Lymphocyte subsets in HIV-exposed uninfected infants and HIV-unexposed uninfected infants

Letter to the Editor Lymphocyte subsets in HIVexposed uninfected infants and HIV-unexposed uninfected infants To the Editor: HIV-exposed uninfected (HEU) infants who undergo fetal development exposed to maternal HIV infection and its treatment might have immunologic and clinical consequences without being HIV infected. HIV and its proteins, which are known to inhibit progenitor cell function and cause progenitor cell apoptosis, cross the placental barrier, potentially leading to infant immunologic deficiency.1 Studies have demonstrated reduced numbers of CD4 T cells, increased numbers of CD8 T cells and CD19 B cells, and increased HIV-specific immune activation among HEU infants compared with HIV-unexposed and uninfected (HUU) infants.1-4 This study compared lymphocyte subsets between HEU and HUU infants in 2 US cohorts (the International Maternal Pediatric

Adolescent AIDS Clinical Trials Group protocol 1025 [P1025] and the Pediatric AIDS Clinical Trials Group protocol 1009 [P1009], respectively) from birth to 12 months of age. We also examined the effects of maternal and neonatal characteristics on levels of lymphocyte subsets among HEU infants. A total of 1338 HEU and 285 HUU infants with at least 1 CD4 or CD8 T-cell measurement were included in this study. Institutional review boards at the participating sites and the Harvard T.H. Chan School of Public Health approved both protocols. Parents provided written informed consent for their own and their children’s participation. Age was classified into categories as follows: 0 to 3, 3 to 6, and 6 to 12 months. HEU infants were more likely to be Hispanic than HUU infants (see Table E1 in this article’s Online Repository at www.jacionline. org). Lymphocyte subsets included absolute counts and percentage of CD4 and CD8 T cells and CD19 B cells. Lymphocyte subsets for P1025 HEU infants were abstracted from medical records;

FIG 1. Lowess curves (with 95% CIs) for change in distribution of lymphocyte subsets with age by HIV exposure status: A, CD4 count; B, CD4 percentage; C, CD8 count; D, CD8 percentage; E, CD19 B-cell count; and F, CD19 B-cell percentage.

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2 LETTER TO THE EDITOR

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TABLE I. Adjusted mean lymphocyte subset levels by HIV exposure status _3 mo Age 0-<

_6 mo Age >3-<

HEU HUU infants infants Lymphocyte (n 5 630) (n 5 102) subsets

CD4 count 2979.5 (cells/mm3) CD4 (%) 51.8 CD8 count 1078.0 (cells/mm3) CD8 (%) 18.7 CD19 B-cell 1108.1 count (cells/mm3) CD19 (%) 19.3

Age >6-12 mo Adjusted mean

Adjusted mean

Adjusted mean Adjusted mean difference

2954.0 25.5 (2196.0 to 247.1) 51.2 0.6 (21.3 to 2.5) 1079.7 21.7 (2116.9 to 113.5) 18.7 20.0 (21.4 to 1.3) 1080.2 27.9 (2138.7 to 194.5) 18.3 0.9 (21.1 to 3.0)

HEU HUU infants infants P value (n 5 786) (n 5 84)

.82

3226.5

.55

44.9

.98

1135.7

.96

15.7

.74

2114.7

.36

29.6

Adjusted mean difference

3015.2 211.3 (240.1to 462.8) 46.6 21.7 (23.5 to 0.1) 1108.0 27.7 (2103.1 to 158.5) 17.2 21.5 (22.8 to 20.2) 1671.0 443.7 (168.6to 718.7) 25.3 4.3 (1.9 to 6.8)

HEU HUU infants infants P value (n 5 643) (n 5 95)

.10

3235.8

.07

44.9

.68

1200.0

.02

16.6

.002 2156.2

<.001

29.2

Adjusted mean difference

P value

2909.2 326.5 .02 (61.4 to 591.6) 45.9 21.0 .29 (22.8 to 0.9) 1081.0 119.0 .11 (225.2 to 263.2) 17.3 20.7 .29 (22.0 to 0.6) 1511.2 645.0 <.001 (312.4to 977.5) 24.4 4.8 (2.6 to 7.1)

