Abstract

Although subclinical mastitis is associated with increased HIV load in breast milk, it is not known whether empirical treatment with antibiotics will reduce breast milk HIV load. We examined the effect of antibiotic treatment for subclinical mastitis on HIV load in breast milk. Seventy-five HIV-infected post-partum women in Malawi with subclinical mastitis were treated with oral amoxicillin/clavulanic acid and were followed between 1 and 24 weeks later. Breastmilk HIV-1 load and sodium concentration were measured and microbiological studies were performed at presentation. At 1 week (n = 34), the proportion of women with elevated breast milk leukocyte counts decreased significantly to 41.2% (p < 0.0001) and there was a nonsignificant increase in breast milk HIV-1 RNA load (p = 0.9264) and sodium concentration (p = 0.08) in the affected breast. At 4 to 12 weeks (n = 63), breast milk HIV-1 RNA load and sodium concentration decreased significantly (p < 0.05) and 17.5% had elevated breast milk leukocyte counts. Treatment with amoxicillin/clavulanic acid was associated with a significant decrease in inflammation of the breast, but breast milk HIV load remained elevated despite a significant decrease from baseline. These findings have important implications regarding how mothers should be counselled on safety of resuming breastfeeding after resolution of subclinical mastitis.

Introduction

Breastfeeding is estimated to account for about one-third of mother-to-child transmission of human immunodeficiency virus (HIV) in sub-Saharan Africa [1] as human milk contains HIV [2, 3]. Subclinical mastitis, which can be diagnosed on the basis of elevated milk leukocyte count or elevated sodium or sodium/potassium ratio, is common during breastfeeding [4, 5] and has been associated with poor infant growth [4], increased HIV load in breast milk and increased risk of mother-to-child transmission of HIV [3, 6]. Treatment for mastitis is largely empirical and is generally aimed at Staphylococcus aureus, the predominant organism cultured from breast milk during mastitis [7].

In many populations in sub-Saharan Africa where there is limited access to clean water, adequate hygiene, and safe formula, HIV-infected women are advised to breastfeed, as the benefits of breastfeeding outweigh the risks [8]. This highlights the need to find interventions that may reduce mother-to-child transmission of HIV in mothers who have no alternatives to breastfeeding. Although subclinical mastitis is associated with increased HIV load in breast milk, it is not known whether empirical treatment with antibiotics will help reduce breast milk HIV load. We examined the effect of antibiotic treatment for subclinical mastitis on HIV load in breast milk among women in Malawi.

Patients and Methods

The study population consisted of HIV-positive breastfeeding women who gave birth at Queen Elizabeth Central Hospital in Blantyre, Malawi, and were participating in a longitudinal study of the epidemiology, microbiology and treatment of subclinical mastitis. Two hundred and fifty HIV-positive women were enrolled in the study at 2 weeks post-partum and were seen at scheduled visits at 4, 6, 10, and 14 weeks, and 6, 9, and 12 months after delivery. Women were eligible for the study if they were HIV-positive, chose to breast-feed, were resident in the Blantyre District, and were at least 18 years of age. Written, informed consent was obtained from all participants enrolled in this study. At each visit, 5–7 ml of breast milk was obtained from each breast by manual expression after carefully sterilizing the nipple and areola with betadine solution. 10 μl of milk was placed on a somatic cell count slide (Bellco, Vineland, NJ), air-dried, and placed in methanol for five seconds. The slides were immersed in LW/NL-X stain containing 0.5 g of methylene blue chloride, 56 ml of 95% ethyl alcohol, 40 ml of xylene, and 4 ml of glacial acetic acid [9]. Slides were dried and then viewed under a microscope at 100× magnification. Women with subclinical mastitis (milk leukocyte counts ≥1 million cells/ml) [5] were treated with oral amoxicillin/clavulanic acid (Augmentin®), 375 mg, three times per day for 10 days. Follow-up visits were scheduled for 2 days and 1 week after diagnosis in order to monitor the presence of inflammation. Women with mastitis were counselled to avoid breastfeeding from the affected breast and to void the affected breast periodically. Microbiological studies were conducted for milk samples with leukocyte counts ≥1 million cells/ml. Milk was plated on blood agar, MacConkey agar, and Sabouraud agar plates and incubated at 35–37°C. Plates were examined at 24, 48, and 72 hours for growth.

