Our results indicate that 79.4% (95%CI: 74.0 – 84.0) of HIV infected mothers shed HIV in breast milk in the absence of ART during continued breastfeeding. As expected, the prevalence of HIV shedding in late breast milk when considering both CAV and CFV is significantly higher than prevalence estimates based on either CAV alone [62.1% (95%CI: 55.9%–67.9%) or CFV alone [65.7% (95%CI: 59.6%–71.4%), however prevalence estimates based on CAV alone and CFV alone did not differ significantly. Our estimates of CAV and CFV shedding are in line with earlier studies in the region.[7,8]
Our results also confirm earlier findings that CD4 count, plasma viral load, and subclinical mastitis are independently associated with HIV shedding in breast milk. In addition to these known determinants, we also found an independent significant negative association between concurrent use of combined oral contraceptive and HIV-1 shedding in breast milk. Of the identified determinants, only subclinical mastitis affects both CFV and CAV shedding and severe SCM, as an indicator of mammary gland inflammation, is by far the most important determinant HIV shedding in breast milk. Our results also indicate that whereas severe SCM is associated with both CFV and CAV, mild SCM is only associated with CFV and has no effect on CAV.
This study was done prior to the introduction of life-long ART for all in Zambia. Since 2016 all pregnant and lactating women living with HIV are started on ART for life (option B+), thus, study participants were not on ART according to national guidelines at the time. Conducting such a study currently would be unethical as all breastfeeding mothers are now supposed to be on ART. Although our results cannot be extrapolated to mothers on ART, they provide some kind of baseline data on wild type HIV that studies on mothers on ART can compare with. Our results however would apply to breastfeeding mothers still not on ART despite option B+ implementation due to suboptimal retention[22,23] or poor coverage in some places. Latest estimates by UNAIDS reported that half of the pediatric HIV infection post-partum could be attributable to mother not on ART.
Despite not being on ART for several months (9 months), we could not detect any HIV (CAV or CAV) in breast milk samples from both breasts of about 20% mothers, suggesting some mothers naturally do not shed HIV in breast milk and therefore their infants may not be exposed to breast milk HIV. Mechanisms governing this resistance to HIV shedding in breast milk are not known. Nevertheless, our results suggest that maternal systemic HIV and sub clinical mastitis, an indicator of mammary gland inflammation, may be involved. Therefore, control of mammary gland inflammation and suppression of systemic HIV may minimize HIV shedding in breast milk.
Our results showed that in the absence of ART, there was a significant positive association between CAV and CFV suggesting local replication of HIV in the mammary gland. Indeed mammary gland CD4 cells and macrophages are more immunologically activated than their blood counterparts making them more permissive to HIV infection and replication. This association is disrupted in mothers on successful ART which suppresses CFV but has little or no effect on CAV[11,12] explaining why studies looking at mothers on ART fail to find significant association between CAV and CFV.
Previous studies have reported association between plasma viral load and breast milk CAV[7,9] but these studies did not take into account breast milk CFV. In our study, breast milk CAV was significantly associated with plasma viral load and CD4 count in univariate analysis. Controlling of milk CFV attenuated this association thereby, indicating that the effect of systemic HIV on breast milk CAV is through breast milk CFV. Since CAV persist in breast milk beyond six months, breastfeeding infants therefore remain at risk of CAV related HIV acquisition[6,7,9,25] even in the presence of successful maternal ART. Additionally, studied on breastfeeding mothers on successful ART have reported episodic detection of CFV in their breast milk over time which puts breastfeeding infants at risk of HIV acquisition too. Therefore, supplementing maternal ART with infant ARV prophylaxis, which is effective against both CFV and CAV[6,19] mediated HIV acquisition, for the entire breastfeeding period, may be more effective in eliminating residual postnatal transmission of HIV.