<.001

For infant age groups, 0 to 3 months was defined as birth to 91 days old, 3 to 6 months was defined as 92 to 182 days, and 6 to 12 months was defined as 183 to 364 days. Models were adjusted for continuous age, sex, and race/ethnicity. Subjects with race/ethnicity in the ‘‘other’’ group were excluded from models because of the extremely low prevalence: 0 to 3 months (n 5 10 [1%]), 3 to 6 months (n 5 23 [3%]), and 6 to 12 months (n 5 15 [2%]). HEU, HIV-exposed uninfected infants from P1025 enrolled between October 2002 and June 2013; HUU, HIV-unexposed uninfected infants from P1009 enrolled between July 2001 and January 2002.

those for P1009 HUU infants were measured as a study procedure in certified laboratories. The local clinical site laboratories using P1025 lymphocytes included 7 Pediatric AIDS Clinical Trials Group–certified immunology laboratories. Virtually all lymphocyte subset determinations were performed with 3-color flow analysis. Similar trends in lymphocyte subsets with age were observed for HEU and HUU infants (Fig 1). Absolute CD4 T-cell counts increased from birth until about 2 months of life and then remained stable through the end of follow-up at 12 months of age. A decreasing trend was observed for CD4 T-cell percentages up to about 3 months of age. There was less variation in absolute counts or percentages of CD8 T cells. Both absolute counts and percentages of CD19 B cells increased dramatically from birth to around 3 months of age and then stayed mostly stable or increased gradually. HEU infants consistently had higher CD19 B-cell percentages and counts than HUU infants through 12 months of age. Multivariable linear regression models were used to compare lymphocyte subsets between HEU and HUU infants within each age group, adjusting for infant age, sex, and race/ethnicity (Table I). Compared with HUU infants at the same age, HEU infants at the age of 6 and 12 months had higher absolute CD4 T-cell counts (adjusted mean, 3236 vs 2909 cells/mm3; P 5.02), HEU infants at the age of 3 to 6 months had slightly lower CD8 T-cell percentages (16% vs 17%, P 5 .02), and HEU infants at the age of 3 to 6 and 6 to 12 months had higher absolute counts and percentages of CD19 B cells (3-6 months: 2115 vs 1671 cells/mm3 [P 5 .002] and 30% vs 25% [P < .001]; 6-12 months: 2156 vs 1511 cells/ mm3 [P < .001] and 29% vs 24%, [P < .001]). There were no differences in CD4 T-cell percentages and absolute CD8 T-cell counts between HEU and HUU infants across all 3 age groups. Among HEU infants, higher measures of CD19 B cells were independently associated with older age (6-12 vs 0-3 months), zidovudine-only prophylaxis (vs zidovudine 1 other antiretroviral medication [ARV]), maternal alcohol use during pregnancy, last maternal CD4 T-cell count of 200 to 349 cells/mm3 (vs

> _350 cells/mm3), maternal Centers for Disease Control and Prevention Category C disease classification, and first reported use of maternal ARV in the third trimester (vs the first trimester, see Table E2 in this article’s Online Repository at www.jacionline. org). The increased CD4 T-cell and CD19 B-cell levels in our study could result from the HIV-specific immune response to transplacental exposure to HIV antigens. HIV-specific immune responses, including cytotoxic T-lymphocyte activity and the CD4 helper T-cell response have been observed among HEU infants.2,5-7 These HIV-specific immune responses could contribute to the protection of HEU infants from HIV infection.8 In one study focusing on the immunostimulatory effect of ARVs in HEU infants, increased CD154 (CD40 ligand) expression was found for both CD4 and CD8 T cells in ARV-exposed HEU infants compared with HUU infants, which persisted after 3 months.9 CD154 is expressed in activated T cells and provides a costimulatory signal for B-cell activation. The increased CD154 expression on TH cells in HEU infants could enhance activation and subsequent proliferation of B cells, resulting in observed increased CD19 B-cell counts. These activated B cells might help protect against HIV infection by facilitating antibodydependent cell-mediated cytotoxicity responses, as evidenced by increased numbers of natural killer cells in HEU infants.3 Additionally, mothers infected with HIV might have more systemic inflammation, inducing higher CD19 B-cell levels in HEU infants by affecting fetal cytokine profiles.10 Because no further phenotypic information other than the overall lymphocyte type was available for P1025 HEU infants, we could not discern whether the observed difference in immune profile for HEU infants compared with that in HUU infants was directly correlated to the HIV-specific cellular response or was simply an epiphenomena of HIV exposure. In conclusion, lymphocyte subsets in HEU infants differed from those of HUU infants. HEU infants had higher CD4 T-cell counts at 6 to 12 months of age and higher absolute numbers and percentages of CD19 B cells at 3 to 12 months of age compared

