidovudine monotherapy. After treatment, the the first 6 months of life. The rapid progressors showed a initial decline in CD4 / cell count seen early in the intermediate significant increase in HIV-1 RNA to ú10 6 HIV-1 RNA cop- progressors was arrested (figure 4D) so that all 5 had CD4 counts ies/ml accompanied by a dramatic loss in CD4 / cells (figure 1500/mm 3 by 1 year of age. HIV-1 RNA levels also continued 4A, B). The level of HIV-1 replication remained high ( 10 5 to decline in the intermediate progressors, who reached levels of HIV-1 RNA copies/ml) in these children throughout followup õ10 5 copies/ml by 2 years of age. These children had no further (median, 95 weeks; 25th and 75th percentiles, 22 and 178) disease progression during follow-up (median, 208 weeks; 25th or until death (n Å 7; median, 69 weeks; 25th and 75th percentiles, and 75th percentiles, 179 and 351). 15 and 100). Effect of antiretroviral therapy on HIV-1 load in infants In contrast, children defined as slow progressors had lower treated from birth. Eight infants born to zidovudine-treated peak levels of plasma viremia (table 3) followed by a decline women became infected with HIV-1, and 4 of these infants in HIV-1 RNA levels (median, 7.5-fold; 25th and 75th percen- were treated with zidovudine for the first 6 weeks following

7 JID 1998;178 (August) RNA Load in Perinatal HIV Disease Progression 381 Figure 3. HIV-1 RNA copy numbers in infected infants over first 4 months of life. Symbols indicate median HIV-1 RNA copy no. among children with rapid (n Å 10), intermediate (n Å 5), or slow (n Å 16) disease progression at each time point. birth (according to the ACTG 076 protocol [20]). The re- had lower levels (529,721 copies/ml; 25th and 75th percentiles, maining 90 infants born to zidovudine-treated mothers were 343,658 and 698,481). uninfected. Of these 90 infants, 59 received oral zidovudine Ten weeks after treatment, the slow progressors showed the during the first 6 weeks of life, according to the ACTG 076 largest median fold-decrease in HIV-1 RNA levels (15-fold; protocol. The infected children treated from birth had relatively 25th and 75th percentiles, 10- and 25-fold) and the lowest low peak HIV-1 RNA levels (median, 689,787 copies/ml; 25th absolute levels of HIV-1 RNA (32,880 copies/ml; 25th and and 75th percentiles, 397,914 and 911,005), and 3 of 4 pro- 75th percentiles, 12,140 and 47,553). Although the rapid and gressed slowly. Of these 4 infants, 2 were infected in utero (1 intermediate progressors were treated at about the same age, progressed rapidly), whereas both of the intrapartum-infected the intermediate progressors showed a greater response to treatinfants progressed slowly. However, the number of infants ment (median, 8.8-fold; 25th and 75th percentiles, 6- and 16- treated from birth in this study is too small to draw any conclu- fold) and decreased to a median of 165,989 HIV-1 RNA copies/ sions. In addition, we do not know if their HIV-1 RNA levels ml; 25th and 75th percentiles, 89,079 and 325,960) 10 weeks would have been higher had they not received zidovudine in after treatment. the first 6 weeks after birth. In contrast, rapid progressors showed only a small drop in No in vitro zidovudine resistance was identified by phenotypic median HIV-1 RNA levels (3.6-fold decrease; 25th and 75th assay in any of the time-of-birth HIV-1 isolates from the treated percentiles, 2.6- and 6.6-fold) to a median absolute level of mothers who transmitted HIV-1 or their infants (IC 50 range Å 469,198 copies/ml (25th and 75th percentiles, 271,908 and mm). We have reported this previously, and our addi- 911,685) 10 weeks after treatment. Two years after the initiational data continue to support that transmission in these cases may tion of treatment, plasma HIV-1 RNA levels were relatively reflect the higher levels of HIV RNA measured in the zidovudine- low for the intermediate (median, 61,074 copies/ml; 25th and treated, HIV-1 transmitting mothers [23]. 