Breast milk HIV load was measured for the breast with subclinical mastitis as well as the contralateral, unaffected breast for all cases detected during the study. Breast milk HIV load was measured in the affected breast at 1 week follow up and at a follow-up visit ≥4 weeks from diagnosis. If breast milk was not available at 1 week, breast milk HIV load was measured at the next follow-up visit ≥4 weeks from diagnosis. Follow-up visits ranged from 1 week to 24 weeks. One week was defined as 5 to 10 days after diagnosis. HIV-1 load was measured in whole milk using quantitative reverse transcriptase polymerase chain reaction (PCR) (Roche Amplicor HIV-1 Monitor version 1.5, Roche Diagnostics, Indianapolis, Indiana) with a sensitivity limit of 400 copies/ml. Breast milk HIV load concentrations reported as undetectable were set at 200 copies/ml, which is the median between the lower limit of detection and zero. For sodium measurement, breast milk samples were centrifuged at 1300 g for 7 min, and the lipid and aqueous portions were separated and removed. The aqueous portion was analyzed for sodium concentration by the use of ion-selective electrodes (Boehringer Mannheim/Hitachi 747 analyzer, Roche/Boehringer Mannheim, Indianapolis) in the Department of Pathology, Johns Hopkins Hospital.

Log10 transformation was used for breast milk HIV load concentrations. Differences in geometric mean HIV loads were compared using a paired t-test. The level of significance in this study was p<0.05.

Results

There were a total of 75 cases of subclinical mastitis that had follow-up visits at either 1 week or at a subsequent scheduled visit. Sixty-three cases had follow-up at 4 to 12 weeks and thirty of those also had follow-up at 1 week. Four cases had follow-up at 1 week only and 8 cases had follow-up at 12 and 24 weeks only. At baseline, the mean log10 HIV load (SD) for all breast milk collected from the breast with subclinical mastitis and the contralateral, unaffected breast was 2.94 (0.82) and 2.55 (0.63), respectively (p<0.0001), or geometric mean of 862 copies/ml and 357 copies/ml, respectively.

The proportion of women with elevated leukocyte counts indicative of subclinical mastitis decreased significantly from baseline (100%) to the 1 week follow-up visit (41.2%) (p<0.0001). Mean log10 HIV load (SD) in the affected breast for cases with 1 week follow-up (n = 34) was 3.11 (0.89), or geometric mean of 1276 copies/ml before treatment and 3.12 (0.89), or geometric mean of 1328 copies/ml (p = 0.93) at 1 week (Table 1). Breast milk sodium concentration (mmol/l) (SD) at baseline and 1 week was 31.8 (32.0) and 40.4 (36.2), respectively (p = 0.08) for the affected breast and 8.4 (6.6) and 9.8 (11.7), respectively, for the control breast (p = 0.53).

Table 1

Laboratory characteristics at baseline and 1 week (n = 34)

 Baseline 1 Week p value 
Elevated leukocyte counta (%) 100 41.2 <0.0001 
Mean log10 HIV load (±SD) 
    Affected breast 3.11 (0.89) 3.12 (0.89) 0.93 
Sodium Concentration (mmol/l) (±SD) 
    Affected breast 31.8 (32.0) 40.4 (36.2) 0.08 
    Control breastb 8.4 (6.6) 9.8 (11.7) 0.52 
 Baseline 1 Week p value 
Elevated leukocyte counta (%) 100 41.2 <0.0001 
Mean log10 HIV load (±SD) 
    Affected breast 3.11 (0.89) 3.12 (0.89) 0.93 
Sodium Concentration (mmol/l) (±SD) 
    Affected breast 31.8 (32.0) 40.4 (36.2) 0.08 
    Control breastb 8.4 (6.6) 9.8 (11.7) 0.52 

aMilk leukocyte counts ≥1 million cells/ml.

bBreast milk sodium concentration at baseline from the control breast was lower than for the affected breast breast (p<0.001).

