Abstract
Subclinical mastitis is common in HIV-infected women and is a risk factor for mother-to-child transmission of HIV. The purpose of this study was to examine the effect of vitamin supplementation [vitamin A + β-carotene, multivitamins (B complex, C, and E), or multivitamins, including vitamin A + β-carotene] on the risk of subclinical mastitis during the first 2 y postpartum among HIV-infected women. The study was a randomized, placebo-controlled, clinical trial including 674 HIV-infected, antiretroviral naïve Tanzanian women who were recruited during pregnancy and followed-up after delivery. Breast milk samples were obtained approximately every 3 mo. Any subclinical mastitis was defined as a ratio of the sodium to potassium (Na:K) breast milk concentrations > 0.6 and further classified as either moderate (Na:K ≥ 0.6 and ≤ 1) or severe (Na:K > 1.0). Fifty-eight percent of women had at least 1 episode of any subclinical mastitis. Women assigned to multivitamins (B complex, C, and E) had a 33% greater risk of any subclinical mastitis (P = 0.005) and a 75% greater risk of severe subclinical mastitis (P = 0.0006) than women who received the placebo. Vitamin A + β-carotene also increased the risk of severe subclinical mastitis by 45% (P = 0.03). Among women with CD4+ T-cell counts ≥ 350 cells/μL, multivitamin intake resulted in a 49% increased risk of any subclinical mastitis (P = 0.006); by contrast, there were no treatment effects among women with CD4+ T-cell counts < 350 cells/μL (P- interaction for treatment × CD4+ T-cell count = 0.10). Supplementation of HIV-infected women with vitamins increased the risk of subclinical mastitis.
Introduction
Subclinical mastitis is an inflammatory response of the mammary tissue that causes tight junctions in the mammary epithelium to open, allowing immune cells and extracellular fluid to flow from plasma to milk (1). Plasma leakage changes the electrolyte composition of milk, increasing the sodium:potassium (Na:K) ratio. An elevated Na:K ratio in breast milk has been used to indicate subclinical mastitis in epidemiological studies (2, 3). Subclinical mastitis is common in HIV-infected women in sub-Saharan Africa (3, 4) and is a risk factor for mother-to-child transmission of HIV (5, 6). Subclinical mastitis presents regardless of the signs of inflammation or infection that are evident in clinical mastitis.
Subclinical mastitis is accompanied by oxidative stress (7). Antioxidant vitamin supplementation may reduce oxidative stress and the inflammatory response during subclinical mastitis. In dairy cows, supplementation with vitamin E (8) or a combination of vitamins A, D, and E (9) reduced the incidence of clinical mastitis. Human trials that examined the effect of vitamin supplementation on the risk of subclinical mastitis have not shown beneficial effects (10–12). However, in 1 trial, there was a trend toward reduced risk of subclinical mastitis among HIV-infected women receiving a multiple micronutrient supplement but not among HIV-uninfected women (11).
We examined the effects of 3 types of vitamin supplementation (vitamin A + β-carotene, vitamin B complex + C + E, vitamin B complex + C + E + vitamin A + β-carotene) or placebo on the risk of subclinical mastitis during the first 2 y postpartum among HIV-infected women who participated in a randomized clinical trial in Tanzania.
Methods
Study design and population.
Starting in April 1995, 1078 HIV-infected women were enrolled into a randomized clinical trial in Dar es Salaam, Tanzania, to examine the effect of vitamin supplements on maternal and child health outcomes. Details of the trial have been described previously (13). In brief, women were enrolled at 12–27 wk of gestation and randomized in a 2-by-2 factorial manner to receive a daily dose of 1 of 4 regimens throughout the first 24 mo postpartum and thereafter: 1) vitamin A (1500 μg RE of retinol) plus 30 mg β-carotene; 2) multivitamins [20 mg thiamin, 20 mg riboflavin, 25 mg vitamin B-6, 100 mg niacin, 50 μg vitamin B-12, 500 mg vitamin C (purified l-ascorbic acid), 30 mg vitamin E (RRR-α- tocopherol acetate), and 0.8 mg folic acid]; 3) multivitamins with vitamin A plus β-carotene; or 4) placebo. All women received 5 mg folic acid and 120 mg ferrous iron daily as part of standard prenatal care. At delivery, women in the 2 groups receiving vitamin A were given an additional dose of 60,000 μg RE of vitamin A, whereas the other 2 groups received placebo. Antiretroviral treatment was not available in this setting at the time of the study.
