Chantry, Caroline J. MD*; Young, Sera L. PhD*; Rennie, Waverly MPH†; Ngonyani, Monica RN†; Mashio, Clara†; Israel-Ballard, Kiersten DrPH‡; Peerson, Janet MS§; Nyambo, Margaret MD†; Matee, Mecky MD, PhD‖; Ash, Deborah PhD¶; Dewey, Kathryn PhD§; Koniz-Booher, Peggy DrPH‡
Of the 430,000 pediatric HIV infections acquired in 2008, 90% were attributable to maternal-to-child transmission (MTCT)1; many of these were caused by breastfeeding.2,3 Yet replacement feedings have not improved HIV-free survival4 and have further resulted in poorer growth5 and greater diarrheal morbidity6,7 and mortality.6 Because of breastmilk's critical importance, current World Health Organization (WHO) recommendations for infants born to HIV-infected (HIV+) mothers in developing countries focus on making breastfeeding safer, for example, by encouraging exclusive breastfeeding (EBF), providing antiretroviral (ARV) prophylaxis, and heat-treating breastmilk.8
Heat-treating expressed breastmilk has been a WHO-recommended infant feeding option in the context of maternal HIV for the past decade.8,9 In the 2010 WHO guidelines, heat-treated breastmilk is recommended as an interim feeding strategy, for example, during mastitis, when prophylactic ARVs are unavailable, or to assist mothers to stop breastfeeding. The guidelines note that programmatic data are scarce and call for more research on the feasibility of implementing and sustaining heat-treatment of breastmilk as a strategy to reduce postnatal MTCT.8
Flash-heating (FH) is a simple method for in-home breastmilk pasteurization. Briefly, a glass jar containing the milk is placed in a pan with water 2 finger widths above the level of the milk. The water is heated over high heat and once the water reaches a rolling boil, the milk is removed from the water and cup-fed to the infant when cooled. Milk typically reaches a peak temperature of 72.9° C. Bacteriologic, virologic, immunologic, and nutritional studies have indicated that it can be a safe feeding method.10–13
The primary objective of this study was to evaluate the feasibility of HIV-infected mothers FH expressed breastmilk to feed their infants upon introduction of complementary foods. Specifically, we measured the uptake, frequency, and duration of use and adherence to the Flash-heat protocol.
Subjects and Design
In this prospective longitudinal study, women older than 18 years who were currently breastfeeding infants aged between 6 weeks and 3 months and who were permanent local residents were recruited. Participants were from 4 community health center child immunization clinics in the Amana Municipal Hospital catchment area in the Ilala District of Dar es Salaam. The investigation, described as an infant feeding study, entailed weekly home visits by community health workers and monthly clinic visits. HIV status and CD4+ counts of mothers were confirmed by review of personal medical records. Women receiving or eligible for ARV therapy based on CD4+ ≤200 were excluded to target infants most likely to benefit from the intervention. Both HIV-infected mothers (n = 101) and those with indeterminate or negative status (n = 43) were enrolled to minimize stigma associated with study participation. HIV-infected participants were encouraged to disclose their status to a household member to gain support and facilitate open communication during home visits.
Written informed consent from all participating mothers was obtained. The study was approved by Institutional Review Boards at the National Institute of Medical Research and Muhimbili University of Health and Allied Sciences (MUHAS) in Tanzania and the University of California Davis and was registered at ClinicalTrials.gov (200513446).
Home-based infant feeding counseling was provided by “peer” counselors (11 females and 1 male) with previous experience as breastfeeding counselors in programs sponsored by the Tanzania Food and Nutrition Center but without biomedical training. For this study, they were provided with a 1-week course on HIV and infant feeding training, based on WHO recommendations and Tanzanian National Policy (2007), which included additional training and demonstrations on FH. Periodic “refresher” sessions were provided.