Eleven women (17.5%) had elevated leukocyte counts indicative of subclinical mastitis at the 4 to 12 week follow-up visit, a significant decrease from baseline (p<0.0001). Mean log10 HIV load (SD) for milk collected from the affected breast of these mothers (n = 63) was 2.99 (0.84) before treatment and 2.76 (0.76) at follow-up (p<0.05), or geometric mean of 964 and 643 copies/ml, respectively (Table 2). Mean log10 HIV load and sodium concentration in breast milk at baseline, 1 week and 4 to 12 weeks are shown in Fig. 1 and Fig. 2, respectively. Breast milk sodium concentration (mmol/l) (SD) at baseline and at 4 to 12 weeks was 36.7 (35.7) and 26.3 (29.1), respectively (p<0.05) for the affected breast and 11.6 (20.6) and 14.8 (24.4), respectively, for the control breast (p = 0.46). For women with follow up at 12–24 weeks (n = 8), mean log10 HIV load (SD) at baseline and follow-up was 2.48 (0.33) and 2.44 (0.40), respectively (p = 0.76), and there were no cases of subclinical mastitis at follow-up. Seven of the 8 women had breast milk HIV loads below the lower limit of detection at baseline and at follow-up.

Table 2

Laboratory characteristics at baseline and 4 to 12 weeks (n = 63)

 Baseline 4 to 12 weeks p value 
Elevated leukocyte counta (%) 100 17.5 <0.0001 
Mean log10 HIV load (±SD) 
    Affected breast 2.99 (0.84) 2.76 (0.76) <0.05 
Sodium Concentration (mmol/l) (±SD) 
    Affected breast 36.7 (35.7) 26.3 (29.1) <0.05 
    Control breastb 11.6 (20.6) 14.8 (24.4) 0.46 
 Baseline 4 to 12 weeks p value 
Elevated leukocyte counta (%) 100 17.5 <0.0001 
Mean log10 HIV load (±SD) 
    Affected breast 2.99 (0.84) 2.76 (0.76) <0.05 
Sodium Concentration (mmol/l) (±SD) 
    Affected breast 36.7 (35.7) 26.3 (29.1) <0.05 
    Control breastb 11.6 (20.6) 14.8 (24.4) 0.46 

aMilk leukocyte counts ≥1 million cells/ml

bBreast milk sodium concentration at baseline from the control breast was significantly lower than for the affected breast (p<0.0001).

Fig. 1.

Mean Breast milk HIV load at baseline, 1 week and 4–12 weeks for mastitic and control breasts.

Fig. 1.

Mean Breast milk HIV load at baseline, 1 week and 4–12 weeks for mastitic and control breasts.

Fig. 2.

Mean breast milk sodium at baseline, 1 week and 4–12 weeks for mastitic and control breasts.

Fig. 2.

Mean breast milk sodium at baseline, 1 week and 4–12 weeks for mastitic and control breasts.

Culture results were available for 56 cases at the time of diagnosis of mastitis, but due to equipment failure, culture results could not be obtained for 19 women. Twenty-seven (48.2%) of the 56 cases had sterile breast milk and 29 (51.8%) had positive breast milk cultures. Of these 29 cases, 20 (69%) had positive cultures for Staphylococcus aureus, 7 (24.1%) had a positive culture for S. epidermidis, and 2 (6.9%) had positive cultures for Staphylococcus but the species was not determined. The proportion of cases with elevated leukocyte counts indicative of subclinical mastitis decreased significantly to 53.8% by 1 week (p<0.01) and 29.2% by 4 to 12 weeks (p<0.0001) for women with positive breast milk cultures before treatment and to 40% by 1 week (p<0.01) and 13.6% by 4–12 weeks (p<0.0001) for mothers with sterile milk at baseline. Mean log10 HIV load in breast milk at baseline was not significantly different for mothers with sterile milk and those with culture positive milk (p = 0.39). There was a significant decrease in mean log10 HIV load in breast milk by 1 week and 4 to 12 weeks for mothers who had sterile milk at diagnosis (p<0.02) but a non-significant decrease for mothers with positive culture results at diagnosis (p > 0.05). Breast milk sodium concentration (SD) for the affected breast of mothers with culture positive breast milk at baseline and 4 to 12 weeks was 45.9 (38.8) and 31.3 (30.4), respectively (p = 0.0698) and 29.6 (30.2) and 15.4 (15.9), respectively (p = 0.0528) for those with sterile milk.

Discussion

This longitudinal study suggests that women with subclinical mastitis who are treated with antibiotics have a reduction in breast milk leukocyte count. However, the effect of inflammation on the alveolar ducts of the affected breast remains several weeks after treatment. The increased presence of plasma components and HIV-1 infected lymphocytes in breast milk resulting from the opening of paracellular pathways between mammary alveolar cells during mastitis are thought to increase HIV load in breast milk [3]. The duration of clinically apparent mastitis is generally seven days without treatment and two days in mastitis treated with antibiotics [7], however, the time it takes for breast milk HIV load to decrease after inflammation has resolved is unknown. In our study, 1 week follow-up after presentation with subclinical mastitis was insufficient to demonstrate a significant reduction in breast milk HIV load despite a greater than 50% reduction in the proportion of mothers with elevated breast milk leukocyte counts. After 4 to 12 weeks, breast milk HIV load decreased significantly from baseline but remained higher than the HIV load in the unaffected breast at baseline.