Baseline information was obtained on sociodemographic characteristics and anthropometry. HIV disease stage was assessed in accordance with the WHO criteria (14). A venous blood specimen was collected into EDTA vacutainer tubes for measurements of hemoglobin, T-cell subsets, micronutrient concentrations, syphilis testing, and malaria smears. Stool samples were obtained for diagnosis of intestinal parasitoses and vaginal specimens for trichomoniasis. Women were followed during pregnancy and after delivery throughout the lactation period at scheduled monthly clinic visits. About 10 mL of breast milk was collected by manual expression from either breast approximately every 3 mo during the first 2 y postpartum. Compliance with the study regimen was assessed at the monthly visits as the proportion of tablets absent from bottles that were returned by each woman from the total number of tablets she should have taken. The median proportion was 83%, with an interquartile range of 79–93, by 2 y of follow-up. Compliance did not differ between treatment arms.
All women provided informed consent for participation before enrollment. The protocol was approved by the Human Subjects Committee of the Harvard School of Public Health and the Research and Publications Committee of Muhimbili University of Health and Allied Sciences.
Laboratory methods.
Hemoglobin was measured using a CBC5 Coulter Counter or cyanmethemoglobin method using a Colorimeter (Corning). Serum retinol and vitamin E were measured using HPLC (15). Stool specimens were examined macroscopically for helminths and microscopically for ova and cysts. Active syphilis was defined as positive results for sera antibodies in both the VDLR (Murex Diagnostic) and Treponema pallidum hemagglutination (Fujirebio) tests. Vaginal and cervical swabs were examined microscopically for infections caused by Trichomonas vaginalis. The presence of malaria parasites was ascertained from Giemsa-stained thick and thin smear blood films. CD4+ T-cell counts were determined with the use of FACSCount and FACSCan systems (Becton Dickinson).
Immediately after breast milk collection, samples were placed on ice and transported to the Microbiology and Immunology Laboratory at Muhimbili National Hospital. On the same day, 1 aliquot was centrifuged at 1500 × g for 12 min at 4°C. The cell-free aqueous milk fraction and milk pellet were separately stored at −70°C until laboratory testing at Children’s Hospital Boston. Flame photometry was used to analyze Na and K concentrations in the aqueous fraction by using the Instrumentation Laboratory 943 Flame Photometer (Instrumentation Laboratory) (16). Each sample was atomized and mixed with propane gas and sprayed into a chimney and ignited. Na produces a characteristic flame with an emission wavelength read at 589 nm; K was read at 776 nm. The day-to-day variations of Na at 78.4 and 14.9 mmol and K at 28.1 and 5.4 mmol were 1.0, 1.3, 1.1, and 1.6%, respectively.
Data analysis.
We defined subclinical mastitis using the ratio of milk Na:K according to previously published categories (2): any subclinical mastitis as Na:K > 0.6, moderate subclinical mastitis as Na:K > 0.6 and ≤ 1, and severe subclinical mastitis as Na:K > 1. We first compared the distribution of baseline characteristics across treatment groups to verify the randomization assumption using the Kruskal-Wallis test for continuous variables and the chi-square test for categorical variables. Next, we conducted intent-to-treat analyses to examine the effect of vitamin supplements on the occurrence of subclinical mastitis. Because each woman provided more than 1 breast milk assessment during follow-up, we used generalized estimating equations to estimate relative risks (RR) of subclinical mastitis throughout the lactation period, comparing each treatment arm against the placebo group. In these models, we specified a binomial distribution, the log-link function, and an exchangeable correlation structure to account for within-person correlation of repeated measurements. In addition, models included a variable indicating timing of the breast milk sample collection (<2 wk from delivery and at 3, 6, 9, 12, and >12 mo postpartum). We first tested for interactions between the vitamin A + β-carotene and the multivitamin arms. There were significant interactions between the vitamin A + β-carotene and the multivitamin arms on any subclinical mastitis (P = 0.02) and severe subclinical mastitis (P = 0.002); thus, results are presented separately for the 4 arms. We examined the effect of treatment over the postpartum period by introducing into the model cross-product terms between indicator variables for treatment groups and time (month postpartum) and testing for significance with the likelihood ratio test. Because the treatment effect could be modified by the women’s stage of HIV disease, we conducted supplemental analyses to examine the effect of supplementation on subclinical mastitis within strata of CD4+ T-cell counts (< vs. ≥350 cells/μL) at baseline. The statistical significance of this interaction was tested with the likelihood ratio test by introducing into the model a cross-product term between an indicator variable for CD4+ T-cell counts < 350 cells/μL and treatment. Data are presented as RR and 95% CI. All P-values were 2-sided. Significance was defined as P < 0.05. Analyses were carried out with SAS version 9.1.
