Clinical mastitis, a bacterial infection of the breast with a symptomatic inflammatory response of the mammary tissue, has been identified as a risk factor for mother-to-child transmission (MTCT) of HIV through breast-feeding.1,2 One potential mechanism to explain this association is that inflammation down-regulates tight junction proteins in the mammary epithelium and allows leakage of leukocytes and cell-free HIV particles from plasma into the lumen.3 Plasma leakage could change the concentration of sodium (Na) and potassium (K) ions in breast milk, and these changes could indicate the presence of mastitis even in the absence of clinical signs and symptoms. Consistent with this hypothesis, Semba and colleagues found that elevated Na concentrations in breast milk were associated with increased risk of MTCT,4 whereas Willumsen et al reported that increased Na/K ratios were related to greater concentrations of cell-free HIV (CFV) in breast milk.5,6 It is unclear, however, whether the Na/K ratio is also associated with MTCT and whether this potential association could be mediated through increased leakage of cell-associated HIV (CAV), in addition to CFV. This is a relevant question, considering that infected cells might play an even more significant role in early breast-feeding MTCT than CFV does.7,8
We examined whether the Na/K ratio in breast milk, as an indicator of subclinical mastitis, was associated with increased risk of breast-feeding MTCT in a case-control study nested within a cohort of HIV-infected Tanzanian women and their infants. We also assessed the cross-sectional associations between the Na/K ratio and the CAV and CFV concentrations in breast milk.
The source population for this nested case-control study was a cohort of 1078 HIV-1-infected mothers who participated in a randomized, double-blind, placebo-controlled trial of vitamins conducted in Dar es Salaam, Tanzania. This cohort has been described previously.9,10 In brief, women were recruited at gestation weeks 12 to 27 and were followed up through pregnancy and the lactation period. Baseline information was obtained on sociodemographic characteristics, clinical and immunological HIV disease stage (including CD4 cell counts), and potential risk factors for transmission of HIV. Data collected during follow-up included the pregnancy outcome and disease progression, morbidity, and mortality of both the mothers and their children. Vertical transmission of HIV was assessed by testing blood samples collected from the child at birth, 6 weeks after birth, and every 3 months thereafter by use of the enzyme-linked immunosorbent assay (ELISA) and/or Western blot analysis, or polymerase chain reaction (PCR) in children who were younger than 18 months (Amplicor HIV detection kit, Roche Diagnostics, Branchburg, NJ). Children who tested negative by 6 weeks and positive thereafter were presumed to have been infected via breast-feeding. Ten mL of breast milk were collected by manual expression from either breast approximately every 3 months postdelivery.
Details on the case-control study design have been published.7 In summary, we included all incident cases of breast-feeding transmission (n = 61). We selected 1 breast milk sample per case to be tested for Na and K concentrations and for CFV and CAV. Two cases did not have a suitable sample for analyses of Na and K; thus, the final number of cases considered was 59. We aimed to select samples that had been collected before the child's first HIV-positive test. In 38 cases, a sample was available between 4 and 24 weeks before the first positive test; in 11 cases, the only breast milk sample available was at the same time as the child's first positive test (n = 8) or within the prior 4 weeks (n = 3), and in another 10 cases, the sample was more than 24 weeks before the first positive test. We randomly selected 1 control per case from the pool of breast-feeding, nontransmitting mothers. Controls were individually matched to cases on the time since delivery when the breast milk sample was collected ±1 week.
After collection, the breast milk samples were centrifuged at 1500 g for 12 minutes at 4°C. The cell-free aqueous milk fraction and milk cell 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 using the Instrumentation Laboratory 943 Flame Photometer (Instrumentation Laboratory, Lexington, MA). Each sample was atomized and mixed with propane gas, and sprayed into a chimney where it was ignited. Na produces a characteristic flame with an emission wavelength read at 589 nm; K was read at 776 nm. The day-to-day variabilities of Na at 78.4 and 14.9 mEq and K at 28.1 and 5.4 mEq were 1.0%, 1.3%, 1.1%, and 1.6%, respectively.