Peer counselors were asked to visit mothers weekly from 2 until 9 months postpartum. They encouraged EBF during the first 6 months of life and appropriate complementary feeding thereafter, instructed mothers on manual expression of breastmilk, and carried illustrated job aids supporting these messages. HIV-infected mothers were considered eligible for counseling on FH if they were breastfeeding an infant documented to be HIV negative at 5 months of age. As an additional safety precaution, mothers were instructed to not feed heated milk to their infant until the first heated sample tested negative for bacterial pathogens. FH was demonstrated to eligible women in their homes, unless the mother requested demonstration at the clinic. Mothers eligible and interested in FH were given a plastic bucket containing an aluminum pan, glass jar, and graduated plastic feeding cup. Those opting to use the method were encouraged to begin before introduction of complementary foods [to avoid mixed feeds (non-EBF)], to heat their milk after each expression and to express and heat their milk as frequently and for as long as possible.
Trained clinic-based nursing staff collected baseline demographic and infant feeding information at study entry and data on infant feeding, growth, and morbidity during subsequent monthly clinic visits. Infant morbidity and FH data were also captured daily by mothers using pictorial logs designed for low literacy. For mothers who were FH breastmilk, peer counselors observed a FH episode biweekly if possible, measured the peak milk temperature, and collected preheat and postheat milk samples for bacterial cultures. Observation checklists of the procedure included noting hand washing, cleansing of jar and cup, appropriate water level in pan, removal of jar of milk from water upon water reaching a rolling boil, and procedure duration. Using a pictorial log, mothers were asked to record milk volume expressed and heated during each episode (estimated by use of graduated cup), length of time required, and whether time was estimated or measured. Biweekly, peer counselors surveyed the mothers in their homes about their FH experiences in the past 24 hours.
Infants with signs or symptoms of illness were referred to clinic staff. Infants testing HIV+ were referred to an HIV care and treatment center and their mothers encouraged to continue breastfeeding. Telephone numbers of study nurses were given to mothers to call with queries or concerns.
Qualitative HIV DNA polymerase chain reaction was performed on dried blood spots collected from infants 5 months old by the National HIV Reference Laboratory in the Department of Microbiology and Immunology at MUHAS utilizing the Amplicor HIV-1 DNA polymerase chain reaction assay version 1.5 (Roche Diagnostics, Branchburg, NJ) according to manufacturer instructions.
Breastmilk aliquots were collected by the counselor in the mother's home using disposable sterile pipettes. The first sample was taken after the community health worker's FH demonstration; subsequent samples were taken after mothers heated their milk. Aliquots of milk (2.0 mL) were placed in sterile vials before heating and again after FH and transported to the bacteriology laboratory at MUHAS on ice within 3 hours of collection. Samples were stored at 2–8°C overnight and then placed at 37°C the following morning. At 0, 3, and 6 hours, aliquots of both unheated and flash-heated breastmilk were each plated on MacConkey's, mannitol salt and blood agars to detect coliforms, Staphylococcus aureus, and total colony counts, respectively.
We estimated a priori that a sample size of 100 HIV-infected women would be necessary to obtain approximately 30 women who were willing to trial FH of an anticipated 75 women who would be eligible for Flash-heat counseling. We hypothesized that at least one-third of women would be willing to try FH. A woman was defined as a “flash-heater” if she chose to use the method after the initial demonstration.
Student t test was used to compare means, and Fisher exact test was used to compare proportions. Growth was compared between groups with analysis of covariance, using change in Z scores at 9 months as the outcome and Z scores at 6 months as the covariate. Mean and median values were calculated for both an individual and by woman-day (unit of 1 woman heating milk for 1 day).
One hundred one HIV-infected Tanzanian women were enrolled between March 1, 2008, and March 31, 2009. Their mean (SD) age was 27.5 (4.8) years (range: 18–41), parity was 2.8 (1.1), and they were 2.4 (0.8) months postpartum (Table 1). Most were married or living with their partner (75.5%), had completed primary school (73.2%), and had disclosed their HIV status to a household member before study entry (75.3%). Most used charcoal for cooking fuel (89.7%), had pit latrines (86.7%), and used either a public tap (49.5%) or shallow well (32.0%) for water.
