Cohort characteristics and follow up
Of 280 breastfeeding women who provided a breast milk sample at the month 1 postpartum visit, 20 women were excluded from the analysis because their infants had positive HIV-1 DNA or RNA assays <48 h after delivery. Thus, 260 women whose infants were HIV-1 uninfected at birth formed the study population (). Women had a median age of 24 years (range 18–39), 215 (83%) were in monogamous marital relationships, and the median duration of the current partnership was 3 years (range 1–23 years). Mean duration of AZT use prior to delivery was 29 days, and 227 (87%) women used AZT antenatally for 14 days or more. Two hundred and fifteen (83%) women had spontaneous vaginal deliveries, 45 (17%) delivered by cesarean section, with only 8 (18%) among these performed prior to the onset of labor. Twenty-seven (10%) women were diagnosed as having clinical mastitis between delivery and month 1, and 92 (37%) had subclinical mastitis at the month 1 visit.
Study population. 1Shading indicates population of interest. 2Infants with positive HIV-1 DNA or RNA assays at <48 h were considered infected in utero and defined as being HIV-1 infected at birth.
Forty (15%) of the 260 infants acquired HIV-1 over the course of follow-up. Twenty-nine (73%) became infected between birth and 1 month of age, and 11 (27%) became infected between the ages of 1 and 12 months. Twenty-four (9%) infants died during follow-up with 13 (54%) among these being HIV-1 infected and 11 (46%) HIV-1 uninfected. Excluding infants who died during follow-up, the mean duration of follow-up was 10 months and 173 (73%) were followed up to the age of 12 months.
Maternal immune status and viral load
At 1 month postpartum, mean maternal CD4+ T cell count was 600 cells/µl and mean maternal CD4 percent was 24%. Thirty-one (14%) women were severely immunosuppressed with CD4 count <200 and/or CD4 percent less than 15. Virus was undetectable in the breast milk of 35 (13%) women and plasma and breast milk viral loads were positively correlated (R = 0.6; p < 0.0001). Mean maternal plasma viral load was 4.7 log10 copies/ml and mean breast milk viral load was 2.9 log10 copies/ml among women with detectable breast milk virus.
Correlates of α-defensins in breast milk
We detected α-defensins in breast milk specimens from 108 (42%) of the 260 women. Among women with detectable α-defensins (≥41 pg/ml), the median α-defensins concentration was 320 pg/ml (range 44–9992 pg/ml) and 13 (12%) women had α-defensins levels >2500 pg/ml.
Levels of breast milk α-defensins were positively correlated with breast milk HIV-1 RNA concentrations (p < 0.001). For every one log increase in α-defensins, breast milk viral load increased 0.2-fold (). Other correlates of elevated α-defensins in breast milk were subclinical mastitis, lower parity, and higher CD4 percent (). In multivariate analysis adjusting for HIV-1 RNA in breast milk, subclinical mastitis, lower parity, and higher CD4 percent remained independently associated with α-defensins levels (p = 0.006, p = 0.01, and p < 0.001, respectively) ().
Correlates of α-Defensins in Month 1 Breast Milk Specimens Obtained from HIV-1-Infected Mothers
When women with detectable breast milk α-defensins were compared with women in whom defensins were not detected, we found higher mean breast milk HIV-1 RNA among those with detectable versus those with undetectable levels of α-defensins (2.9 log10 copies/ml versus 2.5 log10 copies/ml, p = 0.003) (). CC chemokines (MIP-1α, MIP-1β, and RANTES) and the CXC chemokine (SDF-1) were also significantly elevated in breast milk specimens with detectable α-defensins (). The strongest associations were found between α-defensins and the CC chemokines, especially MIP-1α and MIP-1β ().
Comparison between Women with Detectable and Undetectable α-Defensins Levels at 1 Month Postpartum
HIV-1 transmission risk and breast milk α-defensins
Both maternal breast milk HIV-1 RNA and plasma viral load were independently associated with HIV-1 transmission to infants after birth (p < 0.01 for both) (Farquhar et al., in preparation). Thus, in our analysis of the role of α-defensins in breast milk HIV-1 transmission, we adjusted for breast milk HIV-1 viral load. We did not observe a significantly protective effect of α-defensins against infant HIV-1 acquisition (HR 0.7; 95% CI 0.5–1.2; p = 0.2). However, we did observe that when women with detectable α-defensins were compared with women with undetectable α-defensins, the effect of breast milk HIV-1 on transmission risk was attenuated in women with detectable α-defensins. The hazard of infection increased more than 2-fold for every one log increase in breast milk viral load for women with undetectable α-defensins (HR 2.9; 95% CI 1.9–4.4; p < 0.001), whereas for women with detectable α-defensins the change in infection risk for each one log increase in breast milk HIV-1 RNA was less but remained significant (HR 1.7; 95% CI 1.06–2.8; p = 0.03). There was no difference in breast milk α-defensin levels for infants infected early versus those infected after the first month.