75th percentiles, 30,077 and 82,468) and treated slow progressors Antiretroviral treatment after onset of symptoms. Antiretroviral (median, 30,786 copies/ml; 25th and 75th percentiles, therapy was initiated following the onset of HIV-1 12,654, and 37,003). Seventy percent of the rapid progressors related symptoms at a median of 16 weeks of age in treated died by 2 years of age with HIV-1 RNA levels consistently rapid progressors (n Å 8/10; 2 children died of AIDS before ú10 5 copies/ml. The treated rapid progressors who survived treatment), at a median of 15 weeks in intermediate progressors to beyond age 2 (n Å 3) had high, persistent levels of HIV-1 (n Å 5/5), and at a median of 32 weeks in the treated slow RNA in their plasma (median 2 years after treatment was beprogressors (n Å 9/16). At the time of treatment, median HIVand gun, 427,012 copies/ml; 25th and 75th percentiles, 304,128 1 RNA levels were similar in the rapid (2,708,545 copies/ 549,466). ml; 25th and 75th percentiles, 889,380 and 4,894,556) and HIV-1 load over long-term follow-up. Figure 5 shows all intermediate progressors (2,913,680 copies/ml; 25th and 75th HIV-1 RNA levels measured in all surviving infected infants percentiles, 356,673 and 4,015,190), while slow progressors from 4 months to 5 years of age. We calculated each child s

8 382 Dickover et al. JID 1998;178 (August) Figure 4. HIV-1 RNA copy nos. (A, C, E) and CD4 / cell count (B, D, F) over time in infected children with rapid, intermediate, or slow disease progression. Dashed lines indicate onset of antiretroviral therapy. Of children with rapid disease progression, patient 17 ( ) became symptomatic at week 12, started zidovudine at week 28, and was switched to dideoxyinosine (ddi) at week 90; patient 31 ( ) became symptomatic at week 8, started zidovudine at week 18, and was switched to ddi at week 48; patient 1 ( ) became symptomatic at week 5, started zidovudine at week 16, and died at week 68; patient 8 ( ) became symptomatic at week 8, started zidovudine at week 14, and died at week 74. Of children with intermediate disease progression, patient 18 ( ) became symptomatic at week 9 and started zidovudine at week 10; patient 30 ( ) became symptomatic at week 6, started zidovudine at week 16, and was switched to ddi at week 124; patient 19 ( ) became symptomatic at week 16 and started zidovudine at week 16; patient 25 ( ) became symptomatic at week 3, started nevirapine at week 16, and added zidovudine at 22 weeks. Of children with slow disease progression, patient 20 ( ) became symptomatic at week 28, started zidovudine at week 40, and was switched to ddi at week 190; patient 29 (l) was asymptomatic at week 95 and had not begun antiretroviral therapy; patient 15 ( ) became symptomatic at week 40 and had not received antiretroviral therapy as of week 210; patient 28 ( ) became symptomatic at week 26 and started zidovudine at week 26. median HIV-1 RNA copy number beyond their trough through- levels over the follow-up period, suggesting a slow but more out follow-up (median number of determinations, 5; 25th and effective immune response in these children (figure 5). 75th percentiles, 4 and 9; median follow-up, 195 weeks; 25th Time to onset of AIDS (CDC class C) and survival. The and 75th percentiles, 98 and 312). It is important to note that 10 rapid progressors progressed to AIDS by a median of 11 3 rapid progressors died within the first year of life, and 4 died weeks of age (25th and 75th percentiles, 6 and 30), and 7 died between 1 and 2 years of age. Median plasma HIV-1 RNA (median 69 weeks; 25th and 75th percentile, 15 and 100; me- levels were consistently higher beyond 6 months of age in the dian follow-up, 95 weeks; 25th and 75th percentile, 22 and rapid progressors (902,473 copies/ml; 25th and 75th percentiles, 178). Of the 5 intermediate progressors, 1 developed AIDS, 301,837 and 1,390,775) than in the intermediate pro- with lymphoid interstitial pneumonia (335 weeks of age), gressors (83,433 copies/ml; 25th and 75th percentiles, 75,335 which subsequently resolved, and all 5 infants were still alive and 124,770) and slow progressors (34,840 copies/ml; 25th at a median follow-up time of 208 weeks; 25th and 75th percentiles, and 75th percentiles, 21,481 and 56,643) (P õ.001). Following 179 and 351). At the time of writing, only 1 of the 16 the resolution of primary viremia at 6 months, the rapid and slow progressors had developed AIDS (median, 184 weeks, intermediate progressors showed more consistent but still high developmental delay), and all slow progressors were still alive plasma HIV-1 RNA levels. Both treated and untreated slow at a median follow-up time of 264 weeks of age (25th and progressors showed a gradual, persistent decline in HIV-1 RNA 75th percentiles, 106 and 330).