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with HUU infants. B cells could play a protective role in prevention of perinatal HIV transmission, which requires further research. We thank the families for their participation in International Maternal Pediatric Adolescent AIDS Clinical Trials Protocol 1025 and the Pediatric AIDS Clinical Trials Group Protocol 1009 and the subjects and institutions involved in the conduct of the studies. A full list of the study team and participating sites and site personnel is available in this article’s Online Repository at www.jacionline.org. Yanling Huo, MSa Kunjal Patel, DSc, MPHa Gwendolyn B. Scott, MDb Russell B. Van Dyke, MDc George K. Siberry, MDd Sandra K. Burchett, MD, MSe William T. Shearer, MD, PhDf From aCenter for Biostatistics in AIDS Research, Harvard T.H. Chan School of Public Health, Boston, Mass; bthe Division of Pediatric Infectious Disease and Immunology, University of Miami Miller School of Medicine, Miami, Fla; cthe Department of Pediatrics, Tulane University School of Medicine, New Orleans, La; dEunice Kennedy Shriver National Institute of Child Health and Human Development, National Institutes of Health, Bethesda, Md; ethe Division of Infectious Diseases, Boston Children’s Hospital and Harvard Medical School, Boston, Mass; and fthe Department of Immunology Allergy Rheumatology, Baylor College of Medicine/Texas Children’s Hospital, Houston, Tex. E-mail: [email protected]. Overall support for the International Maternal Pediatric Adolescent AIDS Clinical Trials Network (IMPAACT) was provided by the National Institute of Allergy and Infectious Diseases (NIAID) of the National Institutes of Health (NIH) under award nos. UM1AI068632 (IMPAACT LOC), UM1AI068616 (IMPAACT SDMC), and UM1AI106716 (IMPAACT LC), with cofunding from the Eunice Kennedy Shriver National Institute of Child Health and Human Development (NICHD) and the National Institute of Mental Health (NIMH). The Baylor College of Medicine– University of Texas Center for AIDS Research provided support for P1009 (AI36211). The content is solely the responsibility of the authors and does not necessarily represent the official views of the NIH. Disclosure of potential conflict of interest: G. B. Scott receives grant support from the National Institutes of Health (NIH), Merck, Abbvie, GlaxoSmithKline, and Jassen

LETTER TO THE EDITOR 3

and travel support from Merck, Abbvie, GlaxoSmithKline, and Janssen. R. B. Van Dyke receives grant and travel support from the NIH. Y. Huo and K. Patel receive grant and travel support from IMPAACT. The rest of the authors declare that they have no relevant conflicts of interest.