Results
There were 1642 breast milk samples from 674 women available for analyses of Na and K (Supplemental Fig. 1). Each woman contributed a mean of 2.4 ± 1.5 samples. These women did not differ from those who were not included in terms of sociodemographic factors or treatment assignment. Baseline characteristics, including age, education level, plasma concentrations of vitamins A and E, hemoglobin, baseline CD4+ T-cell counts, and infections, did not differ across treatment arms (Table 1).
TABLE 1.
Baseline characteristics of women according to treatment group assignment1
| Characteristic | Placebo, n = 161 | Vitamin A + β-carotene, n = 165 | Multivitamins, n = 175 | Multivitamins and Vitamin A + β-carotene, n = 173 |
| Gestation week at randomization | 20.4 ± 3.7 | 20.1 ± 3.5 | 20.6 ± 3.1 | 20.2 ± 3.1 |
| Age, y | 24.9 ± 4.8 | 25.0 ± 5.2 | 24.9 ± 4.8 | 24.5 ± 4.3 |
| Completed primary education, % (n) | 86.3 (139) | 90.3 (149) | 86.3 (151) | 91.9 (159) |
| Primigravida, % (n) | 28.9 (46) | 34.0 (55) | 36.1 (62) | 30.8 (53) |
| Serum retinol, μmol/L | 0.89 ± 0.42 | 0.83 ± 0.34 | 0.84 ± 0.34 | 0.89 ± 0.29 |
| Serum vitamin E, μmol/L | 10.0 ± 2.9 | 10.0 ± 3.0 | 9.8 ± 3.0 | 10.1 ± 2.7 |
| BMI, kg/m2 | 23.2 ± 3.3 | 23.1 ± 3.5 | 23.7 ± 3.5 | 23.3 ± 3.1 |
| Hemoglobin, g/L | 96 ± 17 | 93 ± 17 | 94 ± 15 | 92 ± 16 |
| Baseline CD4+ T-cell counts, per μL | 415 ± 183 | 401 ± 200 | 428 ± 196 | 413 ± 200 |
| HIV symptomatic,2% (n) | 18.1 (29) | 23.2 (38) | 19.5 (34) | 18.7 (32) |
| Infections, % (n) | ||||
| Malaria | 15.6 (25) | 19.5 (32) | 14.6 (25) | 20.5 (35) |
| Ascaris | 5.9 (8) | 3.6 (5) | 6.3 (9) | 9.3 (13) |
| Hookworm | 8.2 (12) | 10.3 (15) | 11.2 (17) | 11.9 (18) |
| Syphilis | 7.1 (10) | 3.6 (5) | 7.4 (11) | 3.4 (5) |
| Trichomonas vaginalis | 25.5 (40) | 21.3 (34) | 23.8 (41) | 27.8 (47) |
Values are mean ± SD unless noted otherwise. Women's characteristics did not differ by treatment group.
Symptomatic women were at stages 2 or 3 according to the WHO staging system of HIV disease (14).
The proportions of women with at least 1 sample < 2 wk from delivery and at 3, 6, 9, 12, and >12 mo were 49, 70, 45, 30, 18, and 31%, respectively. The lack of availability of breast milk samples was primarily due to missed clinic visits, low breast milk output at the time of collection, or weaning. The mean number of samples available did not differ by treatment assignment (P = 0.54). Overall, 58% of the women had at least 1 episode of any subclinical mastitis, 36% had at least 1 episode of moderate subclinical mastitis, and 32% had at least 1 episode of severe subclinical mastitis. The point prevalence of subclinical mastitis within 2 wk of delivery and at 3, 6, 9, 12, and >12 mo was 66, 33, 26, 20, 20, and 25%.