CAV load in milk was quantified from the milk cells pellet after DNA extraction by use of real-time PCR (FastStart DNA Master SYBR Green I mix and LightCycler, Roche Diagnostics) and expressed as the proportion of infected cells per 10,000 cells. CFV RNA was isolated from the aqueous milk fraction using the High Pure Viral RNA Kit (Roche Diagnostics, Indianapolis, IN), after implementing an ultrasensitive protocol of the Amplicor HIV Monitor test version 1.5 (Roche Diagnostics, Branchburg, NJ), as detailed previously.7
Subclinical mastitis, the exposure of interest, was defined using the Na/K ratio distribution according to previously published categories5: a Na/K ratio ≤0.6 is considered to be normal, >0.6 to 1.0 is considered to be moderately raised, and >1.0 is considered to be highly raised. The highest ratio category of >1.0 is indicative of severe subclinical mastitis, whereas a Na/K ratio >0.6 to 1.0 indicates subclinical inflammation. The outcomes of interests were mother-to-child breast-feeding transmission of HIV-1 and the levels of CFV and CAV in breast milk.
Baseline characteristics of cases and controls were compared using the signed-rank test for continuous variables and the McNemar test for categorical variables. The association between ordinal categories of Na/K ratio and transmission of HIV was examined by fitting univariate and multivariate conditional logistic regression models. Odds ratios (OR) and 95% confidence intervals (CI) were obtained from these models by using the lowest Na/K ratio category as the reference and adjusting for the CD4 cell count at baseline and assignment to the vitamin A supplementation arm of the trial, because this was previously reported to increase the risk of MTCT.9 To test for a dose-response association between the severity of mastitis and transmission, we introduced an ordinal variable representing the Na/K ratio categories into the regression models and tested its statistical significance by use of the Wald test.
High CFV and high CAV concentrations in breast milk were defined dichotomously, using the median among the cases as the cutoff point.7 The proportions of women with high CFV or CAV were compared across Na/K ratio categories by using the Cochran-Armitage test for trend. Adjusted relative risks for high CFV or CAV shedding in relation to mastitis were estimated from multivariate binomial regression models adjusted for CD4 cell counts and assignment to vitamin A supplements. The Statistical Analysis System software version 9.1 (SAS Institute, Cary, NC) was used for all analyses.
The study was approved by the Human Subjects Committee of the Harvard School of Public Health and the Research and Publications Committee of Muhimbili University College of Health Sciences.
Maternal age, parity, height, weight, clinical stage of HIV disease, or hemoglobin concentrations at baseline did not differ significantly between cases and controls, as previously reported.7 Cases were more likely to have been assigned to the vitamin A arm of the trial (59%) than controls (46%; P = 0.12), and had lower average CD4 cell counts/mm3 (383, standard deviation [SD] = 235 vs. 456, SD = 191 in controls; P = 0.01). Mean CFV (copies/mL) and CAV (copies/10,000 cells) in breast milk were each significantly higher in cases (CFV = 2526, SD = 5179; CAV = 58, SD = 202) compared to controls (CFV = 633, SD = 1808, P = 0.002; CAV = 12, SD = 43, P = 0.001).
Average Na and K concentrations (mmol/L) were 7.5 (SD = 6.2) and 11.4 (SD = 2.2) in cases and 6.7 (SD = 9.3) and 12.4 (SD = 2.9) in controls (P = 0.05 for Na and P = 0.01 for K). The mean Na/K ratio was 0.66 (SD = 0.53) in cases and 0.61 (SD = 1.22) in controls (P = 0.01). In univariate analyses, compared with a normal Na/K ratio, a moderately raised Na/K ratio was positively, although not significantly, associated with transmission, whereas a highly raised Na/K ratio was associated with a 9-fold statistically significant increase in transmission (P = 0.04) (Table 1). There was evidence for a dose-response association (P, test for trend = 0.01). The results did not change substantially after adjusting for maternal CD4 cell counts at baseline and assignment to the vitamin A arm of the trial.