REFERENCES 1. Nielsen SD, Jeppesen DL, Kolte L, Clark DR, Sorensen TU, Dreves AM, et al. Impaired progenitor cell function in HIV-negative infants of HIV-positive mothers results in decreased thymic output and low CD4 counts. Blood 2001;98:398-404. 2. Clerici M, Saresalla M, Colombo F, Fossati S, Sala N, Bricalli D, et al. T-lymphocyte maturation abnormalities in uninfected newborns and children with vertical exposure to HIV. Blood 2000;96:3866-71. 3. Ono E, Nunes dos Santos AM, de Menezes Succi RC, Machado DM, de Angelis DSA, Salomao R, et al. Imbalance of naive and memory T lymphocytes with sustained high cellular activation during the first year of life from uninfected children born to HIV-1-infected mothers on HAART. Braz J Med Biol Res 2008;41:700-8. 4. Borger-Almeida E, Milanez HM, Vilela MMS, Cunha FGP, Abramczuk BM, Reis-Alves SC, et al. The impact of maternal HIV infection on cord blood lymphocyte subsets and cytokine profile in exposed non-infected newborns. BMC Infect Dis 2011;11:38. 5. Kuhn L, Coutsoudis A, Moodley D, Trabattoni D, Mngqundaniso N, Shearer GM, et al. T-helper cell responses to HIV envelope peptides in cord blood: protection against intrapartum and breastfeeding transmission. AIDS 2001;15:1-9. 6. Legrand FA, Nixon DF, Loo CP, Ono E, Chapman JM, Miyamoto M, et al. Strong HIV-1-specific T cell responses in HIV-1-exposed uninfected infants and neonates revealed after regulatory T cell removal. PLoS One 2006;1:e102. 7. John-Stewart GC, Mbori-Ngacha D, Payne BL, Farquhar C, Richardson BA, Emery S, et al. HIV-1-specific cytotoxic T lymphocytes and breast milk HIV-1 transmission. J Infect Dis 2009;199:889-98. 8. Afran L, Knight MG, Nduati E, Urban BC, Heyderman RS, Rowland-Jones SL. HIV-exposed uninfected children: a growing population with a vulnerable immune system? Clin Exp Immunol 2014;176:11-22. 9. Romano MF, Buffolano W, Bisogni R, Russo R, Liuzzi R, Bunders M, et al. Increased CD154 expression in uninfected infants born to HIV-positive mothers exposed to antiretroviral prophylaxis. Viral Immunol 2006;19:363-72. 10. Bunders M, Pembrey L, Kuijpers T, Newell ML. Evidence of impact of maternal HIV infection on immunoglobulin levels in HIV-exposed uninfected children. AIDS Res Hum Retroviruses 2010;26:967-75. http://dx.doi.org/10.1016/j.jaci.2017.01.033

3.e1 LETTER TO THE EDITOR

LIST OF P1025 TEAM MEMBERS, PARTICIPATING SITES, AND PERSONNEL P1025 Team: Gwendolyn B. Scott, MD, University of Miami School of Medicine, Miami, Florida; Elizabeth Smith, MD, National Institute of Allergy and Infectious Diseases Division of AIDS, Pediatric Medicine Branch, Bethesda, Maryland; Heather Watts, MD, National Institute of Child Health and Human Development, Pediatric, Adolescent, and Maternal AIDS (PAMA) Branch, Bethesda, Maryland; KaSaundra M. Oden, MHS, International Maternal Pediatric Adolescent AIDS Clinical Trials Group, Silver Spring, Maryland; Yanling Huo, MS, Harvard School of Public Health, Boston, Massachusetts; Kunjal Patel, DSc, MPH, Harvard School of Public Health, Boston, Massachusetts; Emily A. Barr, CPNP, CNM, MSN, University of Colorado Denver, The Children’s Hospital, Denver, Colorado; Diane W. Wara, MD, University of California at San Francisco, San Francisco, California; Sandra K. Burchett, MD, MSc, Boston Children’s Hospital and Harvard Medical School, Boston, Massachusetts; Jenny Gutierrez, MD, Bronx-Lebanon Hospital, Bronx, New York; Kathleen Malee, PhD, Ann and Robert H. Lurie Children’s Hospital of Chicago, Chicago, Illinois; Patricia Tanjutco, MD, Washington Hospital Center, Washington, DC; Yvonne Bryson, MD, David Geffen School of Medicine, University of California, Los Angeles, California; Michael T. Basar, BS, Frontier Science & Technology Research Foundation, Amherst, New York; Adriane Hernandez, MA, Frontier Science & Technology Research Foundation, Amherst, New York; Amy Jennings, BS, Frontier Science & Technology Research Foundation, Amherst, New York; Tim R. Cressey, PhD, BSc, Program for HIV Prevention & Treatment, Chang Mai, Thailand; Jennifer Bryant, MPA, Westat, Rockville, Maryland. Participating sites and site personnel: 5041 Children’s Hospital of Michigan NICHD CRS (Theodore B. Jones, MD; Ernestine Brown, RN; Natalie Woods, RD); 5052 University of Colorado Denver NICHD CRS (Alisa Katai, MHA; Tara Kennedy, FNP-BC; Kay Kinzie, MSN, FNP-BC; Jenna Wallace, MSW; CTSI grant no. UL1 TR000154); 5031 San Juan City Hospital PR NICHD CRS (Rodrigo Diaz-Velasco, MD, FACOG, AAHIVS; Midnela Acevedo-Flores, MD, MT; Elvia Perez-Hernandez, BS, MEd, MA, MPH; Antonio RodriguezMimoso, MD; FACOG); 5048 USC LA NICHD CRS (Franc¸oise Kramer, MD; LaShonda Spencer, MD; James Homans, MD; Andrea Kovacs, MD); 4601 UCSD Maternal, Child, and Adolescent HIV CRS (Andrew Hull, MD; Mary Caffery, RN, MSN; Jean M. Manning RN, BSN; Stephen A. Spector, MD); 4101 Columbia IMPAACT CRS; 4201 University of Miami Pediatric Perinatal HIV/AIDS CRS (Charles D. Mitchell, MD;