Eighty-six infants of mothers known to be HIV-infected were alive and in follow-up at 5 months of age when infants were tested for HIV (Fig. 1). Of these, 72 tested HIV negative, 13 tested HIV positive, and 1 infant had no record of testing. Fifty-four of the 72 (75%) mothers eligible for counseling on FH agreed to express milk and observe a flash-heat demonstration.
The mean (SD) number of counselor visits before the infant reached 6 months was 4.5 (6.2). Mothers eligible for FH received 9.7 (5.4) visits. Of those, mothers who chose to flash-heat received 10.5 (5.7) visits versus 8.9 (4.9) for those who opted not to. After 6 months, flash-heaters received an additional 5.3 (4.4) visits for a total of 15.8 (8.1) visits between 2 and 9 months.
Uptake of Flash-Heat
Thirty-seven of the 72 eligible mothers (51.4%) chose FH after their infant reached 6 months (Fig. 1). One infant died at 6.0 months before the mother commenced FH. Mothers who elected to use FH did not differ in age, religion, parity, or education from those who chose not to use FH but may have had lower socioeconomic status as reflected by less use of kerosene stoves, P = 0.042 (Table 1) and anecdotal reports of the counselors. An increasing proportion of eligible mothers chose FH as the study progressed (P = 0.003), that is, 38.8% enrolled in the first 6 months of the study became flash-heaters compared with 78.2% of women enrolled in the subsequent 7 months.
Data on FH
Data on FH behaviors are reported from daily logs and 24-hour recalls and observations. Of the 36 women with FH experience, data are available from logs from 35 women covering 1566 days of FH, from recalled experiences of 27 mothers covering 83 24-hour periods (Table 2) and peer counselors' observations of 80 episodes among 30 mothers expressing and FH milk (Table 3).
Duration of FH
Median (range) duration of FH documented by daily log use during the study was 9.6 weeks (range 1 day to 15.6 weeks); mean (SD) duration of FH during the study was 8.7 (5.3) weeks. Notably, at least 4 women reported FH beyond the period of study follow-up (3 months), 1 of whom continued to do so for an additional 9 months, until her infant was 18 months old. Thus, the reported mean duration of FH during the study is shorter than mean duration of actual use.
In 24-hour recalls, all mothers reported cleansing the utensils. During FH sessions observed by counselors, mothers washed their hands with soap and water 100% of the time. Utensils were washed 95% of the time, with soap and/or boiling water in 78.8% of episodes, and with only running water 16.2% of the time.
During FH observations, milk was expressed into the jar used for heating (as recommended) 21.5% of the time and into a cup or glass during all other episodes. Mean time for expression was 16.7 (7.5) minutes.
The median frequency of milk expression was 3 times daily: daily mean (SD) was 2.6 (0.8) based on log results and 3.3 (1.5) times based on 24-hour recall.
Median milk volume per episode was 120 mL based on daily logs and 24-hour recall and 100 mL during observation. According to recall data, median volumes were higher for fourth and fifth daily expressions (180 mL) than for the first through third expressions (100 mL), that is, women who expressed more frequently also expressed greater volumes. The linear association between total daily milk volume and number of expressions daily was statistically significant, P = 0.006 (Fig. 2). Median (range) total daily volumes were 300 (25–1120) mL from logs and 360 (10–1200) mL from recalls. When controlling for differences between individuals, there was no significant within-individual relationship between age of infant and daily milk volume expressed.
Heating and Feeding Expressed Milk
Mothers heated and fed milk as frequently as they expressed, suggesting milk was not combined from more than 1 expression for heating. Reported time lapse between expressing and heating milk ranged from 10 minutes to 7.5 hours (79% of intervals were measured; 21% estimated).
During FH sessions observed by counselors, kerosene was the most frequently used fuel (42.3%), followed by charcoal. In all but 2 observations, the water in the pan was at the proper level; the milk was removed from the pan before the water boiling in only 1 episode. Observed mean time required for heating was 12.0 (4.6) minutes, and mean peak milk temperature was 79.3 (4.4)°C (range: 68.0–88.8).
