Laterally CSs were compared with 2 groups: DSs and CNSs. At 1 week, CSs had a lower CD4 count (<200 cells/μL; P = 0.022) and were more likely to have a maternal pVL >100,000 copies/mL (P = 0.009) than DSs. Similar but nonsignificant associations comparing CSs with DSs with respect to pVL and CD4 cell count were observed at 1 and 4 months. CSs were more likely than CNSs to have a low CD4 count (<200 cells/μL) and high pVL (>100,000 copies/mL) at 1 week (CD4 cell count, P < 0.0001; pVL, P < 0.0001), 1 month (CD4 cell count, P = 0.0046; pVL, P = 0.017), and 4 months (CD4 cell count, P = 0.06; pVL, P = 0.005).
Among a subgroup of HIV-infected and uninfected women, sodium levels were significantly correlated across breasts at the 1-week and 1-month visits (see Table 2B; see Fig. 2B). Only 18 sodium levels from both breasts were available at the 4-month visit, limiting our capacity to distinguish if lateral correlation was present at this time point.
Breast Milk Viral Shedding Over Time
Of the 138 HIV-infected women, breast milk viral load was detectable in 44.3% of 1-week samples (n = 106, median = 487), 67.6% of 1-month samples (n = 74, median = 774), and 57.1% of 4-month samples (n = 98, median = 364). Breast milk viral shedding at 1 week was correlated with shedding at 1 month (n = 50, ρ = 0.67, P < 0.0001) and 4 months (n = 71, ρ = 0.51, P < 0.0001), and 1-month viral shedding correlated with shedding at 4 months (n = 54, ρ = 0.69, P < 0.0001). Maternal CD4 cell count but not pVL was significantly correlated with breast milk viral load at 1 week (CD4 cell count: ρ = −0.21, P = 0.03; pVL: ρ = −0.038, P = 0.70). Both factors were significantly associated with breast milk viral load at 1 month (CD4 cell count: ρ = −0.38, P = 0.0008; pVL: ρ = 0.25, P = 0.03), however, and most strongly at 4 months (CD4 cell count: ρ = −0.30, P = 0.0025; pVL: ρ = 0.33, P = 0.0010).
In regression models, after adjusting for pVL, CD4 cell count predicted breast milk viral load at 1 week (P = 0.058) and 1 month (P = 0.0007). After adjustment for maternal CD4 cell count, pVL predicted breast milk viral load at 1 month (P = 0.0181) and 4 months (P < 0.0001).
Of the 104 HIV-infected women with multiple breast milk viral load measurements over time, 39.4% had detectable virus in every breast milk sample, 30.8% had inconsistent viral shedding patterns, and 29.8% had an undetectable viral load in all the samples tested. Breast milk viral load from temporally consistent shedders was higher (median = 523 copies/mL at 1 week, median = 5775 copies/mL at 1 month, and median = 609 copies/mL at 4 months) than from inconsistent shedders (median = 180 copies/mL at 1 week [P = 0.25], median = 324 copies/mL at 1 month [P = 0.01], and median = 322 copies/mL at 4 months [P = 0.15]). Compared with inconsistent shedders, women who consistently shed virus in breast milk were more likely to have CD4 counts <200 cells/μL (46.3% vs. 12.5%; P = 0.0042) and pVLs >100,000 copies/mL (58.5% vs. 21.9%; P = 0.013). This was even more pronounced when comparing consistent shedders with consistent nonshedders (CD4 count <200 cells/μL [46.3% vs. 0%], P < 0.0001; pVL >100,000 copies/mL [58.5% vs. 4.4%], P < 0.0001).
Elevated Sodium in Breast Milk
Among 161 (HIV-infected and HIV-uninfected) women, the proportion with elevated breast milk sodium (≥13 mmol/L) declined sharply from 41.8% at 1 week to 20.0% at 1 month and further to 8.7% at 4 months. During the first week of lactation, sodium levels <16 mmol/L are considered normal; at this threshold, 30.3% had elevated sodium levels at 1 week (Table 3).26 Sodium levels at 1 week and 1 month were positively correlated with 4-month levels (1 week to 4 months: n = 85, ρ = 0.30, P = 0.04; 1 month to 4 months: n = 61, ρ = 0.32, P = 0.01). Sodium levels at 1 week were positively correlated with 1-month levels (n = 57, ρ = 0.27, P = 0.04).
Regardless of sampling time, breast milk sodium levels did not differ by maternal HIV infection status (see Table 3). The slightly higher proportion of HIV-negative women with elevated sodium at 4 months was probably explained by a small excess of HIV-negative mothers with first-born children. In our cohort, lower parity was associated with increased risk of elevated sodium in breast milk.
Among HIV-infected women (n = 138), breast milk sodium concentrations were moderately correlated with breast milk viral load at all visits (1 week: n = 104, ρ = 0.29, P = 0.003; 1 month: n = 69, ρ = 0.35, P = 0.003), with the strongest correlation observed at the 4-month visit (n = 97, ρ = 0.42, P < 0.0001). Lower CD4 cell count and higher pVL were associated with elevated sodium in breast milk at 1 and 4 months, but neither was at 1 week (see Table 3).