Breast milk normally contains 5–6 mmol/l of sodium [10–12]. Elevated breast milk sodium concentration is considered a sensitive indicator of mastitis [10, 13, 14] and occurs when paracellular pathways between mammary alveolar cells open up due to inflammation, allowing extracellular fluid containing sodium to enter the milk [13–17]. In our study, sodium concentration in the affected breast did not decrease significantly until 4 to 12 weeks, paralleling the changes observed in HIV viral load in the same breast. Although reduced, the breast milk sodium concentration at 4 to 12 weeks was still elevated indicating residual increased permeability in the affected breast. These findings suggest that while inflammation of the mammary glands may decrease rapidly after treatment with antibiotics, the opened paracellular pathways between alveolar cells may take longer to regain their integrity and therefore continue to allow plasma components and extracellular fluid to enter the breast milk. Although mothers were counselled to void the affected breast periodically, it is possible that incomplete emptying may have added to high breast milk sodium concentration at 4 to 12 weeks.

Previous studies of clinical mastitis have shown that Staphylococcus aureus is isolated in about 40–50% of cases [15, 18–20] and normal skin and respiratory flora are isolated in about half the cases [21]. In this study, Staphylococcus aureus was the predominant organism isolated from cases with positive cultures at diagnosis however, half of our cases had sterile milk at baseline. It has been postulated that ‘non-infectious’ mastitis may be due to blocked ducts, breast engorgement or physical injury to the breast which may result in a local inflammatory response [22]. In the present study, there was a significant decrease in the proportion of cases with elevated leukocyte counts indicative of subclinical mastitis after treatment with amoxicillin/clavulanic acid among mothers with sterile breast milk at diagnosis, suggesting these cases may represent early or low grade infections and not inflammatory mastitis due to obstructed milk drainage. Breast milk leukocyte count was detected at prescheduled follow-up visits, not on the basis of symptoms of mastitis, and it is possible that certain cases had progressed longer than others by the time they were detected.

Although the culture findings of Staphylococcus aureus in a substantial proportion of our cases suggests that amoxicillin/clavulanic acid is an appropriate empirical treatment, women with positive breast milk cultures at baseline did not have a significant decrease in breast milk HIV load. It is possible that these women had more extensive disruption of cellular integrity at the time of presentation and that HIV load takes longer to decrease in these women. A limitation of this study is that it was not a placebo-controlled clinical trial of amoxicillin/clavulanic acid, and thus decreases in leukocyte counts and breast milk HIV load cannot be attributed with certainty to antibiotic treatment.

Formula feeding, maternal antiretroviral therapy, and infant antiretroviral prophylaxis could reduce the risk of mother-to-child transmission of HIV, especially for infants whose mothers do not experience any symptoms and therefore do not seek treatment. These options, unfortunately, are not viable for many mothers, especially in sub-Saharan Africa [8] and more feasible interventions are needed. The findings of this study have important implications regarding how mothers should be counselled on the safety of resuming breastfeeding after resolution of subclinical mastitis since a notable decrease in inflammation preceded a significant decrease in HIV viral load in breast milk. Further investigation is needed to evaluate appropriate management and treatment of mastitis in HIV-positive women to minimize the risk of vertical transmission through breastfeeding until antiretroviral medications become more widely available for mothers in sub-Saharan Africa.