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Salih Yasin, MD; Safia Khan, MD); 5083 Rush University Cook County Hospital Chicago NICHD CRS (Mariam Aziz, MD; Latania Logan, MD; Julie Schmidt, MD; Helen Cejtin, MD); 5096 University of Alabama Birmingham NICHD CRS (Marilyn Crain, MPH, MD; Sharan Robbins, BA; Mickey Parks, CRNP; Yvonne Gamble Duke, MA); 6901 Bronx-Lebanon Hospital IMPAACT CRS (Murli Purswani, MD; Stefan Hagmann, MD, MSc, FAAP; Mary Vachon, LMSW, MPH); 5012 NYU School of Medicine NICHD CRS (William Borkowsky, MD; Maryam Minter, RN; Aditya Kaul, MD; Nagamah Deygoo, MS); 3801 Texas Children’s Hospital CRS (Shelley Buschur, RN, NMV; Kathleen Pitts, CPNP; Chivon McMullen-Jackson, BSN, RN; Theresa Aldape, LMSW; grant no. AI069441); 4001 Chicago Children’s CRS (Donna McGregor, RN); 5009 Children’s Hospital of Boston NICHD CRS (Arlene Buck, RN; Catherine Kneut, RN, CPNP); 5018 USF-Tampa NICHD CRS (Patricia Emmanuel, MD; Karen Bruder, MD; Gail Lewis, RN); 6501 St Jude/UTHSC CRS (Katherine Knapp, MD; Edwin Thorpe, MD; Nina Sublette, FNP, PhD; Pam Finnie, MSN); 2802 NJ Medical School CRS (Charmane CalilapBernardo, RN; Linda Bettica, RN); 3601 UCLA-Los Angeles/ Brazil AIDS Consortium (LABAC) CRS (Jaime G. Deville, MD; Karin Nielsen-Saines, MD; Nicole Falgout, RN; Michele Carter, RN); 4005 Mt Sinai Hospital Med Center, Women’s & Children’s HIV Program (Brenda Wolfe, APN; Molly Hartrich, MPH); 5017 Seattle Children’s Hospital CRS; 5023 Washington Hospital Center NICHD CRS (Steven Zeichner, MD, PhD; Sara R. Parker, MD; Vanessa Emmanuel, BA); 5028 University of Illinois College of Medicine at Chicago, Department of Pediatrics (Kenneth Rich, MD; Karen Hayani, MD; Julia Camacho, RN); 5051 University of Florida College of Medicine, Jacksonville (Mobeen Rathore, MD; Ayesha Mirza, MD; Nizar Maraqa, MD; Kathleen Thoma, MA, CCRP); 5094 University of Maryland Baltimore NICHD CRS (Douglas Watson, MD; Corinda Hilyard); 6601 University of Puerto Rico Pediatric HIV/AIDS Research Program CRS (Irma L. Febo, MD; Vivian Tamayo, MD; Ruth Santos, RN, MPH; Maritza CruzRodriguez); 5003 Metropolitan Hospital NICHD CRS; 5013 Jacobi Medical Center Bronx NICHD CRS (Susan Gross, MD; Michael Moore, MD; Carmen Caines, RN); 5038 Yale University School of Medicine; 5045 Harbor UCLA Medical Center NICHD CRS (Margaret Keller, MD; Spring Wettgen, RN, PNP; Judy Hayes, RN; Yolanda Gonzalez, RN); 5095 Tulane University New Orleans NICHD CRS (Yvette Luster, RN; Robert Maupin, MD; Chi Dola, MD; Margarita Silio, MD); 6701 The Children’s Hospital of Philadelphia IMPAACT CRS(Steven D. Douglas, MD; Richard M. Rutstein, MD; Carol A. Vincent, CRNP, MSN).