Our results suggest that COC may inhibit CFV shedding in breast milk. COC contains synthetic estrogen and progestin. In our study progestin-only ICs were neither associated with CAV nor CFV in breast milk. Our results therefore, suggest that the inhibitory effect of COC on breast milk CFV may be due to estrogen. Since the rate of detecting HIV-1 in plasma (P = .638) did not differ significantly between COC users and non-users, our results also suggest that estrogen selectively inhibits HIV-1 in the mammary gland. Earlier studies reported a similar tissue specific inhibitory effect of estrogen on HIV-1 replication in the cervico-vaginal tract despite similar plasma viral load. Literature indicates that estrogen inhibits HIV-1 replication by suppressing HIV-1 long terminal repeats (LTR) promoter activity[27,28] thereby inhibiting HIV-1 transcription and by reducing susceptibility of CD4+ lymphocytes and macrophages to HIV-1 infection.[29,30] Further studies are needed to confirm this association.
Infant sex has not been previously associated with CFV shedding in breast milk. Although marginally significant (P = .055), our study suggests that mothers of female infants may shed more breast milk CFV compared to those of male infants. Recent findings indicate that infant's gender may influence the composition of human breast milk[31–33] and breastfeeding practice, both of which would affect HIV shedding in breast milk. In line with our findings, earlier studies[35,36] reported higher risks of early postnatal acquisition of HIV in female infants. This was attributed to possible higher susceptibility to HIV infection in female infants than in males arising from genetic, immunological, hormonal and environmental factors. Our results provide an additional mechanism to explain higher rates of postnatal HIV transmission in female infants. However, contrary to our results, a large meta-analysis reported significantly higher risks of late postnatal acquisition of HIV in male infants. This study, however, did not consider maternal plasma viral load thereby ignoring an important confounder to this association.
Our study had several strengths. Firstly, our study had largest number of breast milk samples collected at the same time point compared to similar studies. Secondly, HIV shedding was estimated based on both breasts and both CAV and CFV thereby giving a true reflection of HIV shedding in breast milk. Thirdly, a rigorous sampling and randomization process in the parent trial means that we had representative sample and confounders were dealt with. Lastly, mothers included in this study were not on ART therefore it would not be possible to conduct such a study currently. Limitations of our study were, firstly, this was a secondary analysis of a trial designed for a different purpose. Secondly, contraceptive use data was collected by self-report. However, this data was collected during family planning counseling at each monthly visit and therefore mothers were very familiar contraceptive methods. Thirdly, study participants were selected from a cohort of HIV-1 infected breastfeeding mothers with high CD4+ count and not on ART. Therefore, our results may not be extrapolated to mothers on ART or with low CD4 count. This being a cross sectional study, the causal nature of the association between HIV shedding and its determinants could not be assessed. Lastly wide confidence intervals for some of our estimates may indicate the need for a larger sample size to improve accuracy of our estimates.
This study estimated that about 80% of HIV infected mothers not on ART shed HIV in breast milk during continued breastfeeding. Major factors driving this shedding were low CD4 count, unsuppressed plasma viral load and severe sub-clinical mastitis. The inverse relationship between breast milk HIV and use of combined oral contraceptives needs further clarification. Continued shedding of CAV may contribute to residual postnatal transmission of HIV in mothers on successful ART.