Breast problems (defined as engorgement, red/shiny breasts, cracked/bleeding nipples, painful breast[s], blocked duct[s], abscess, and/or symptoms of Candida) were detected in 15 (11.3%) of 132 HIV-positive women at the 1-week postpartum visit. Breast problems were unilateral in 10 of 15 women at 1 week and in all women at 1 month (n = 11) and 4 months (n = 3). Of these HIV-infected women with breast problems, 13 of 15 had their sodium level assessed at 1 week, 7 of 11 at 1 month, and 2 of 3 at 4 months. At 1 and 4 months postpartum, elevated sodium was more common (20.3% at 1 month and 7.2% at 4 months) than reported or observed breast problems (9.5% at 1 month and 2.8% at 4 months) (see Table 3). Strong associations were observed between elevated sodium and clinical breast problems at 4 months, but there were no significant associations at the earlier visits.
Mother-to-Child Transmission of HIV
Breast milk viral shedding at all visits was significantly related to MTCT of HIV (Table 4). In 3 separate multivariate logistic regression models for each visit adjusting for CD4 cell count and pVL, detectable breast milk viral load at 1 week (odds ratio [OR] = 3.50, 95% confidence interval [CI]: 1.71 to 7.17), at 1 month (OR = 3.22, 95% CI: 1.52 to 6.80), and at 4 months (OR = 5.49, 95% CI: 2.13 to 14.23) was associated with HIV transmission. CD4 cell count was also significantly associated with transmission after adjusting for breast milk viral load at 1 week and 4 months (1 week: β = −0.004, P = 0.013; 4 months: β = −0.0052, P = 0.037). pVL was not associated with transmission after adjusting for CD4 cell count and breast milk viral load in these models.
Consistency of viral shedding laterally across breasts and temporally across visits was associated with increased risk of transmission (see Table 4). Women with detectable viral loads in both breasts at more than 1 time point were more likely to transmit HIV to their children (OR = 3.4, 95% CI: 1.08 to 10.79; P = 0.03). In a regression model, including multiple time points, detectable breast milk viral load at more than 1 time point was predictive of transmission (OR = 3.35, β = 1.21; P = 0.0393) independent of breast milk viral load at individual time points.
Elevated breast milk sodium at 4 months was related to HIV transmission (P = 0.001), but elevated sodium at 1 week and 1 month was not. Excluding women with breast problems, the significant association between elevated sodium at 4 months and transmission remained (P = 0.002). There was a nonsignificant trend toward increased HIV transmission with unilateral breast problems.
Breast milk viral load was elevated among all transmitters regardless of the route of transmission (Table 5). There was a suggestion that the association between breast milk viral shedding and intrauterine transmission was slightly weaker than that observed for intrapartum and postnatal transmission. Nearly all (93% to 100%) transmitting mothers whose children had negative results by PCR at birth had high levels of HIV in breast milk at 1 and 4 months postpartum.
Our data examining breast milk viral shedding and sodium concentrations at multiple visits over the first 4 months of breast-feeding provide a more refined understanding of the role of elevated sodium concentrations and the influence of breast milk viral load in postnatal HIV transmission. Many studies have identified the importance of breast milk viral load on the risk of postnatal MTCT of HIV, demonstrating that high viral load increases the risk of transmission.9,11,13,20 This analysis has the added benefit of assessing the temporal and lateral dynamics of breast milk HIV RNA and sodium levels, however. Breast milk composition and breast permeability change dramatically from the early breast-feeding period to mature milk; understanding these changes in relation to HIV over time is important to the development of appropriate counseling and risk analysis.
In our cohort, breast milk HIV viral load was strongly correlated between breasts and consistent viral shedding was the most predictive marker of transmission. Because of the relatively common occurrence of intermittent viral shedding even among transmitting women, however, the clinical utility of a single breast milk viral load measurement is limited.
Breast milk sodium levels reflect epithelial permeability and have been shown to be elevated when milk volumes are low, such as during the first week of lactation, weaning, and with breast inflammation. More than 40% of women reported here had sodium concentrations >13 mmol/L at 1 week postpartum (30.3% at the 16-mmol/L cutoff). Because of the dynamic nature of breast milk composition during the first days of lactation, sodium at 1 week was not associated with maternal CD4 cell count, maternal pVL, or HIV transmission. As breast-feeding stabilizes, sodium levels recede and become strongly associated with breast milk viral shedding and HIV transmission at 4 months, irrespective of CD4 cell count and pVL. In mature milk, sodium levels average 6 mmol/L and persistently elevated concentrations during later breast-feeding signify inflammatory processes that may manifest as clinically detectable breast pathologic conditions in some women.21,25 We observed elevated sodium levels in the absence of observed or recently reported breast problems in a substantial proportion of women, indicating the presence of subclinical mastitis. Thus, breast milk sodium measurement at later postnatal ages may be a sensitive, more rapid, and less costly surrogate marker of viral shedding in milk than direct HIV viral load testing. The pattern of breast milk sodium at 1 month was intermediate between the 1-week and 4-month observations, suggesting that sodium levels are less useful potential surrogates for subclinical mastitis, increased viral load, and risk of HIV transmission.