Although most often mothers fed the heated milk to the infant immediately (71.6% of reports), heated milk was stored in the jar used for heating (9.9%), a cup (12.3%), or bottle (1.2%) anywhere from 5 minutes to 7.7 hours. Stored milk was reported to be covered 78.6% of the time. Mother reported reusing the water 95% of the time, most often for cooking (50.0%) or bathing (40.8%).
During observed FH sessions, infants were fed the milk with a cup in all cases except once each using a spoon and bottle; water was added to the milk in 3.8% of observations, always after heating the milk.
Bacterial cultures were performed on 105 preheated and postheated samples from 61 mothers. Of the 105 samples collected before heating, 44 (41.9%) samples had bacterial growth with a median colony-forming units (CFU)/mL of 2.4 × 103 (range 80- too many too count). All but 1 of the contaminated samples were positive for coliforms; median coliform count was 2.0 × 103 CFU/mL (range 50- too many too count). Thirty-two samples (30.5%) contained pathogens. Twenty-six samples (24.8%) were positive for S. aureus [median concentration 3.0 × 103 (range: 90–7.8 × 103)]; additional pathogens isolated included Escherichia coli and Klebsiella and Bacillus species. One sample was negative before heating and positive for Staphylococcus epidermedis and coliforms after heating, (total colony count 3.4 × 103 CFU/mL). All other 104 heated samples yielded negative bacterial cultures. Of the 44 mothers who provided more than 1 sample, 3 mothers' samples were contaminated on multiple occasions.
To evaluate the risk for staphylococcal food poisoning if unheated samples were stored before heating, the growth rate of S. aureus was calculated over 6 hours at 37°C. Mean (SD) log CFU/mL of those samples testing positive for S. aureus was 3.22 (0.04) at 0 hours; and at 6 hours, it was 3.44 (0.26), P < 0.003. No samples reached a concentration of 104 CFU/mL.
Mothers reported hiding milk expression in 78.3% of the time and FH in 76.0% of the time; 92.6% of mothers hid these practices at some point—usually from neighbors (78.5% of reports of hiding expression and 65.0% of those hiding FH), with fewer reports of hiding from family (21.5%, 26.6%) or friends (7.7%, 16.7%). Most (71.2%) mothers reported that someone in their family or household knew they were FH; most often this was the infant's father (64.9%) with the most common reactions being “supportive” (41.1%), “neutral” (35.7%), and “curious” (21.4%). Forty-six percent of mothers surveyed explained to others why they FH milk; half of these mothers gave the reason of killing HIV and one-third said it was because of a breast infection or problem. Ten percent of mothers reported newly disclosing their HIV status during the study.
There was no statistically significant difference in number of morbidities (number of days ill with each symptoms as follows: diarrhea, cough, runny nose, fever, vomiting, rash, or ear infection) between 6 and 9 months in infants who received versus those who did not receive flash-heated breastmilk, but there was a trend toward fewer ear infections in those receiving heated breastmilk, P = 0.08. Growth was also not significantly different between the 2 groups of infants.
Breast and Nipple Pain
After 6 months postpartum, there were 4 reports of breast or nipple pain within the past 2 weeks from 3 mothers; 2 who did not flash-heat compared with 1 flash-heater P = 0.58.
Dietary data from 24-hour recalls (n = 217) after 6 months of age for the 34 infants fed heated breastmilk versus the 28 who were not revealed no difference in the mean daily number of servings of meat [0.46 (0.39) vs. 0.45 (0.49), P = 0.56], meat stew [0.05 (0.18) vs. 0.11 (0.27), P = 0.46] or eggs [0.01 (0.09) vs. 0.06 (0.20), P = 0.24]. However, infants not receiving expressed heated breastmilk received a slightly greater mean (SD) number of daily servings of other animal milks than did those receiving human milk, 1.5 (0.9) versus 1.0 (0.7), P = 0.03.
In 3 of 83 (3.6%) surveys of flash-heaters, the infant was fed directly at the breast in the last week, from 2 to 10 times.