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TABLE E1. Distributions of background characteristics by study Characteristics

Male sex Race/ethnicity White non-Hispanic Black non-Hispanic Hispanic (regardless of race) Other Unknown Black race Hispanic Preterm (<37 weeks’ gestation) Low birth weight (<2500 g) Gestational age (wk) Mean (SD) Median (IQR) Neonatal ARV prophylaxis No ARV ZDV 1 other ZDV only Maternal age (y) at delivery Mean (SD) Median (IQR) _35 y Maternal age at delivery > Last maternal CD4 cell count (cells/mm3) at/before delivery <200 200-349 > _350 Last maternal RNA at/before delivery (copies/mL) >1000 401-1000 < _400 Last maternal CDC classification during pregnancy: category C Used cigarettes during pregnancy Used alcohol during pregnancy Used illicit drugs during pregnancy Maternal ARV exposure during pregnancy cARV Non-cARV No ARV exposure Most intensive maternal ARV exposure during pregnancy PI-containing cARV NNRTI-containing cARV Other cARV regimen Non-cARV regimen No ARV exposure Trimester of first reported ARV use 1st 2nd 3rd No ARV exposure Duration (wk) of ARV use during pregnancy Mean (SD) Median (IQR)

HEU infants (n 5 1338)

HUU infants (n 5 285)*

656 (49%)

146 (51%)

80 671 542 29 16 727 542 253 207

41 160 80 4

(6%) (50%) (41%) (2%) (1%) (54%) (41%) (19%) (15%)

(14%) (56%) (28%) (1%)

38.0 (2.0) 38.1 (37.3-39.1) 1 (0%) 148 (11%) 1,189 (89%) 27.9 (6.0) 28 (23-32) 215 (16%) 116 (9%) 248 (19%) 943 (70%) 164 59 1084 131 185 306 159

(12%) (4%) (81%) (10%) (14%) (23%) (12%)

1308 (98%) 16 (1%) 10 (1%) 1,018 125 165 16 10

(76%) (9%) (12%) (1%) (1%)

693 535 96 10

(52%) (40%) (7%) (1%)

25.2 (10.6) 24.0 (17.7-36.7)

Missing data among the overall sample: race (8%), ethnicity (1%), low birth weight (4%); last maternal CD4 measurement (2%), last maternal RNA measurement (2%), last maternal Centers for Disease Control and Prevention category classification (1%), used cigarettes during pregnancy (30%), used alcohol during pregnancy (10%), used illicit drugs during pregnancy (28%), and maternal ARV exposure during pregnancy (0.3%). cARV, Combination ARV regimen containing 3 or more drugs; CDC, Centers for Disease Control and Prevention; IQR, interquartile range; NNRTI, non-nucleoside reverse transcriptase inhibitor; PI, protease inhibitor; ZDV, zidovudine. *Only limited demographic characteristics were obtained in the P1009 study.

