Between November 1997 and January 2000, mothers and their newborn infants at 14 maternity clinics and hospitals in greater Harare, Zimbabwe, were enrolled within 96 hours of delivery.11 12
Mother-infant pairs were eligible if the infant was a singleton with birth weight greater than or equal to 1500 g, the mother planned to stay in Harare after delivery, and neither mother nor infant had an acutely life threatening condition. Mothers provided written informed consent. Detailed description of eligibility and consent rates has been previously published.15
Baseline characteristics were collected by questionnaire or from hospital records. Maternal blood and breast milk, and infant blood, were collected at baseline. Aliquots of maternal plasma and breast milk supernatant, and infant plasma and cell pellets (Amplicor whole blood polymerase chain reaction (PCR) sample preparation method; Roche Diagnostic Systems, Alameda, CA), were archived at −70 °C. Baseline maternal HIV status was determined by two enzyme linked immunosorbent assays (ELISAs) run in parallel; discordant ELISA results were repeated and then resolved by western blot if necessary.
Follow-up was conducted at six weeks, three months, and then three monthly for 12 to 24 months. For all HIV positive mothers and their infants, and a representative subsample of HIV negative women and their infants (52% of total), maternal blood and breast milk, and infant blood, were collected and archived at all visits. For the remaining 48% of women who were HIV negative at baseline and their infants, maternal samples were archived at six and 12 months, and infant blood at 12 months. Women who were HIV negative at baseline were retested for HIV at all subsequent blood collections. Cell pellets were archived at all time points for infants of HIV positive mothers and at the time of the first positive maternal ELISA and at all subsequent infant blood collections for infants whose mothers seroconverted postnatally.
Among women who seroconverted during follow-up but had a last negative ELISA test less than three months post partum, late prenatal infections could not be distinguished from early postnatal infections on the basis of ELISA results because the interval between infection and seroconversion can be up to three months. This group included women who tested negative with ELISA at baseline and then either tested positive with ELISA at six weeks or did not provide blood samples before testing positive at three months or later. To further characterise timing of infection for these women, archived baseline plasma samples were assayed for HIV load by quantitative HIV RNA testing (Roche Amplicor HIV-1 Monitor test, version 1.5; Roche Diagnostic Systems).
HIV testing for exposed infants was conducted after all patient contact was completed. In the infants of mothers who tested HIV positive at baseline, the last available infant specimen was tested by ELISA for plasma samples collected at 18 months or later (GeneScreen HIV 1/2; Sanofi Diagnostics Pasteur, Johannesburg, South Africa) or by PCR assay for cell pellets collected at less than 18 months (Roche Amplicor HIV-1 Monitor test, version 1.5). If negative, the infant was classified as uninfected; if positive, samples collected at earlier time points were tested to establish the timing of infection. All available specimens from infants of women who seroconverted were tested for HIV by PCR.
For mothers who seroconverted, breast milk supernatant and plasma samples collected at the time of the mother’s last negative ELISA test and at all subsequent time points were tested for HIV load using the ultrasensitive (milk) and standard (plasma) methods (detection limits 50 and 400 copies/mL, respectively). Plasma and breast milk HIV load was also measured for a representative sample of women who tested HIV positive at baseline.
Infant feeding histories were ascertained and breastfeeding exclusivity defined as previously described.12
Throughout the trial, medical care, counselling,16 17
and condoms were freely provided. Education and counselling of HIV negative women included HIV prevention strategies and the message that maternal seroconversion during breast feeding is associated with an increased risk of breastfeeding associated transmission. Antenatal HIV testing, antiretroviral prophylaxis for prevention of mother to child transmission, and highly active antiretroviral therapy were not available in public sector facilities during the time of the trial.