First and for most we would like to thank the mothers who participated in the ANRS12174 trial. We would like to thank Pr M. Laroque for providing us with access to a flame spectrometer, University Teaching Hospital; Lusaka and University of Montpellier for providing lab space and staff at the Centre for International health and UMR 1058 for their support and cooperation. We would also like to acknowledge the contribution of the ANRS 12174 trial group: ANRS 12174 trial group: University of Montpellier 1 (France): Roselyne Vallo, Valerie Marechal, Dorine Neveu, Vincent Foulongne, Michel Segondy; University of Paris V (France): Stephane Blanche, Jean-Marc Treluyer, Deborah Hirt; Makerere University (Uganda): James K. Tumwine, Grace Ndeezi, Charles Karamagi, Philippa Musoke, Proscovia M. Mugaba, Mary Kwagala, Joan Murungi, Hawa Nabuuma Muweesi, Evelyn Ninsiima, Simon Baryeija, Frederic Juma, Caleb Bwengye Kata, Stuart Katushabe; University of Ouagadougou (Burkina Faso): Nicolas Meda, Rasmata Ouedraogo, Diarra Ye, Eric Some, Hugues A. Traore, Christelle Nadembega, Justin Konate, Arsene Zongo, Abass Ouedraogo, Desire Neboua, Aissatou Belemvire, Armel Bambara, Justine Boncoungou; Danielle University of Western Cape (South Africa): Cheryl Nikodem, Justus Hofmeyr, Kim Harper, Debra Jackson, David Sanders, Mandisa Singata, Amwe Aku, Collins Okegbe-Eze, Xoliswa Williams, Nolundi Mshweshwe, Vatiswa Henge, Fikiswa Gomba, Tapiwa Gundu, Oswell Khandwa; University of Zambia (Zambia): Mildred Lusaka, Mary Chizyuka, Mary Phiri, Billies Imakando, Mwenechanya Musaku, Monica Kapasa, Gondwe Clement, Hilton Mwila Mwaba, Japhet Matoba, Chafye Siuluta, Katai Chola, Patricia Mwamutanda; University of Bergen (Norway): Halvor Sommerfelt, Ingunn Engebretsen, Jorn Klungsoyr, Jan van den Broeck, Jorn Blume; INSERM-ANRS (France): Claire Rekacewicz.
Conceptualization: David Gatsinzi Rutagwera, Jean-Pierre Molès, Chipepo Kankasa, Mwiya Mwiya, Edouard Tuaillon, Nicolas Nagot, Philippe Van de Perre, Thorkild Tylleskär.
Data curation: David Gatsinzi Rutagwera, Jean-Pierre Molès, Chipepo Kankasa, Mwiya Mwiya.
Formal analysis: David Gatsinzi Rutagwera, Marianne Peries, Nicolas Nagot.
Funding acquisition: Chipepo Kankasa, Philippe Van de Perre, Thorkild Tylleskär.
Investigation: David Gatsinzi Rutagwera, Jean-Pierre Molès, Chipepo Kankasa, Mwiya Mwiya, Philippe Van de Perre, Thorkild Tylleskär.
Methodology: David Gatsinzi Rutagwera, Jean-Pierre Molès, Chipepo Kankasa, Mwiya Mwiya, Edouard Tuaillon, Marianne Peries, Nicolas Nagot, Philippe Van de Perre, Thorkild Tylleskär.
Project administration: David Gatsinzi Rutagwera.
Resources: Chipepo Kankasa, Mwiya Mwiya, Edouard Tuaillon, Marianne Peries, Nicolas Nagot, Philippe Van de Perre.
Software: David Gatsinzi Rutagwera, Marianne Peries.
Supervision: Jean-Pierre Molès, Chipepo Kankasa, Edouard Tuaillon, Philippe Van de Perre, Thorkild Tylleskär.
Visualization: David Gatsinzi Rutagwera.
Writing – original draft: David Gatsinzi Rutagwera.
Writing – review & editing: Jean-Pierre Molès, Chipepo Kankasa, Mwiya Mwiya, Edouard Tuaillon, Marianne Peries, Nicolas Nagot, Philippe Van de Perre, Thorkild Tylleskär.
David Gatsinzi Rutagwera orcid: 0000-0001-7092-9307.
David Gatsinzi Rutagwera orcid: 0000-0001-7092-9307.
. Zambia Consolidated Guidelines for Treatment and Prevention of, HIV Infection. Lusaka: Ministry of Health; 2018.
. Consolidated guidelines on the use of antiretroviral drugs for treating and preventing, HIV infection: recommendation for a public health, approach. 2 edGeneva: World Health Organisation; 2016.