We next examined whether HIV shedding was associated with the Na/K ratio in breast milk, regardless of the women's case/control status (Table 1). The proportion of women with high CAV in breast milk increased significantly by Na/K ratio categories, whereas the association with CFV was not statistically significant. In multivariate analyses, women with highly raised Na/K were 3.4 times more likely to have high CAV concentrations in breast milk compared to women with a normal ratio (P = 0.007). The association was only slightly attenuated after further adjustment for high CFV, with prevalence ratios of 2.13 (95% CI: 1.03, 4.43) for moderately raised Na/K and 2.96 (95% CI: 1.21, 7.23) for highly raised Na/K, compared to normal (P, test for trend = 0.003).
In this nested case-control study, subclinical mastitis, as indicated by an elevated Na/K ratio in breast milk, was positively associated with breast-feeding transmission of HIV-1. In addition, the Na/K ratio was directly related to cell-associated HIV shedding in breast milk.
We selected only 1 breast milk sample per woman to ascertain the association between mastitis indicators and MTCT. This is a potential limitation, given that the composition of breast milk could vary substantially during lactation.6 To overcome the distorting effect of breast milk composition over time, we individually matched cases to controls on the age of the breast milk sample at the time of collection. Another potential limitation is that milk samples were collected from either breast; given that mastitis is frequently unilateral,6,11 the presence of subclinical mastitis may have been underestimated.
Previous studies from the Ivory Coast,1 Kenya,2,12 and Tanzania13 had suggested that clinical signs of mastitis were related to increased risk of breast-feeding MTCT. In addition, a study from Malawi4 indicated that subclinical mastitis, measured as an elevated Na concentration in breast milk, was also related to MTCT. Increased Na/K ratios were associated with greater CFV in a study from South Africa,5,6 but no data were available regarding the relation between the Na/K ratio and MTCT. Our finding of a positive association between the Na/K ratio and MTCT is in line with the Malawi report. However, in contrast with the South Africa study, we found only a weak, statistically nonsignificant association between the Na/K ratio and CFV load, but a strong and significant relation with CAV shedding. One possible reason for this discrepancy is that the South Africa report included only women during the first 14 weeks postpartum,6 whereas our case-control study included samples obtained during the first 2 years. We have previously shown that CFV shedding in milk seems to be a stronger predictor of late rather than early breast-feeding MTCT, whereas CAV shedding is a risk factor for transmission throughout lactation.7 Another potential reason for the discrepancy may include differences in the distribution of HIV subtypes between the populations; some subtypes might be preferentially associated to CFV or CAV shedding.
The association between an elevated Na/K ratio and increased CAV shedding suggests that passage of HIV-infected macrophages and lymphocytes through the basement membrane of the mammary epithelium into the alveolar lumina could contribute to increased risk of HIV transmission during episodes of mastitis.
It is uncertain whether increased mammary permeability, as indicated by elevated Na/K ratios, can be due to HIV infection even in the absence of bacterial mastitis. One recent study in Zambia found that increased Na/K was more frequent among HIV-infected than HIV-uninfected women and correlated with breast symptoms independent of HIV infection,14 suggesting that HIV infection alone may not necessarily explain increased Na/K ratios.
Among HIV-infected women, antibiotic treatment of mastitis episodes could result in reductions in the number of breast milk leukocytes within 1 week.15 Because these HIV-infected leukocytes in breast milk could mediate the association between mastitis and MTCT, according to our findings, opportune and complete treatment of mastitis episodes might contribute to decrease the burden of infant HIV infection through breast-feeding. Prospective studies are warranted to determine the timing after a treated episode of mastitis when it would be safest to resume breast-feeding from the affected breast, because increased mammary permeability seems to persist for several weeks after treatment.15 Future studies should also consider the validation of simpler methods to diagnose subclinical mastitis against the Na/K ratio in breast milk, inasmuch as flame photometry may not be widely available in the countries most heavily affected by the HIV epidemic.
The relatively low prevalence of exposure to severe subclinical mastitis in our cases (15%) indicates that risk factors other than mastitis are likely to be at play in the pathogenesis of MTCT in this population; investigation of such factors is urgently needed. Ascertaining the efficacy of providing antiretroviral treatment to lactating HIV-infected women and/or their breast-feeding infants in preventing MTCT remains an important public health research priority.
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