In the absence of antiretrovirals, approximately 10%–15% of infants breast-fed by their HIV-infected mothers will acquire infection via this route.1 Risk factors for breast milk transmission include low maternal CD4 count, high plasma or breast milk viral load, and presence of breast pathology, including subclinical and clinical mastitis and abscess.2–5 Subclinical mastitis has been associated with increased levels of breast milk HIV-1 RNA and increase in milk inflammatory cytokines that could facilitate transmission in the infant by cellular activation.6–9 Because of the increased risk of transmission during mastitis and because most mastitis is unilateral (ie, confined to only one breast), the World Health Organization (WHO) recommends that HIV-infected women with mastitis or other unilateral breast pathology stop breastfeeding on the affected breast and feed on the contralateral (or unaffected) breast; once symptoms resolve, women should again feed from both breasts.10 Although the quantity of HIV in breast milk and subclinical mastitis has been correlated,11,12 no longitudinal data on viral dynamics in breast milk before, during, and after clinically detectable breast pathology are available. Thus, WHO guidelines are based on 2 unsubstantiated assumptions. First, the recommendation assumes that breast milk viral quantity declines to premastitis levels after clinical symptom resolution. Second, that breast milk viral quantity in the contralateral breast does not increase. Theoretically, fever, inflammation, and other constitutional signs of clinical mastitis could lead to high HIV RNA in the clinically unaffected breast.13 Empirical data on the temporal and lateral dynamics of breast milk HIV RNA before and after mastitis are needed to develop evidence-based counseling for HIV-infected, breastfeeding women with mastitis or abscess.
Mastitis can be classified as either clinical or subclinical. Clinical mastitis is characterized by observable signs and symptoms, for example, engorgement, redness, induration, pain, and fever. Clinical mastitis and other overt breast pathology, including abscess, have been associated with increased risk of mother-to-child HIV transmission (MTCT), but studies have not directly measured changes in breast milk HIV RNA with clinical mastitis or abscess.4,8 Subclinical mastitis is generally defined by markers of breast epithelium permeability—sodium or ratio of sodium:potassium concentrations. Subclinical mastitis tends to correlate with breast milk HIV concentrations6,14 and with inflammation,15 possibly explaining association between subclinical mastitis and increased HIV transmission. Interpretations of studies on subclinical mastitis are complicated by normal developmental changes over time in these biochemical markers as breastfeeding is established.16 Moreover, these biochemical changes may not necessarily be directly relevant to the clinical circumstance of overt breast morbidity.
To address the clinical problem of how to counsel HIV-infected, breastfeeding women with mastitis or other generally unilateral breast pathology, this study examined longitudinal patterns of breast milk viral shedding in affected and unaffected contralateral breasts before, during, and after, clinically detectable breast pathology.
Breast milk samples were selected from HIV-infected women with mastitis or abscess enrolled in the Zambia Exclusive Breastfeeding Study (ZEBS) in Lusaka, Zambia. ZEBS was approved by all participating institutions' ethical review boards and is described in detail elsewhere.17,18 In brief, ZEBS was a randomized trial to assess the impact of exclusive breastfeeding with abrupt weaning at 4 months on postnatal HIV transmission and child survival. All women were counseled to exclusively breastfeed until 4 months; half were randomized to stop all breastfeeding at 4 months and the other half to continue breastfeeding for a duration of their own choice. This analysis is restricted to 943 randomized women who initiated breastfeeding and did not receive antiretroviral therapy other than single-dose Nevirapine at onset of labor. Antiretroviral therapy was not available in Zambia for most of the study period. Bilateral maternal breast milk samples were collected at 1 week, 1, 4, 4.5, 6, 9, 12, 15, 18, 21, and 24 months postpartum, and as long as breastfeeding continued. A protocol change in September 2004 restricted milk collection to 1, 4, 4.5, and 12 months postpartum. Single-dose Nevirapine is known to affect HIV RNA quantity in breast milk; thus, samples from both breasts were examined as the effect of Nevirapine would be bilateral, yet the effect of mastitis is unilateral.19,20
Trained study nurses undertook a clinical breast examination and structured symptom history at clinic visits scheduled at 1 week, 1, 2, 3, 4, 4.5, 5, 6, 9, 12, 15, 18, 21, and 24 months postpartum. A diagnosis of unilateral clinical mastitis or abscess was made in 86/943 (9.1%) women and at 110/10,987 (1.0%) visits. Clinical mastitis was defined as recent or current fever (>37°C) and at least one of the following signs and symptoms: engorgement, red/shiny breasts, blocked duct(s), and/or recent or current painful breasts. At each visit, nurses determined presence or absence of breast abscess, a clinically evident pathology characterized by a pus-filled, hard mass in the breast tissue. Women were also asked whether they had visited a clinic or hospital because of illness since the prior visit.