9 JID 1998;178 (August) RNA Load in Perinatal HIV Disease Progression 383 Figure 5. HIV-1 RNA copy nos. in infected infants with rapid, intermediate, or slow progression. Average of 4 samples/child over 4 months to 5 years of age. The Kaplan-Meier plots of time to AIDS, stratified according time of initiation of antiretrovirals was not significantly predictive to the time of transmission, and the peak in HIV-1 RNA levels of the time to onset of symptoms, AIDS, or death most are shown in figure 6. Among 18 infants infected in utero, 8 probably because treatment was initiated after the onset of (44.4%) developed AIDS by 1 year of age. In contrast, only 1 symptoms, and in some cases, the presenting symptom was (7.7%) of 13 intrapartum-infected infants (HIV-1 RNA negative AIDS. Similarly, the Cox proportional hazards model of infant at birth) had developed AIDS by 1 year of age (P Å.03; figure CD4 cell count at 6 months of age was only predictive of time 6A). Children with peak HIV-1 RNA levels ú10 6 copies/ml to death, probably because many infants had onset of symptoms during primary viremia (õ6 months of age) progressed to AIDS or AIDS (or both) by 6 months of age (figures 1, 6). significantly faster than children with peak HIV-1 RNA levels of õ10 6 copies/ml (P Å.008; figure 6B). All 7 infected infants who died from AIDS were infected in utero, and 6 (86%) had peak Discussion HIV-1 RNA levels ú10 6 copies/ml during primary viremia. This prospective study of infants perinatally exposed to and Cox proportional modeling analysis was done to evaluate infected with HIV-1 shows that both the time of infection in potential early prognostic indicators for the risk of developing infants and the pattern and magnitude of HIV-1 virus load during HIV-1 related symptoms or of progressing to AIDS or death. early primary infection have significant prognostic value for long- A 1-log increase in HIV-1 RNA copy number at birth, a 1-log term outcome. These data extend previous studies that observed increase in HIV-1 RNA peak copy number, and a 1-log increase an increased risk of rapid progression in infants who had HIV- in maternal HIV-1 RNA copy number at birth were each inde- 1 detected at birth or who had high levels of HIV-1 DNA in the pendently associated with the risks of developing symptoms first few weeks of life [6, 9]. In agreement with a recent study or AIDS or of progressing to death (table 4) over the follow- by Shearer et. al. [17], we found that children who had HIV-1 up period of up to 8 years. In contrast, a 1-log increase in the RNA present at birth had a higher risk of having early onset of infant s first CD4 cell count (median, 2 weeks; 25th and 75th symptoms and rapid disease progression compared with those percentiles, 1 and 9) was not associated with a decreased risk who were negative for HIV-1 RNA at birth. of developing symptoms or AIDS or of progressing to death Cross-sectional studies have shown that perinatally infected (table 4). However, the infant s CD4 cell count at 6 months infants have very high levels of HIV-1 RNA during the first was independently associated with the risk of death in those year of life and that some children have a slow drop in infants who died after 6 months of age (n Å 4, table 4). HIV-1 RNA levels over years 2 5 [10, 29, 30]. Studies in The infant s sex, gestational age, and birth weight were not adults with primary infection have shown that HIV-1 RNA independently associated with the risk of developing symptoms levels rapidly increase in the early weeks following infection or AIDS or of progressing to death. Antiretroviral treatment and that plasma RNA levels after 120 days are predictive of status of the mother during gestation or of the infant immediately ultimate disease progression [12, 14, 31]. Primary HIV-1 infec- following birth were also not predictive of time to symp- tion in perinatally infected infants may be more complicated toms, AIDS, or death; however, the numbers were small. The because of the influence of the time of infection and the fact