This study has several important findings. Foremost, a substantial percent (51.4%) of HIV-infected mothers whose infants tested HIV negative at 5 months were willing to express and flash-heat their breastmilk upon introduction of complementary foods. This was a greater proportion than the 33% we anticipated. It is notable that the percentage of mothers that chose to flash-heat dramatically increased over the course of the study, possibly as a result of the counselors' greater experience. Second, we observed that mothers were able to successfully follow the FH protocol, and the heated milk was bacteriologically safe. Third, the amount of breastmilk provided to these infants substantially contributed to their diet. The mean daily volume of 322 mL constitutes approximately 34% of caloric needs for an average 6 month infant (weight 7.6 kg)14 and increased by 2-fold to 3-fold the mean amount of animal milk they otherwise received (1 serving daily). Fourth, most mothers sustained this practice for at least 2 months, with duration varying widely from 1 day to 1 year. Last, these mothers succeeded in FH with only modest support from trained peer counselors rather than healthcare providers, suggesting this counseling could be sustainable in terms of costs and workforce capacity at a larger scale in resource-limited settings.
In addition to the wide variation in duration of method use, mothers in our study reported a huge range of milk volume expressed daily, from a mean of only 46 mL to nearly a liter. Anti-infective effects of breastmilk are dose responsive, and even partial breastfeeding has been documented to decrease infections in infants.15,16 There may be a threshold effect, however, and the lower amounts of daily breastmilk expressed by some mothers in this study may not provide significant immunoprotection although the greater amounts almost certainly would.15 Of note, this feasibility study was not powered to detect differences in growth and morbidity and should not be used to infer that clinically significant differences would not occur in a larger trial.
Milk volumes at the lower end of the range expressed by mothers in our study likely relate, in part, to relatively infrequent expression (mean 2.3 episodes recorded daily) as milk production declines if the breasts are not emptied regularly and thoroughly.17,18 Accordingly, we expect that mothers with more frequent expression would be better able to maintain a robust milk supply, producing greater mean milk volumes, and sustaining milk production for longer durations. This expectation is supported by our data whereby mean reported volumes of milk expression for the fourth and fifth episodes in the previous day were greater than mean reported volumes for the first through third episodes, that is, women expressing more frequently had both larger volumes per expression and larger daily volumes.
One possible strategy that may enable mothers to express milk more frequently would be for them to “batch heat” milk from 2 or more expressions. This method, involving heating more than 1 jar of milk in a pan simultaneously, is currently under investigation (K. Israel-Ballard, DrPh, personal communication March 15, 2011). However, it will be necessary to document if peak milk temperatures with “batch heating” are similar to those attained when heating a single jar (the method used during virologic safety studies) before recommending this method.
In all contaminated specimens that we tested, bacterial growth that occurred during 6 hours of storage was minimal, albeit statistically significant, and did not result in bacterial counts considered potentially unsafe (105 CFU/mL)17; heating eliminated all pathogens. We surmise that the 1 sample that was positive for bacteria after heating was contaminated after heating, as the preheated sample was bacteriologically negative. Nevertheless, if milk is to be stored, we recommend that milk be heated before storage, when possible, to further minimize the “theoretical” risk of staphylococcal food poisoning. It is also important to note that current recommendations are that expressed human milk may be stored for 6–8 hours at ambient temperatures up to 25°C,19 with conflicting evidence regarding storage safety at higher temperatures.20–22 We have previously demonstrated that FH breastmilk does not significantly impact the antimicrobial properties of the milk23 but note the need for more work to determine the “shelf-life” of expressed breastmilk, heated or unheated, at ambient temperatures above 25°C.