Of the 86 women diagnosed with unilateral clinical mastitis/abscess diagnoses at 110 visits, 38/86 (44.2%) women had breast milk samples taken from both breasts before, during, and after diagnosis. The remaining 72 cases of mastitis or abscess in 48 women had missing samples because of the standardized milk collection protocol, missed visits, or cessation of breastfeeding (see Figure S1, Supplemental Digital Content, http://links.lww.com/QAI/A375). Prepathology samples were taken from the clinic visit completed just before the visit when the pathology was first diagnosed. Mastitis and abscess have rapid onset21,22 and are typically caused by noninfectious factors, namely, milk stasis.10 Therefore, a visit completed at least 2 weeks before should be free of pathology. Samples were defined as being during the condition if collected at the same visit when mastitis or abscess was diagnosed as described above. Data on the duration of symptoms were not collected. Resolution of mastitis varies according to the severity of symptoms; in the absence of antibiotics, symptoms typically resolve within 24 hours to 8 days.10,22–25 Postpathology samples were taken from the next available milk sample after diagnosis when breast problems were not reported and a minimum of 9 days after the diagnosis. Of 228 samples selected for testing (left and right breast milk samples at 3 time points), 211 (92.5%) had valid results obtained. Of the 38 women, 37 (97.3%) had results in the affected breast at the pre-, 31 (81.6%) during-, and 34 (89.5%) postdiagnosis visits. Of the 38 women, 36 (94.7%) had results in the unaffected, contralateral breast pre-, 36 (94.7%) during, and 37 (97.4%) postdiagnosis.
The 211 breast milk samples in 38 women with breast pathology were compared with 115 milk samples in 115 women who did not experience breast pathology. These 115 women were selected as those without breast pathology from the 138 HIV-infected women with breast milk samples collected at 1 week, 1 month, and 4 months postpartum analyzed in a previous report describing breast milk HIV RNA across breasts.16
Infant HIV infection was determined by HIV DNA polymerase chain reaction testing on dried blood samples collected at birth, 1 week, and 1, 2, 3, 4, 4.5, 5, 6, 9, 12, 15, 18, 21, and 24 months postpartum. All positive results were confirmed on a second sample if possible. All negative results were confirmed negative by quantification of betaglobulin to ensure sample adequacy.
Copies of HIV-1 RNA per milliliter of breast milk were measured with the Roche Amplicor Ultrasensitive HIV-1 1.5 kit (Roche Molecular Systems, Inc., Branchburg, NJ) with a lower detection limit of 50 copies per milliliter. The assay has been validated for quantitation of HIV-1 RNA in breast milk.26 As RNA quantity was nonnormally distributed, log-transformed data were used for analyses. For samples below the detection limit of the assay, a value of 49 copies per milliliter was imputed.