10 384 Dickover et al. JID 1998;178 (August) Figure 6. Kaplan-Meier plots showing proportion of infected infants who were free of AIDS, according to detection of HIV-1 RNA at birth (A) or peak level of HIV-1 in plasma during primary viremia (B). that the infection is occurring in an immature host infant or fetus who is also growing at a rapid rate. Using the sensitive HIV-1 RNA PCR assay, we were not able to detect virus in birth specimens of children previously categorized as intrapartum infected. In contrast, all infants who had positive results for HIV-1 cocultures or DNA PCRs at birth had detectable HIV-1 RNA, with levels ranging from 100 to 2.9 and 11 million HIV-1 RNA copies/ml in twins who both died with AIDS and Pneumocystis carinii pneumonia within 3 months of birth. Most in utero infected children had relatively low levels of HIV-1 RNA at birth, however, suggesting that either infection in utero occurred late in gestation or that virus replication was somehow inhibited in the fetus. This, however, does not exclude the possibility of early in utero virus transmission and replication in privileged sites, such as the brain or gastrointestinal tract. We found that plasma viremia as measured by HIV-1 RNA PCR showed distinct patterns in the first weeks of life that differed in children who were ultimately characterized as rapid, slow, or intermediate progressors on the basis of clinical criteria. The most striking finding was the rapid and continued rise in plasma viremia in the rapid progressors; the rise did not reach a peak until a

11 JID 1998;178 (August) RNA Load in Perinatal HIV Disease Progression 385 Table 4. Cox proportional hazards models for the evaluation of potential early prognostic indicators for the risk of developing HIV-1 related symptoms or of progression to AIDS or death. HIV-1 related Variable symptoms P AIDS P Death P Log infant HIV RNA copy no. at birth 1.4 ( ) ( ) ( ).023 Log infant peak HIV RNA copy no. 3.4 ( ) ( ) ( ).04 Log infant HIV RNA copy no. at 6 months NS 6.2 ( ).03 NS Log first infant CD4 cell count NS NS NS Log infant CD4 cell count at 6 months NS NS 0.05 ( ).006 Log mother HIV RNA copy no. at birth 8.1 ( ) ( ) ( ).02 NOTE. Data are relative hazard (95% confidence interval). Relative hazards are those associated with log 10 increase in HIV RNA copy no. or CD4 / cell count. P values were determined using x 2 test. NS, not significant (P ú.10). median of 12 weeks of age and was accompanied by a profound drop in HIV-1 RNA levels after treatment and a continued gradual drop in CD4 cell count. In comparison, the slow progressors, who decline in HIV-1 RNA levels during the follow-up period of up had low or undetectable levels of HIV-1 RNA at birth, showed to 8 years, and no slow progressor died (median follow-up 264 a lower but more rapid peak in plasma viremia and a distinct weeks; 25th and 75th percentiles, 106 and 331). drop in levels even prior to initiation of antiviral treatment. In Because most rapid progressors were infected in utero, quescontrast to studies of primary viremia in adults, there was no tions remain as to whether perturbations of the fetal thymus or definitive early set point ; however, all infected infants showed other immune progenitor cells were caused by HIV-1 infection, their lowest HIV-1 RNA levels in the first year of life at Ç6 thereby blunting the development of an immune response [32]. months of age [14, 31]. The lowest trough HIV-1 RNA levels Studies by Uittenboggart et al. [33] have shown that certain HIV seen in these children were still significantly higher than those isolates have a greater predilection for infection and destruction previously reported in adults (figure 5) [14]. In intermediate and of early progenitor cells in the in vitro human fetal thymus model. slow progressors, the trough in HIV RNA copy number was Others have shown that many perinatally infected infants with followed first by a brief increase in HIV-1 RNA levels and then rapid disease progression have a lymphocyte profile similar to by a gradual decrease over time. that of infants with congenital thymic dysfunction [32]. All rapid progressors had persistently high levels of We evaluated a variety of virologic and immunologic param- HIV-1 RNA ( 10 5 copies/ml) throughout the follow-up pe- eters as early prognostic indicators for the onset of HIV-1 riod (median, 95 weeks; 25th and 75th percentile, 22 and 178) related symptoms, AIDS, and death. While HIV-1 RNA levels or until their death, despite antiretroviral treatment with zido- at birth were significantly associated with the risk of developing vudine or dideoxyinosine monotherapy. In contrast, intermedi- symptoms or AIDS and of progression to death, the infant s ate progressors appeared to show a greater response coincident first CD4 cell count at a median of 2 weeks of age was not a with antiretroviral therapy, followed by resolution of initial prognostic indicator for these outcomes. We found that the HIV-1 related symptoms, an increase in CD4 cell count in the mother s HIV-1 RNA level at delivery was an excellent progfirst year of life, and subsequently stable CD4 cell counts, and nostic indicator for the risk