. Field CJ. The immunological components of human milk and their effect on immune development in infants. J Nutr 2005;135:1–4.
. Coovadia HM, Rollins NC, Bland RM, et al. Mother-to-child transmission of HIV-1 infection during exclusive breastfeeding in the first 6 months of life: an intervention cohort study. Lancet (London, England) 2007;369:1107–16.
. Ndirangu J, Newell ML, Thorne C, et al. Treating HIV-infected mothers reduces under 5 years of age mortality rates to levels seen in children of HIV-uninfected mothers in rural South Africa. Antivir Ther 2012;17:81–90.
. Van de Perre P, Rubbo PA, Viljoen J, et al. HIV-1 reservoirs in breast milk
and challenges to elimination of breast-feeding transmission of HIV-1. Sci Transl Med 2012;4:143sr143.
. Rousseau CM, Nduati RW, Richardson BA, et al. Association of levels of HIV-1-infected breast milk
cells and risk of mother-to-child transmission. J Infect Dis 2004;190:1880–8.
. Rousseau CM, Nduati RW, Richardson BA, et al. Longitudinal analysis of human immunodeficiency virus type 1 RNA in breast milk
and of its relationship to infant infection and maternal disease. J Infect Dis 2003;187:741–7.
. Ndirangu J, Viljoen J, Bland RM, et al. Cell-free (RNA) and cell-associated (DNA) HIV-1 and postnatal transmission through breastfeeding. PloS One 2012;7:e51493.
. Koulinska IN, Villamor E, Chaplin B, et al. Transmission of cell-free and cell-associated HIV
-1 through breast-feeding. J Acquir Immune Defic Syndr (1999) 2006;41:93–9.
. Slyker JA, Chung MH, Lehman DA, et al. Incidence and correlates of HIV-1 RNA detection in the breast milk
of women receiving HAART for the prevention of HIV-1 transmission. PloS One 2012;7:e29777.
. Lehman DA, Chung MH, John-Stewart GC, et al. HIV-1 persists in breast milk
cells despite antiretroviral treatment to prevent mother-to-child transmission. AIDS (London, England) 2008;22:1475–85.
. Shapiro RL, Ndung’u T, Lockman S, et al. Highly active antiretroviral therapy started during pregnancy or postpartum suppresses HIV-1 RNA, but not DNA, in breast milk
. J Infect Dis 2005;192:713–9.
. Salazar-Gonzalez JF, Salazar MG, Learn GH, et al. Origin and evolution of HIV-1 in breast milk
determined by single-genome amplification and sequencing. J Virol 2011;85:2751–63.
. Gantt S, Carlsson J, Heath L, et al. Genetic analyses of HIV-1 env sequences demonstrate limited compartmentalization in breast milk
and suggest viral replication within the breast that increases with mastitis. J Virol 2010;84:10812–9.
. Lyimo MA, Mosi MN, Housman ML, et al. Breast milk
from Tanzanian women has divergent effects on cell-free and cell-associated HIV
-1 infection in vitro. PloS One 2012;7:e43815.
. Lunney KM, Iliff P, Mutasa K, et al. Associations between breast milk
viral load, mastitis, exclusive breast-feeding, and postnatal transmission of HIV. Clin Infect Dis 2010;50:762–9.
. Kuhn L, Kim HY, Walter J, et al. HIV-1 concentrations in human breast milk
before and after weaning. Sci Transl Med 2013;5:181ra151.
. Nagot N, Kankasa C, Tumwine JK, et al. Extended pre-exposure prophylaxis with lopinavir-ritonavir versus lamivudine to prevent HIV-1 transmission through breastfeeding up to 50 weeks in infants in Africa (ANRS 12174): a randomised controlled trial. Lancet (London, England) 2016;387:566–73.
. Taha TE, Hoover DR, Kumwenda NI, et al. Late postnatal transmission of HIV-1 and associated factors. J Infect Dis 2007;196:10–4.