First, the analysis included a within-person, within-breast comparison of HIV RNA in breast milk across the 3 time points. Second, the contralateral breast at the same time point was compared. Third, comparisons with women who did not experience breast pathology were conducted. Crude analyses compared the distributions of HIV RNA in breast milk over time, across breasts, and between the groups. Relationships among breast pathology, breast milk HIV RNA and infant feeding patterns, acute maternal illness, and postnatal age of the child were examined. Generalized estimating equations were used to compare breast milk HIV RNA quantity between groups adjusting for repeat measures per subject. Change in HIV RNA quantity among visits was compared with generalized linear models (PROC GLM). All analyses were completed with SAS, version 9.1.3 (SAS Institute, Inc., Cary, NC).
Of the 86 women with unilateral mastitis or abscess, 38 (42.2%) had breast milk samples collected at a visit before, during, and after diagnosis. The 38 women with samples available were similar to the 48 who had breast pathology but lacked milk samples (Table 1). Women included in the analysis who had breast pathology had a mean CD4 count of 322 cells per microliter (23.6% CD4 count <200 cells per microliter) and mean plasma viral load of log10 4.62 copies per milliliter at enrollment during pregnancy. Four (11.8%) women reported a history of breast mastitis or abscess during previous experiences of breastfeeding.
Timing and Symptoms of Mastitis and Abscess
The timing and symptoms associated with mastitis or abscess are presented in Table 2. The majority of events occurred early in the postpartum period; most women were exclusively breastfeeding at this time. Women had a mixture of symptoms, defined by number and severity, associated with clinical mastitis and abscess. Women excluded from this analysis had mastitis diagnosed earlier and more often had pathology on the left side; otherwise, women were similar (Table 2).
Breast Milk HIV RNA in the Affected Breast
Breast milk HIV RNA during mastitis or abscess (log10 2.93 copies per milliliter) was significantly higher than before diagnosis (log10 2.56 copies per milliliter, P = 0.03). After resolution of symptoms, mean HIV RNA quantity decreased to levels comparable with those observed prepathology (log10 2.46 copies per milliliter, P = 0.52) (Table 3; see Figure S2, Supplemental Digital Content, http://links.lww.com/QAI/A375). Differences were slightly stronger when the analysis was restricted to women who had HIV RNA >50 copies per milliliter (Table 3). However, if considered simply as above or below the threshold for detection (50 copies per milliliter), there were no significant differences.
Mean change in HIV RNA concentrations from the pre- to the during-pathology visit was log10 0.45 copies per milliliter (95% CI: 0.16 to 0.74) in the affected breast (Table 4). With resolution of mastitis/abscess, breast milk HIV RNA decreased on average log10 −0.54 (95% CI: −0.19 to −0.89) copies per milliliter in the affected breast. Breast milk HIV RNA concentrations were similar in the samples collected before and after resolution of symptoms [change of log10 −0.04 (95% CI: −0.33 to 0.25) copies per milliliter in the affected breast].
Breast Milk HIV RNA in the Contralateral, Unaffected Breast
In the contralateral breast, HIV RNA did not significantly change among the pre- (log10 2.52 copies per milliliter), during- (log10 2.60 copies per milliliter, P = 0.56), or postdiagnosis periods (log10 2.36 copies per milliliter, P = 0.20) (Table 3; see Figure S2, Supplemental Digital Content, http://links.lww.com/QAI/A375). There were no significant increases or decreases in viremia before and after diagnosis of pathology in the unaffected breast (Table 4). Nor were there changes in the contralateral, unaffected breast in the proportion with detectable HIV RNA before, during, or after pathology.
At the visit when mastitis or abscess was diagnosed, the affected breast (log10 2.93 copies per milliliter) had significantly higher breast milk HIV RNA concentrations than the contralateral, unaffected breast (log10 2.60 copies per milliliter, P = 0.05). At the visits before and after the diagnosis, breast milk HIV RNA was not different across breasts (P = 0.55).