We expected to observe increased breast milk viral shedding at 1 week with increased mammary gland permeability during the establishment of lactation, but we did not. Because concentrations of innate immune factors in breast milk tend to be highest in early versus later milk,27 these anti-infective soluble factors may help to counteract the increased permeability that exists at this time, which would ordinarily result in higher breast milk viral load.
Alternatively, low viral load at 1 week might be explained by the continued antiviral activity of short-course NVP taken during labor on breast milk viral load at 1 week. NVP (half-life of 6 days) was consumed during labor by 93% of the HIV-infected women in the cohort, thus suppressing the 1-week breast milk viral load sample.28 Long-term highly active antiretroviral therapy (HAART) has been shown to reduce breast milk viral load, and breast milk viral load reductions from single-dose NVP have been reported in a study in Kenya.29-31 Although these data do not establish the impact of NVP on breast milk viral load at the 1-week visit, we observed similar proportions of undetectable breast milk viral load compared with other studies among similar populations in the absence of NVP exposure.7,12,32
Studies in Malawi and South Africa have reported associations between elevated breast milk sodium levels and increased risk of HIV transmission; however, they did not report the temporal variations.12,20 In the Malawi study, only sodium levels from 1 point in time (6 weeks postpartum) were available and clinical data on breast problems were not reported.20 In the South African study, sodium levels were aggregated over the first 14 weeks of breast-feeding without distinguishing between earlier and later samples.12 Our data refine these observations to highlight the developmental specificity of sodium as a marker in breast milk in relation to HIV transmission. Elevated sodium in early milk (1) reflects the physiologic onset of normal lactation, (2) is a poor predictor of HIV transmission, and (3) may correlate with soluble anti-infective factors of breast milk. Elevated sodium in later milk likely reflects clinical or subclinical mucosal pathologic change linked to an increased risk of HIV transmission. Later but not earlier measurements of sodium may be a useful and simple predictor of increased risk of postnatal HIV transmission.
Several studies that have measured viral shedding in mucosal compartments, including breast milk and genital tract secretions, have observed stronger associations between viral shedding at the mucosa and MTCT of HIV than between systemic pVL and transmission.33-35 In addition, others have shown cell-associated virus to be a stronger predictor of transmission.13 In our cohort, breast milk viral shedding at 1 week, 1 month, and 4 months was associated with increased risk of HIV transmission through all routes: in utero, intrapartum, and postpartum transmission. High breast milk viral loads may be an indicator of greater mucosal shedding at other sites. It is perhaps for this reason that breast milk viral load correlated more strongly than pVL with intrapartum HIV transmission, which is also thought to be attributable to exposure to HIV in mucosal fluids.36-38
Our findings demonstrate the capacity of the breast epithelium to prevent entry of HIV into breast milk in approximately one third of untreated women at individual times. If present, however, HIV remains in milk at relatively stable levels across breasts and over time. Breast milk viral load levels were highly concordant across breasts at each time point, suggesting the influence of systemic factors rather than local factors or inflammation in the regulation of breast epithelial cell permeability to HIV. Similarly, sodium levels were correlated across breasts but not strongly, suggesting the impact of local factors on sodium level. Our data confirm previous findings of escalation of HIV transmission with increased breast milk viral loads. Elevated sodium concentration in breast milk during established breast-feeding but not early (<1 month) breast-feeding was also a significant predictor of HIV transmission. It is important to investigate interventions, such as good lactation practices, that may reduce the likelihood of inflammatory breast pathologic findings associated with elevated sodium and viral load. Simple methods to determine sodium concentrations in breast milk at later postnatal ages may also be helpful to guide in counseling breast-feeding HIV-infected women.
The authors thank the staff of the ZEBS for their tireless efforts, the study participants for their dedication to the study, and Don Decker for his technical assistance in the laboratory assays.
K. Semrau was responsible for data analysis, writing, and editing. M. Ghosh was responsible for data generation (laboratory testing). C. Kankasa was responsible for data acquisition from the ZEBS and editing. M. Sinkala was responsible for data acquisition from the ZEBS and editing. P. Kasonde was responsible for data acquisition from the ZEBS and editing. M. Mwiya was responsible for data acquisition from the ZEBS and editing. D. M. Thea was responsible for writing and editing. L. Kuhn was responsible for study conception and design, data analysis, writing, and editing. G. M. Aldrovandi was responsible for study conception and design, writing, and editing.
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Keywords:© 2008 Lippincott Williams & Wilkins, Inc.
breast-feeding; breast milk sodium; HIV; lactation; mastitis; mother-to-child transmission; viral load