of developing AIDS in the infant. none of the infants died (median follow-up, 208 weeks; 25th This agrees with our previous findings that mothers with higher and 75th percentiles, ). The reasons for the difference HIV-1 RNA levels at delivery are more likely to transmit in apparent treatment response of these 2 groups are unclear HIV-1 to their infants in utero, and these infants were more since both the timing of treatment and the median HIV-1 RNA likely to show rapid disease progression [9, 23]. Blanche et al. copy number prior to treatment were similar. In addition, only [34] also reported that infected infants born to mothers with 2 intermediate progressors received a more potent antiviral AIDS and infants with positive DNA PCR results at birth were regimen than zidovudine monotherapy, including a combina- more likely to show rapid disease progression. tion with nevirapine. This suggests that host or viral factors A recent report on a large cohort of perinatally HIV-1 could also account for the observed differences in disease pro- infected infants ú1 month of age found that baseline HIV-1 gression between rapid and intermediate progressors. RNA levels of ú10 5 copies/ml serum were associated with The initially encouraging results seen in the intermediate pro- an increased risk of mortality [10]. While the number of perinagressors who first presented like rapid progressors but changed tally infected infants followed from birth in this study is relacourse, suggest that more potent antiviral regimens may have an tively small, our data clearly suggest that maternal HIV-1 RNA even greater impact. The slow progressors showed the largest levels at delivery, infant HIV-1 RNA levels at birth, and the

12 386 Dickover et al. JID 1998;178 (August) peak infant HIV-1 RNA level during the first 6 months of life References may be used as indicators of the risk of disease progression to 1. Scott GB, Hutto C, Makuch RW, et al. Survival in children with perinatally AIDS in perinatally infected infants. Our data also indicate that acquired human immunodeficiency virus type 1 infection. N Engl J Med perinatally infected infants with peak HIV-1 RNA levels 10 6 / 1989;321: ml during the first 6 months of life and persistent counts of 2. Blanche S, Tardieu M, Duliege A, et al. Longitudinal study of 94 symptom /ml thereafter are at the greatest risk of rapid disease atic infants with perinatally acquired human immunodeficiency virus infection. Evidence for a bimodal expression of clinical and biological progression. In addition, Shearer et al. [17] reported that the symptoms. Am J Dis Child 1990;144: median HIV-1 RNA levels over the first 2 months of life in 3. 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Virologic and immunologic characterization of symptomatic and primary HIV-1 infection. J Acquir Immune Defic Syndr pears possible in perinatally infected infants if therapy with Hum Retrovirol 1995;9: potent combinations of antiretrovirals is started early. Our 14. Mellors JW, Rinaldo CR, Gupta P, White RM, Todd JA, Kingsley LA. study, along with a growing body of data, suggests that the Prognosis in HIV-1 infection predicted by the quantity of virus in the early pattern of primary infection in infants may be the most plasma. Science 1996;272: critical for determining long-term outcome. Careful evaluation 15. Galetto-Lacour A, Yerly S, Perneger TV, et al. Prognostic value of viremia in patients with long standing human immunodeficiency virus infection. of early aggressive antiretroviral therapy in perinatally infected J Infect Dis 1996;173: infants to address these important questions is urgently needed. 16. O Brien TR, Blattner WA, Waters D, et al. Serum HIV-1 RNA levels and time to development of AIDS in the Multicenter Hemophilia Cohort Study. JAMA 1996;276: Shearer WT, Quinn TC, LaRussa P, Lew JF, et al. Viral load and disease Acknowledgments progression in infants infected with human immunodeficiency virus type 1. N Engl J Med 1997;336: Bryson YJ, Luzuriaga K, Sullivan JL, Wara DW. Proposed definitions for We thank Arceli Gagajena and Coreen Correa for their assis- in utero versus intrapartum transmission of HIV-1. N Engl J Med 1992; tance and follow-up of the patients; Victor Wong and Steven Tay- 327: lor for providing us with infant samples and clinical follow-up; 19. Centers for Disease Control Revised classification system for human Lian Wei, Eileen Garratty, and Joanna Camba-Colon for laboratory immunodeficiency virus infection in children less than 13 years of age. assistance; and Diana Liao for data management. MMWR Morb Mortal Wkly Rep 1994;43:1 9.