Signs, Symptoms, and Breast Milk HIV RNA
Each of the signs and symptoms associated with mastitis and abscess were examined separately for impact on HIV RNA breast milk concentrations. Mean breast milk HIV RNA quantity from the sample taken during pathology in the affected breast was significantly higher in those women who had a red/shiny breast (log10 3.47 vs. log10 2.65 copies per milliliter, P = 0.05) or abscess (log10 3.48 vs. log10 2.62 copies per milliliter, P = 0.03). Women who reported painful breasts also tended to have higher viral concentrations at the time of pathology (log10 3.05 vs. log10 2.17 copies per milliliter, P = 0.07). Mean change in HIV RNA quantity from the pre- to the during-pathology visit varied by specific sign or symptom (see Figure S3, Supplemental Digital Content, http://links.lww.com/QAI/A375). Women with red/shiny breasts had a log10 0.82 increase in HIV RNA concentration compared with women without this sign (log10 0.26 copies per milliliter, P = 0.08). Women with abscess had the largest increase in breast milk HIV RNA (log10 0.89 vs. log10 0.18 copies per milliliter, P = 0.03).
Comparison of Women With Mastitis to Women Without Mastitis
The 38 women with mastitis or abscess were compared with 115 women without breast pathology. The 38 women with mastitis or abscess were older (27.4 ± 6.1 vs. 25.0 ± 4.7 years, P = 0.01) and had lower CD4 count at enrollment (322 ± 149 cells per microliter vs. 403 ± 207 cells per microliter, P = 0.03). Women with mastitis or abscess were more likely, at enrollment, to report swollen glands in more than one place (10.5%) compared with nonmastitis women (1.7%) (P = 0.03). However, HIV-1 plasma viral load at enrollment was similar (log10 4.62 ± 0.80 vs. log10 4.46 ± 0.83 copies per milliliter, P = 0.44) between the groups (Table 1).
Of the breast milk samples from women with mastitis or abscess, 164/211 (77.7%) had detectable HIV RNA ≥50 copies per milliliter compared with 61/115 (54.9%) breast milk samples from women without breast pathology (P < 0.01, odds ratio = 2.86; 95% CI: 1.74 to 4.69). The mean breast milk HIV RNA concentration in the affected breast milk sample at the time of breast pathology diagnosis (log10 2.93 ± 0.19 copies per milliliter) was significantly higher than among women without breast pathology (log10 2.41 ± 0.09 copies per milliliter, P = 0.01). Breast milk HIV RNA concentrations in samples collected from women without breast pathology were similar to the breast milk HIV RNA concentrations in samples collected from the unaffected breasts at all time points and from the affected breast at pre- and postdiagnosis time points (Table 5).
MTCT of HIV
Of the 38 pairs, 8 (21.1%) children were HIV infected by 24 months of age. The overall rate of MTCT is similar to that of the larger cohort. Among the 8 infected children, 2 were infected in utero, 3 intrapartum or in the early postnatal period (first positive at 1 month), and 3 during breastfeeding after 1 month of age. Of the 6 non-intrauterine infections, 4 (66.7%) were found to be infected after mastitis or abscess occurred in the mother. The 4 post-breast pathology transmitters were compared with the 30 nontransmitting women. Transmitters had higher plasma viral load than nontransmitters (log10 4.78 vs. log10 4.20 copies per milliliter, P = 0.05), nonsignificantly lower CD4 counts (282 vs. 328, P = 0.55), and higher breast milk HIV RNA quantity before (log10 3.50 vs. log10 2.32 copies per milliliter, P = 0.03) and after mastitis/abscess (log10 3.53 vs. log10 2.08 copies per milliliter, P = 0.02). Additionally, mean breast milk HIV RNA quantity at the time of mastitis/abscess tended to be higher in the transmitters (log10 3.60 vs. log10 2.73 copies per milliliter, P = 0.24), although it was not statistically significant.