Women who were assigned to multivitamins without vitamin A + β-carotene had a 33% greater risk of any subclinical mastitis (P = 0.005) and a 75% greater risk of severe subclinical mastitis (P = 0.0006) compared with women who received placebo (Table 2). Vitamin A + β-carotene increased the risk of severe subclinical mastitis by 45% (P = 0.03). The effects of multivitamins did not vary over the postpartum period for either moderate subclinical mastitis (P-interaction = 0.90) or severe subclinical mastitis (P-interaction = 0.53).
TABLE 2.
Effect of vitamin supplements on risk of subclinical mastitis among HIV-infected women in Tanzania
| Subclinical mastitis outcome | Placebo, n = 161 | Vitamin A + β-carotene, n = 165 | Multivitamins, n = 175 | Multivitamins and Vitamin A + β-carotene, n = 173 |
| Any (either moderate or severe; Na:K > 0.6) | ||||
| ≥1 positive sample, % | 49.7 | 60.6 | 60.6 | 60.1 |
| RR (95% CI) | 1.0 | 1.22 (0.99, 1.49) | 1.33 (1.09, 1.61) | 1.18 (0.96, 1.45) |
| Moderate (Na:K > 0.6 and ≤ 1) | ||||
| ≥1 positive sample, % | 31.7 | 36.4 | 32.6 | 42.8 |
| RR (95% CI)1 | 1.0 | 1.22 (0.90, 1.66) | 1.25 (0.92, 1.71) | 1.28 (0.96, 1.72) |
| Severe (Na:K > 1) | ||||
| ≥1 positive sample, % | 23.0 | 35.8 | 37.7 | 31.1 |
| RR (95% CI)2 | 1.0 | 1.45 (1.04, 2.03) | 1.75 (1.27, 2.41) | 1.29 (0.91, 1.83) |
Cases of severe subclinical mastitis were excluded; estimates represent treatment effects for moderate subclinical mastitis vs. no subclinical mastitis.
Cases of moderate subclinical mastitis were excluded; estimates represent treatment effects for severe subclinical mastitis vs. no subclinical mastitis.
Among women with CD4+ T-cell counts ≥ 350 cells/μL, multivitamins resulted in a 49% increased risk of any subclinical mastitis (P = 0.006); by contrast, multivitamins had no effect in women with lower CD4+ T-cell counts (P-interaction = 0.10) (Table 3).
TABLE 3.
Effect of vitamin supplements on risk of subclinical mastitis according to baseline CD4+ T-cell counts among HIV-infected women in Tanzania1
| Any (either moderate or severe) subclinical mastitis (Na:K > 0.6) | Placebo | Vitamin A + β-carotene | Multivitamins | Multivitamins and Vitamin A + β-carotene |
| Lower CD4+ T-cell count (<350/μL) | ||||
| n | 54 | 70 | 53 | 62 |
| ≥1 positive sample, % | 57.4 | 68.6 | 66.0 | 62.9 |
| RR (95% CI) | 1.0 | 1.13 (0.86, 1.49) | 1.18 (0.89, 1.56) | 0.98 (0.73, 1.31) |
| Higher CD4+ T-cell count (≥350/μL) | ||||
| n | 96 | 87 | 113 | 100 |
| ≥1 positive sample, % | 43.8 | 54.0 | 58.4 | 57.0 |
| RR (95% CI) | 1.0 | 1.26 (0.93, 1.71) | 1.49 (1.12, 1.98) | 1.31 (0.97, 1.79) |
The -interaction for CD4+ T-cell count and treatment group is 0.10.
Discussion
In this randomized trial, we found that supplementation with vitamin B-complex, C, and E to HIV-infected lactating women was related to increased risk of subclinical mastitis as measured by the Na:K ratio in breast milk. This effect was apparent among women with higher baseline CD4+ T-cell counts but not in women at more advanced stages of HIV disease. Vitamin A + β-carotene supplementation also increased the risk of severe subclinical mastitis. The prevalence of subclinical mastitis in this cohort of women was high, with 58% of women having at least 1 episode.