The proportion of mothers who chose to express and heat treat their milk was somewhat less than the 66% of 30 eligible mothers who did so in a similar study in rural Zimbabwe.24 The high rate of uptake in the Zimbabwean study may have been due, in part, to a social marketing campaign that promoted expression and heat-treatment of breastmilk for any mother (even those who are HIV-negative). Our study was performed in the absence of such generalized messages. Although that approach likely decreases stigma, such messages may have unintended consequences, eg, creating a barrier for HIV-negative women who might otherwise leave fresh expressed breastmilk for their infant when separated. Another salient difference between our study and that of Mbuya et al24 is that in our study, education and support about FH was provided by lay counselors with a much less intensive home visit schedule (mean of 15.8 visits over 7 m) than did the nurse counselors in Zimbabwe (21 visits over 8 weeks). Use of lay counselors could be a more cost-effective and sustainable practice given the dearth of health care workers in countries hardest hit by HIV/AIDS25 and where limited resources make breastfeeding imperative.26
There are several limitations to this study. This population was self-selected as we enrolled willing participants in a research study on infant feeding, and therefore does not necessarily represent the population of all HIV-infected mothers in the area. Women with the most supportive home environments may have enrolled, potentially resulting in a greater proportion of mothers opting to try FH. Alternatively, the burden of study participation may have deterred mothers who would otherwise have chosen to provide Flash-heated breastmilk to their uninfected infants. Further, some of the questionnaires were administered by the counselors who were supporting EBF and FH, possibly resulting in reporting bias.
In summary, our findings add significantly to understanding the feasibility of heat-treatment of breastmilk and reinforce those from Zimbabwe that suggest FH is feasible for many HIV-infected women. Although extended ARV prophylaxis for either the breastfeeding mother or her breastfed child is currently considered to be the optimal strategy for prevention of mother-to-child transmission (PMTCT),8 the low coverage of ARV prophylaxis in sub-Saharan Africa27 suggests the need for complementary PMTCT strategies. As with all medical interventions in resource-limited settings, ARV agents are sometimes temporarily unavailable due to “stock-outs”; it will be important for mothers to be aware of the WHO-recommended option to express and heat treat their breastmilk during this time of increased transmission risk. In addition to the 4 scenarios for which use of heat-treated breastmilk is currently recommended by WHO,8 Flash-heated breastmilk could also be provided after ARV cessation at 12 months to further prolong provision of safe breastmilk to these vulnerable infants. Indeed, breastmilk continues to have dual nutritional and immunoprotective importance beyond the first year, the latter evidenced by the 1.6-fold increase in mortality from infectious causes amongst nonbreastfed infants in resource-poor settings compared with their breastfed counterparts in the second year of life.28
Based on the feasibility data to date, a clinical trial of the effects of flash-heated breastmilk on infant health outcomes is warranted. Only with such information will it be possible to provide both PMTCT programs and the families they serve with evidence-based recommendations. Finally, as countries seek to implement the 2010 WHO guidance, rigorous monitoring and evaluation around their inclusion of heat-treatment within PMTCT programs is necessary to appropriately guide scale-up of such strategies.
The authors gratefully acknowledge funding from the National Institutes of Health (NIH R01HD057602) and the Thrasher Research Fund. The authors warmly thank Makilika study staff and the mothers and babies who participated in the study.
1. UNAIDS. AIDS Epidemic Update. Geneva, Switzerland: WHO; 2009.
2. Horvath T, Madi BC, Iuppa IM, et al.. Interventions for preventing late postnatal mother-to-child transmission of HIV. Cochrane Database Syst Rev. 2009; CD006734.
3. Kourtis AP, Lee FK, Abrams EJ, et al.. Mother-to-child transmission of HIV-1: timing and implications for prevention. Lancet Infect Dis. 2006;6:726–732.
4. Kuhn L, Sinkala M, Semrau K, et al.. Elevations in mortality associated with weaning persist into the second year of life among uninfected children born to HIV-infected mothers. Clin Infect Dis. 2010;50:437–444.
5. Arpadi S, Fawzy A, Aldrovandi GM, et al.. Growth faltering due to breastfeeding cessation in uninfected children born to HIV-infected mothers in Zambia. Am J Clin Nutr. 2009;90:344–353.
6. Kafulafula G, Hoover DR, Taha TE, et al.. Frequency of gastroenteritis and gastroenteritis-associated mortality with early weaning in HIV-1-uninfected children born to HIV-infected women in Malawi. J Acquir Immune Defic Syndr. 2010;53:6–13.
7. Onyango-Makumbi C, Bagenda D, Mwatha A, et al.. Early weaning of HIV-exposed uninfected infants and risk of serious gastroenteritis: findings from two perinatal HIV prevention trials in Kampala, Uganda. J Acquir Immune Defic Syndr. 2010;53:20–27.