Women who ever tested HIV positive and their infants were classified into four groups according to the risk period for mother to child transmission:
- 1 Baseline positive: mother tested HIV positive at baseline with ELISA or western blot (mother infected before delivery; infant at risk of intrauterine, intrapartum, and breastfeeding associated transmission);
- 2 Baseline positive and at risk of breastfeeding associated transmission (baseline positive at risk of BFT): mother tested HIV positive with ELISA or western blot at baseline and her infant tested negative with PCR at six weeks (mother infected before delivery; infant escaped intrauterine and intrapartum infection but at risk of breastfeeding associated transmission);
- 3 Postnatal seroconverter: mother tested HIV negative with ELISA or western blot at three months postpartum or later or her baseline plasma had undetectable HIV RNA, and mother tested positive with ELISA or western blot at a subsequent visit (mother seroconverted postnatally; infant at risk of breastfeeding associated transmission after maternal infection while still breast feeding);
- 4 Baseline seroconverter: mother’s baseline plasma sample tested HIV negative with ELISA or western blot but the same sample positive with RNA PCR (mother infected during late pregnancy and was seroconverting during delivery; infant at risk of late intrauterine, intrapartum, and breastfeeding associated transmission).
We compared mother to child transmission rates for seroconverting women with women who had tested HIV positive at baseline and whose infants were at risk of HIV infection during the same periods. Breastfeeding associated transmission rates were compared between the baseline positive at risk of BFT group and the postnatal seroconverter group because the infants in both groups were at risk of breastfeeding associated transmission only. Total transmission rates were compared between the baseline seroconverter group and the baseline positive group because these women’s infants were at risk of intrauterine, intrapartum, and breastfeeding associated transmission. Baseline characteristics were compared between the baseline positive at risk of BFT group and the postnatal seroconverter group, and between the baseline positive group and the baseline seroconverter group. Differences were tested by Kruskal-Wallis tests or χ2 tests, as appropriate.
Kaplan-Meier methods were used to estimate breastfeeding associated transmission rates for baseline positive at risk of BFT pairs and postnatal seroconverter pairs, with timelines beginning at six weeks post partum for baseline positive at risk of BFT mothers and at maternal HIV infection (midpoint between last negative and first positive tests) for postnatal seroconverters. Among women in the postnatal seroconverter group, the median number of days (interquartile range) between the mother’s last negative and first positive ELISA tests (hereafter termed “seroconversion interval”) was 181 (range 92 to 323) days. Thus, for some mothers, our estimated time of infection was relatively imprecise. To more closely examine the temporal relation between maternal infection and breastfeeding associated transmission, we repeated the analysis restricting postnatal seroconverter mothers to those with seroconversion intervals of less than or equal to 90 days (hereafter termed “short seroconversion interval”). Kaplan-Meier methods were also used to estimate and compare rates of total transmission and total transmission or death for infants of baseline positive and baseline seroconverter mothers.
In all Kaplan-Meier analyses, infants who never had a positive HIV test were censored at the time of their last negative result, 60 days after breastfeeding cessation or their mother’s date of death, or at 24 months of age, whichever occurred first. Differences were tested using the log rank test. Smoothed hazard functions were calculated using an Epanechnikov kernel function in Stata.
We calculated rates of breastfeeding associated transmission per 100 child years of breast feeding for three month intervals over the first 12 months of HIV exposure. These intervals represented time after six weeks of age for infants of baseline positive at risk of BFT women and time after maternal HIV infection for infants of postnatal seroconverter women. We examined whether infant age at the time of maternal infection modified the effect of maternal seroconversion on breastfeeding associated transmission, first by Poisson regression and then with a Cox model that included child age at maternal primary infection stratified in three month intervals; transmissions per 100 child years of breastfeeding exposure were calculated for each stratum.
Concentrations of breast milk and plasma HIV load were log10 transformed. Among baseline positive at risk of BFT women, we calculated the proportions of plasma and breast milk samples with detectable virus loads, and the median (intra(?er?)quartile range) HIV load among detectable samples for each specimen type at each visit during the postpartum year. For postnatal seroconverter women, we calculated the same statistics for available plasma and breast milk samples collected at the time of the mother’s last negative ELISA test and at time points less than 30 days, 31-90 days, 91-180 days, 181-270 days, and 271-365 days after maternal infection.