References

1
Van de Perre
P
Mother-to-child transmission of HIV-1: the ‘all mucosal’ hypothesis as a predominant mechanism of transmission
AIDS
 , 
1999
, vol. 
13
 (pg. 
1133
-
6
)
2
Pillay
K
Coutsoudis
A
York
D
, et al.  . 
Cell-free virus in breast milk of HIV-1-seropositive women
J Acquir Immune Defic Syndr
 , 
2000
, vol. 
24
 (pg. 
330
-
6
)
3
Semba
RD
Kumwenda
N
Hoover
DR
, et al.  . 
Human immunodeficiency virus load in breast milk, mastitis, and mother-to-child transmission of human immunodeficiency virus type 1
J Infect Dis
 , 
1999
, vol. 
180
 (pg. 
93
-
8
)
4
Filteau
SM
Rice
AL
Ball
JJ
, et al.  . 
Breast milk immune factors in Bangladeshi women supplemented postpartum with retinol or β-carotene
Am J Clin Nutr
 , 
1999
, vol. 
69
 (pg. 
953
-
8
)
5
Semba
RD
Neville
MC
Breast-feeding, mastitis, and HIV transmission: nutritional implications
Nutr Rev
 , 
1999
, vol. 
57
 (pg. 
146
-
53
)
6
Willumsen
JF
Filteau
SM
Coutsoudis
A
, et al.  . 
Subclinical mastitis as a risk factor for mother-infant HIV transmission
Adv Exp Med Biol
 , 
2000
, vol. 
478
 (pg. 
211
-
23
)
7
Semba
RD
Mastitis and vertical transmission of HIV among breastfeeding women
Prenat Neonat Med
 , 
1999
, vol. 
4
 (pg. 
370
-
5
)
8
Georgeson
JC
Filteau
SM
Physiology, immunology, and disease transmission in human breast milk
AIDS Patient Care STDs
 , 
2000
, vol. 
14
 (pg. 
533
-
9
)
9
Packard
VS
Tatini
S
Fugua
R
, et al.  . 
Marshall
RT
Direct Microscopic methods for bacteria or somatic cells
Standard Methods for the Examination of Dairy Products
 , 
1992
16th
Washington D.C
American Public Health Association
(pg. 
318
-
9
)
10
Neville
MC
Keller
RP
Seacat
J
, et al.  . 
Studies on human lactation. I. Within feed and between-breast variation in selected components of human milk
Am J Clin Nutr
 , 
1984
, vol. 
40
 (pg. 
635
-
46
)
11
Ereman
RR
Lönnerdal
B
Dewey
KG
Maternal sodium intake does not affect postprandial sodium concentrations in human milk
J Nutr
 , 
1987
, vol. 
117
 (pg. 
1154
-
7
)
12
Aperia
A
Broberger
O
Herin
P
, et al.  . 
Salt content in human breast milk during the three first weeks after delivery
Acta Paediatr Scand
 , 
1979
, vol. 
68
 (pg. 
441
-
2
)
13
Neville
MC
Allen
JC
Archer
PC
, et al.  . 
Studies in human lactation: milk volume and nutrient composition during weaning and lactogenesis
Am J Clin Nutr
 , 
1991
, vol. 
54
 (pg. 
81
-
92
)
14
Connor
AE
Elevated levels of sodium and chloride in milk from mastitis breast
Pediatrics
 , 
1979
, vol. 
63
 (pg. 
910
-
1
)
15
Thomsen
AC
Espersen
T
Maigaard
S
Course and treatment of milk stasis, noninfectious inflammation of the breast, and infectious mastitis in nursing women
Am J Obstet Gynecol
 , 
1984
, vol. 
149
 (pg. 
492
-
5
)
16
Lawrence
RA
Breastfeeding: a guide for the medical profession
 , 
1994
St. Louis
Mosby
17
Morton
JA
The clinical usefulness of breast milk sodium in the assessment of lactogenesis
Pediatrics
 , 
1994
, vol. 
93
 (pg. 
802
-
6
)
18
Devereux
WP
Acute puerperal mastitis
Am J Obstet Gynecol
 , 
1970
, vol. 
108
 (pg. 
78
-
81
)
19
Niebyl
JR
Spence
MR
Parmley
TH
Sporadic (nonepidemic) puerperal mastitis
J Reprod Med
 , 
1978
, vol. 
20
 (pg. 
97
-
100
)
20
Matheson
I
Aursnes
I
Horgen
M
, et al.  . 
Bacteriological findings and clinical symptoms in relation to clinical outcome in puerperal mastitis
Acta Obstet Gynecol Scand
 , 
1988
, vol. 
67
 (pg. 
723
-
6
)
21
Willumsen
JF
Filteau
SM
Coutsoudis
A
, et al.  . 
Breastmilk RNA viral load in HIV-infected South African women: effects of subclinical mastitis and infant feeding
AIDS
 , 
2003
, vol. 
17
 (pg. 
407
-
14
)
22
Fetherston
C
Risk factors for lactation mastitis
J Hum Lact
 , 
1998
, vol. 
14
 (pg. 
101
-
9
)

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