Vertical Cytomegalovirus Transmission From HIV-Infected Women Randomized to Formula-Feed or Breastfeed Their Infants

Background. Cytomegalovirus (CMV) is associated with morbidity and mortality in human immunodeficiency virus (HIV)-exposed infants. We assessed the effect of and relative contribution of breastfeeding to CMV acquisition among infants delivered by HIV-infected mothers. Methods. Between 1993 and 1998 pregnant, HIV-infected women in Nairobi, Kenya, were randomly assigned to breastfeed or formula-feed their infants in an HIV transmission study. Women were allocated equally between treatment arms, and the study was not blinded. The primary endpoint of this nested study was time to infant CMV infection. Results. CMV infection was assessed in 138 breastfed and 134 formula-fed infants. Baseline characteristics were similar between arms. Breastfed infants acquired CMV earlier than formula-fed infants (median age of acquisition, 4.26 vs 9.87 months; P <. 001) and had a higher 1-year probability of CMV infection (0.89 vs 0.69; P <. 001). Breastfeeding was associated with a 1.6-fold increased risk of infant CMV acquisition independent of infant HIV status (multivariable hazard ratio, 1.61; 95% confidence interval, 1.20-2.16; P =. 002). Approximately one third of CMV infections occurred during the peripartum period, with 40% acquired through breastfeeding and the remainder acquired through modes other than breast milk. Conclusions. Preventing CMV acquisition may be a priority for HIV-exposed infants, but there is a narrow window of opportunity for intervention. Approaches that reduce maternal cervical and breast milk CMV reactivation may help delay infant infection. The study was approved by the Institutional Review Board of the University of Washington and the Ethics and Research Committee of Kenyatta National Hospital. All participants provided written informed consent for participation, and human experimentation guidelines of the University of Washington were followed. This is a retrospective analysis of specimens collected during a randomized controlled trial of breastfeeding versus formula feeding and HIV transmission, conducted in Nairobi, Kenya, between 1993 and 1998 [22]. We assumed the rate of CMV acquisition in the breastfeeding arm would be similar to that in our previous observational study in Kenya, which was 90% by 6 months of age [9]. With equal allocation to study arms and estimating 300 infants would meet eligibility criteria, the study was powered to detect a 30% difference in transmission between arms, using Cox regression with β = 0.80 and α = 0.05, assuming a 2-sided test. Eligibility criteria for the randomized controlled trial included pregnancy of <32 weeks gestation, willingness to have feeding modality randomly assigned, access to clean municipal water, and intent to reside in Nairobi for >2 years. For the purpose of this substudy, we included all infants who had dried blood spot (DBS) specimens collected before 14 weeks of age. This trial was conducted before antiretroviral therapy (ART) for prevention of mother-to-child HIV transmission (PMTCT) became available in Kenya; all women and infants were ART naive. At enrollment, caregivers provided sociodemographic characteristics and medical histories and underwent a clinical examination. At approximately week 32 of gestation, women were randomly assigned to the breastfeeding or formula-feeding arm at a 1:1 ratio, using computer-generated block randomization. Treatment allocation was not blinded to study staff; assignment to study arm was revealed to women and clinicians via presealed envelopes. In the formula group, safe formula preparation was demonstrated during home visits, and women were provided with free dried milk formula throughout the study. Women and infants were assessed in clinic monthly in the first year and quarterly during the second year of the infant’s life and as needed for illness. Postnatal visits included assessment of both mother’s and baby’s clinical status, as well as infant growth and feeding. Infant blood specimens were collected at birth, at 6 weeks of age, at 14 weeks of age, and every 3 months thereafter until 24 months of age for HIV testing [22]. Women delivering at home or another facility were asked to bring their newborns to the study clinic for clinical examination and HIV testing as soon as possible following delivery. Infant HIV diagnosis was conducted in this study using polymerase chain reaction (PCR) to detect HIV gag DNA in peripheral blood mononuclear cells or DBS as previously described [22, 23]. Due to the high incidence of infant HIV infection in the breast fed arm, in April 1998 the data safety and monitoring board (DSMB) closed the study early, and recommended that breast feeding women be advised to stop breast feeding and be given formula [22]. All CMV loads were measured in DBS as previously described [21]; plasma specimens from this study have been extensively used in vertical transmission studies over the past decade and were no longer were available for longitudinal CMV assessments. In our assay, when CMV loads are >300 copies/mL in plasma, the 2 assays are comparable for CMV detection, although they return lower CMV load measurements (Atkinson, unpublished data). CMV loads were measured in all specimens for each infant up to 12 months of age. The lower limit of detection was 100 copies/mL. Infants with no detection of CMV DNA throughout follow-up underwent serologic testing of their last-collected plasma specimen closest to 12 months of age, to determine final infection status; only plasma specimens collected beyond 6 months of age were included for serologic analysis, to avoid confounding by maternal antibodies. Enzyme-linked immunosorbent assay (ELISA) was used to detect a panel of antibodies targeting CMV immunoglobulin G (CMV ELISA II test kit Wampole Laboratories, Princeton, New Jersey). All analyses were intent-to-treat and were performed using SPSS, version 20.0 (IBM). Baseline characteristics for infants included in the CMV analyses were compared between randomization arms using Mann–Whitney U tests (for continuous variables) and χ2 tests (for categorical variables). HIV-exposed uninfected infants were defined as infants with their last CMV test at or after their last HIV-negative test and who never had HIV infection diagnosed. The proportion of infants infected with CMV at birth were compared between arms using Fisher’s exact test; the proportion with CMV DNA detection and the proportion with CMV antibody detection were compared between arms using Pearson χ2 tests. Time to CMV infection was compared between arms using Kaplan–Meier curves and the log-rank test. z tests were used to compare median time to CMV infection and the probability of CMV infection at 1 year. Cox proportional hazards models were used to assess the independent effects of time-varying HIV infection status and feeding modality, as well as any potential effect modification of these 2 variables on CMV acquisition. In addition to the intent-to-treat analyses described above, all analyses were performed categorizing infants as breastfed or formula fed, based on the mother’s self-reported feeding history. For these analyses, infants whose mothers ever reported breastfeeding were classified as breastfed, and infants whose mothers never reported breastfeeding were classified as formula fed. Estimates of the proportion of CMV infections attributable to different modes of transmission in the first year of life were obtained by comparing the proportions of infants with CMV DNA detection in the breastfed and formula-fed infants at defined time points. Because of interval sampling at delivery and 6 weeks of age, we were unable to discriminate in utero from intrapartum infections; we thus combined infections detected at ≤6 weeks of age as “peripartum” transmissions, which are presumed to primarily include in utero and intrapartum and potentially very early breast milk or saliva transmissions. Infections due to saliva and/or urine exposure were defined as those infections occurring after 6 weeks of age in the formula-fed infants, and infections due to breast milk exposure were defined as the excess infections between the breastfed and formula-fed infants at 1 year of age. Because 30% of the women randomly assigned to the formula-feeding arm elected to breastfeed [22], this analysis was conducted as as-treated.