The apparent adverse effect of multivitamins was unexpected. Increased Na or Na:K has been associated with higher concentrations of immunological and inflammatory factors in breast milk (10, 17, 18). Thus, it is possible that multivitamins enhanced the women’s inflammatory response to the infection. This “paradoxical” effect was previously reported in individuals who received high doses of vitamin E and developed more severe symptoms associated with respiratory infections (19). Some of the primary etiological agents of mastitis infection, including Staphylococcus aureus and Escherichia coli, elicit the production of cytokines such as interferon-γ and interleukin-8, which enhance the oxidative burst capacity of neutrophils (20). It has been reported that vitamin C also enhances some aspects of neutrophil function (21). Increased intake of ascorbic acid in the presence of S. aureus or E. coli infections might result in excessive oxidation via enhanced neutrophil activity.
A negative effect of vitamin A + β-carotene on subclinical mastitis could help explain the previously reported adverse effect of supplementation with these nutrients on mother-to-child transmission of HIV through breast-feeding (22). Subclinical mastitis is a strong risk factor for mother-to-child transmission of HIV (5, 6) through increases in breast milk viral shedding due to leakage from plasma. The effect of vitamin A supplementation on severe subclinical mastitis might also represent an enhancement of inflammatory responses due to vitamin A. In some trials among children, vitamin A supplementation was associated with increased incidence of respiratory symptoms (23, 24). β-Carotene administered in high doses (≥20 mg/d) has been associated with adverse outcomes, including increased risk of lung cancer (25, 26), which might be related to prooxidant activity. It is possible that a similar mechanism could explain the adverse effect of vitamin A and β-carotene supplementation on mastitis, but we cannot distinguish the effect of β-carotene alone, because both nutrients were given in the same arm.
Previous micronutrient supplementation trials found no effect on the risk of subclinical mastitis (Supplemental Table 1) (10–12). In 1 small trial, there was a suggestion of a beneficial effect among HIV-infected women but not in those who were not infected (11). The vitamin doses in that trial were lower than the doses in our study; in addition, the supplement also included other micronutrients such as vitamin D, zinc, copper, and selenium. It is possible that high doses of vitamins enhance the inflammatory response to mastitis, whereas smaller doses have little effect on mastitis among immuno-compromised women.
We have previously reported that supplementation with vitamin B-complex, C, and E to HIV-infected women resulted in improved pregnancy outcomes (27), delayed HIV disease progression (13), and improved infant growth (28). In addition, among women with poor immunological and nutritional status, multivitamins appeared to decrease the risk of mother-to-child transmission (22). Multivitamins have benefits in this population and the potential risks of subclinical mastitis need further exploration.
There was a high prevalence of subclinical mastitis among the women in our study, with 58% of women experiencing at least 1 episode. Another study among HIV-infected women in Malawi found that 27% of women had at least 1 episode of subclinical mastitis in the first year postpartum (4). In non-HIV–infected women from Ghana, 45% had severe subclinical mastitis at 3 mo postpartum (29). The different rates of subclinical mastitis could be attributed to differences in health care or breast-feeding practices that could affect milk stasis (30).
Our study has some limitations. We did not assess clinical mastitis. We also do not have detailed information on clinical breast symptoms or breast-feeding practices among the women in our trial. This information could add insight into the high rates of subclinical mastitis overall. Also, potential differences in these variables among treatment groups could be a confounder in our study. However, the randomized design of our trial provides strength to our results.
Prevention, early detection, and effective treatment of mastitis are important, because in addition to increasing the risk of mother-to-child transmission of HIV, mastitis also adversely affects lactation performance (31) and possibly infant growth (11, 31). Future studies should examine specific risk factors for mastitis and the effect of mastitis on infant breast milk intake, growth, and morbidity. Also warranted are studies to evaluate the correlations between subclinical mastitis indicators and the presence of specific etiological microorganisms in mammary tissue.
Supplementary Material
Acknowledgments
J.E.A. designed the analysis plan, analyzed and interpreted the data, and wrote the first draft of the manuscript; S.A. and K.P.M. contributed to field activities, identification of breast milk samples, and interpretation of results; W.W.F. contributed to the study design, data collection, and interpretation of results; and E.V. designed the current research project and contributed to data analysis and interpretation. All authors read and approved the final manuscript.
Footnotes
Supported by grant R01HD045134 from the NIH. J.E.A. was supported by NIH training grant T32DK07703.
Supplemental Figure 1 and Table 1 are available with the online posting of thispaper at jn.nutrition.org.
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