. Nagot N, Kankasa C, Meda N, et al. Lopinavir/Ritonavir versus Lamivudine peri-exposure prophylaxis to prevent HIV-1 transmission by breastfeeding: the PROMISE-PEP trial Protocol ANRS 12174. BMC Infect Dis 2012;12:246.
. Kiwanuka G, Kiwanuka N, Muneza F, et al. Retention of HIV infected pregnant and breastfeeding women on option B+ in Gomba District, Uganda: a retrospective cohort study. BMC Infect Dis 2018;18:533.
. Haas AD, Tenthani L, Msukwa MT, et al. Retention in care during the first 3 years of antiretroviral therapy for women in Malawi's option B+ programme: an observational cohort study. Lancet HIV 2016;3:e175–82.
. Haas AD, Msukwa MT, Egger M, et al. Adherence to antiretroviral therapy during and after pregnancy: cohort study on women receiving care in Malawi's Option B+ Program. Clin Infect Dis 2016;63:1227–35.
. Milligan C, Overbaugh J. The role of cell-associated virus in mother-to-child HIV transmission. J Infect Dis 2014;210(Suppl 3):S631–640.
. Money DM, Arikan YY, Remple V, et al. Genital tract and plasma human immunodeficiency virus viral load throughout the menstrual cycle in women who are infected with ovulatory human immunodeficiency virus. Am J Obstet Gynecol 2003;188:122–8.
. Szotek EL, Narasipura SD, Al-Harthi L. 17beta-Estradiol inhibits HIV-1 by inducing a complex formation between beta-catenin and estrogen receptor alpha on the HIV promoter to suppress HIV transcription. Virology 2013;443:375–83.
. Narasipura SD, Henderson LJ, Fu SW, et al. Role of beta-catenin and TCF/LEF family members in transcriptional activity of HIV in astrocytes. J Virol 2012;86:1911–21.
. Tasker C, Ding J, Schmolke M, et al. 17beta-estradiol protects primary macrophages against HIV infection through induction of interferon-alpha. Viral Immunol 2014;27:140–50.
. Rodriguez-Garcia M, Biswas N, Patel MV, et al. Estradiol reduces susceptibility of CD4+ T cells and macrophages to HIV-infection. PloS One 2013;8:e62069.
. Galante L, Milan AM, Reynolds CM, et al. Sex-specific human milk composition: the role of infant sex in determining early life nutrition. Nutrients 2018;10:
. van Sadelhoff JHJ, van de Heijning BJM, Stahl B, et al. Longitudinal variation of amino acid levels in human milk and their associations with infant gender. Nutrients 2018;10:
. Hahn WH, Song JH, Song S, et al. Do gender and birth height of infant affect calorie of human milk? An association study between human milk macronutrient and various birth factors. J Matern Fetal Neonatal Med 2017;30:1608–12.
. Shafer EF, Hawkins SS. The impact of sex of child on breastfeeding in the United States. Matern Child Health J 2017;21:2114–21.
. Taha TE, Nour S, Kumwenda NI, et al. Gender differences in perinatal HIV acquisition among African infants. Pediatrics 2005;115:e167–72.
. Temmerman M, Nyong’o AO, Bwayo J, et al. Risk factors for mother-to-child transmission of human immunodeficiency virus-1 infection. Am J Obstetr Gynecol 1995;172(2 Pt 1):700–5.
. Coutsoudis A, Dabis F, Fawzi W, et al. Late postnatal transmission of HIV-1 in breast-fed children: an individual patient data meta-analysis. J Infect Dis 2004;189:2154–66.
Keywords:Copyright © 2019 the Author(s). Published by Wolters Kluwer Health, Inc.
breast milk; cell-associated HIV; cell-free HIV; continued breastfeeding; deoxyribonucleic acid; ribonucleic acid; sub-clinical mastitis