CD4 counts

Covariates

Estimated difference* (95% CI)

CD4 (%) P value

Estimated difference* (95% CI)

CD8 counts P value

Estimated difference* (95% CI)

CD8 (%) P value

Estimated difference* (95% CI)

CD19 counts P value

Estimated difference* (95% CI)

CD19 (%) P value

Estimated difference* (95% CI)

Age at the time of measuring lymphocyte subsets (reference: >6-12 mo) _3 mo 0-< 2205.3 (2309.5 <.001 6.7 (6.0to 7.5) <.001 276.7 (2135.2 .01 2.6 (2.0 to 3.3) <.001 2839.1 (21091.1 <.001 28.8 (210.4 to 2101.0) to 218.1) to 2587.1) to 27.2) _6 mo >3-< 225.9 (2120.8 .59 0.5 (20.1 to 1.1) .08 296.4 (2153.7 <.001 21.0 (21.6 <.001 249.1 (2233.7 .60 0.5 (20.7 to 69.0) to 239.1) to 20.4) to 135.5) to 1.6) .001 2213.4 (2275.9 <.001 21.3 (21.9 <.001 Race: Black 2437.1 (2559.5 <.001 21.5 (22.4 to 20.6) to 2150.9) to 20.7) to 2314.7) Sex: Male 2136.4 (2248.2 .02 21.2 (22.0 to 20.4) .01 0.9 (0.4 to 1.5) .001 to 224.7) Gestational age (wk): per 33.9 (5.7 to 62.1) .02 0.2 (0.0 to 0.5) .02 23.7 (11.3 to 36.2) <.001 0.2 (0.0 to 0.3) .02 1-wk increase Neonatal ARV prophylaxis: 2202.3 (2392.0 .04 ZDV 1 other (reference: to 212.6) ZDV only) Used alcohol during pregnancy 2.5 (0.9to 4.1) _350 cells/mm3]) Last maternal CD4 counts during pregnancy (cells/mm3 [reference > <200 2252.1 (2439.4 .01 22.8 (24.3 to 21.2) <.001 2.9 (20.1 to 5.8) to 264.8) 200-349 2196.2 (2335.7 .01 21.6 (22.6 to 20.5) .004 2.1 (0.2to 3.9) to 256.8) Last maternal CDC classification 461.2 (102.4 .01 2.8 (0.0to 5.6) during pregnancy: Category C to 820.0) Trimester of first reported ARV use (reference: 1st trimester) Second trimester 2108.7 (2230.6 .08 21.1 (22.0 to 20.2) .02 0.3 (20.3to 1.0) .30 125.8 (242.7 .14 1.4 (20.1 to 2.8) to 13.2) to 294.4) Third trimester 2205.7 (2427.0 .07 21.6 (23.4 to 0.2) .07 1.4 (0.3 to 2.5) .02 436.3 (117.0 .01 4.3 (1.1to 7.4) to 15.7) to 755.6)

P value

3.e3 LETTER TO THE EDITOR

TABLE E2. Adjusted associations of maternal and neonatal characteristics with lymphocyte subsets among P1025 HEU infants

<.001 .45

.002 .06 .03 .05

.07 .01

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Covariates with a P value of less than .1 from univariable analysis were kept in a multivariable model, which was further reduced, containing only covariates with P values of less than .1 as the final regression model. An indicator of missing data was created and included in multivariable analysis: race (8%) and alcohol use during pregnancy (9%). Covariates with greater than 20% missing data were excluded from multivariable analysis: cigarettes (29%) and illicit drug use (27%) during pregnancy. Infants who did not receive any neonatal ARV prophylaxis (n 5 1) or whose mother did not receive any ARV during pregnancy (n 5 10 for CD4 and CD8 analyses; n 52 for CD19 analyses) were excluded from multivariable analyses. CDC, Centers for Disease Control and Prevention; ZDV, zidovudine. *Estimated difference in adjusted mean level of lymphocyte subset between participants with versus without a specific characteristic.