Breastfeeding complications, including mastitis and abscess are common, occurring among an estimated 10%–33% of all nursing women.10,27 Breast pathology can be painful, and many women stop breastfeeding after experiencing mastitis.28 Mastitis and abscess have the added risk among HIV-infected women of increasing the risk of postnatal MTCT.3,4 WHO recommendations for breastfeeding in the presence of mastitis/abscess in HIV-infected women encourage women to continue to breastfeed only from the contralateral, unaffected breast until symptoms resolve. Our study provides the first empirical data to support this recommendation. In this cohort of 38 women with mastitis or abscess, significant, albeit modest, increases in breast milk HIV RNA were observed in the affected breast. It is important to note that even small changes in viral load seem to impact HIV heterosexual transmission; similar trends are likely to be true in breast milk MTCT.29 In the majority of women, breast milk HIV RNA concentrations in the affected breast decreased from the elevated level at the time of symptoms to its baseline level or lower with symptom resolution. In the contralateral breast, breast milk HIV RNA did not change over the period of mastitis.
Our results suggest that there is no long-lasting impact of unilateral mastitis on breast milk viral shedding in either breast. This is consistent with the results from studies of HIV dynamics during acute illness and vaccination. In both circumstances, acute increases in HIV RNA are transient with a return to baseline levels.30,31 Our results also demonstrate that breast milk HIV RNA concentrations are not elevated in the affected breast before the mastitis event. Furthermore, it seems that the regulation of HIV entry into breast milk is independent between breasts because local pathology in one breast does not negatively impact the contralateral breast. Thus, the influence of mastitis and abscess on the risk of transmission to the child is temporary and confined to the affected breast.
Our data further indicated that the degree of clinical symptomatology correlated with HIV concentrations in breast milk. Women with abscess or red/shiny breast had greater change in HIV RNA concentrations from baseline to the symptomatic visit in the affected breast. Potentially, the severity of the symptoms is an indicator of increased breast permeability or inflammation, allowing entry into or increased replication of virus in the breast compartment. Given the data presented and the important correlation between increased RNA quantity and HIV transmission, lactation counselors and nurses counseling HIV-infected women should support appropriate breastfeeding techniques and encourage mastitis-affected women to feed from the contralateral breast only. Improvements in breastfeeding counseling have been shown to correlate with reduced breast problems; therefore, enhanced counseling could both prevent problems developing and provide appropriate advice for those with mastitis.28
The strength of this study is the utilization of women as their own control, which essentially eliminates confounding on fixed and time-dependent variables. The study is further strengthened by the consistency of the results from the within-person analysis to the results from conventional 2 exposure group comparisons. Our study has the added advantage of milk samples from both breasts over time allowing within-person comparisons at single time points.
The study is limited by small sample size, although this is one of the only cohorts with longitudinal milk samples collected from both breasts. Second, because of the length of time between the mastitis/abscess visit and postpathology visit (average of 92 days), it was not possible to determine how quickly breast milk HIV RNA returned to baseline levels. Third, measures of breast milk cell–associated HIV, a predictor of breast milk transmission of HIV, were not available.11,32 Fourth, we had only limited data on some time-varying variables that may be potential confounders, such as other acute illnesses in the mother. We were able to examine if clinic attendance among visits modified the study results, and they did not. Finally, handedness may have been helpful to determine if mastitis was more likely to occur in one breast or the other in our population, as has been demonstrated in other studies.33
With respect to breast complications, such as mastitis and abscess, in HIV-infected women, the current WHO recommendations seem to be appropriate. Breast milk HIV RNA concentrations increase during a period of mastitis or abscess in the affected breast, thus placing the child at increased risk of MTCT. HIV-infected women should refrain from breastfeeding on the affected breast but can continue to breastfeed from the contralateral breast. Breast milk HIV RNA concentrations in the contralateral breast did not significantly increase despite breast pathology in the other breast. Further studies of the physiology of mastitis and abscess and their relationships with breast milk virological dynamics are required to enhance the current guidelines.
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