10. Israel-Ballard K, Coutsoudis A, Chantry CJ, et al.. Bacterial safety of flash-heated and unheated expressed breastmilk during storage. J Trop Pediatr. 2006;52:399–405.
11. Israel-Ballard K, Donovan R, Chantry C, et al.. Flash-heat inactivation of HIV-1 in human milk: a potential method to reduce postnatal transmission in developing countries. J Acquir Immune Defic Syndr. 2007;45:318–323.
12. Chantry CJ, Israel-Ballard K, Moldoveanu Z, et al.. Effect of flash-heat treatment on immunoglobulins in breast milk. J Acquir Immune Defic Syndr. 2009;51:264–267.
13. Israel-Ballard KA, Abrams BF, Coutsoudis A, et al.. Vitamin content of breast milk from HIV-1-infected mothers before and after flash-heat treatment. J Acquir Immune Defic Syndr. 2008;48:444–449.
14. Food and Agricultural Organization of the United Nations. Human Energy Requirements: Report of a Joint FAO/WHO/UNU Expert Consultation. October 17–24, 2001;Rome, Italy. 2004. Available at: ftp://ftp.fao.org/docrep/fao/007/y5686e/y5686e00.pdf
. Accessed June 13, 2011.
15. Raisler J, Alexander C, O'Campo P. Breast-feeding and infant illness: a dose-response relationship? Am J Public Health. 1999;89:25–30.
16. Bilenko N, Ghosh R, Levy A, et al.. Partial breastfeeding protects Bedouin infants from infection and morbidity: prospective cohort study. Asia Pac J Clin Nutr. 2008;17:243–249.
17. Lawrence RA, Lawrence LR. Breastfeeding: A Guide for the Medical Profession. 7th ed. Philadelphia, PA: Elsevier Mosby; 2010.
18. Academy of Breastfeeding Medicine Protocol Committee. ABM Clinical Protocol #9: Use of Galactogogues in Initiating or Augmenting the Rate of Maternal Milk Secretion (First Revision January 2011). Breastfeed Med. 2011;6:41–49.
19. Academy of Breastfeeding Protocl Committee, Eglash, A. ABM clinical protocol #8: human milk storage information for home use for full-term infants (original protocol March 2004; revision #1 March 2010). Breastfeed Med. 2010;5:127–130.
20. Hamosh M, Ellis LA, Pollock DR, et al.. Breastfeeding and the working mother: effect of time and temperature of short-term storage on proteolysis, lipolysis, and bacterial growth in milk. Pediatrics. 1996;97:492–498.
21. Ajusi JD, Onyango FE, Mutanda LN, et al.. Bacteriology of unheated expressed breast milk stored at room temperature. East Afr Med J. 1989;66:381–387.
22. Igumbor EO, Mukura RD, Makandiramba B, et al.. Storage of breast milk: effect of temperature and storage duration on microbial growth. Cent Afr J Med. 2000;46:247–251.
23. Chantry CJ, Wiedeman J, Buehring G, et al.. Effect of flash-heat treatment on antimicrobial activity of breastmilk. Breastfeed Med. 2011;6:111–116.
24. Mbuya MN, Humphrey JH, Majo F, et al.. Heat treatment of expressed breast milk is a feasible option for feeding HIV-exposed, uninfected children after 6 months of age in rural Zimbabwe. J Nutr. 2010;140:1481–1488.
25. Nakakeeto ONL, Umaranayake L. The global strategy to eliminate HIV infection in infants and young children: a seven-country assessment of costs and feasibility. AIDS. 2009;23:987–995.
26. Young SL, Mbuya MN, Chantry CJ, et al.. Current knowledge and future research on infant feeding in the context of HIV: basic, clinical, behavioral, and programmatic perspectives. Adv Nutr. 2011;2:225–243.
28. WHO Collaborative Study Team on the Role of Breastfeeding on the Prevention of Infant Mortality. Effect of breastfeeding on infant and child mortality due to infectious diseases in less developed countries: a pooled analysis. Lancet. 2000;355:451–455.
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