Copyright © 2015 Wolters Kluwer Health, Inc. All rights reserved. Cytomegalovirus, and possibly Epstein–Barr virus, shedding in breast milk is associated with HIV-1 transmission by breastfeeding Johannes Viljoen a,b , Edouard Tuaillon b,c , Nicolas Nagot b,c , Siva Danaviah a , Marianne Peries b,c , Prevashinee Padayachee a , Vincent Foulongne b,c , Ruth Bland a,d , Nigel Rollins e,f , Marie-Louise Newell a,g,M and Philippe van de Perre b,c,M Objective: Postnatal HIV-1 mother-to-child transmission (MTCT) occurs in spite of antiretroviral therapy. Co-infections in breast milk with cytomegalovirus (CMV) and Epstein – Barr virus (EBV) are associated with increased HIV-1 shedding in this compart- ment. We investigated CMV levels and EBV detection in breast milk as potential risk factors for MTCT of HIV-1 via breastfeeding. Methods: Cell-free HIV-1 RNA, cell-associated HIV-1 DNA, CMV and EBV DNA were quantified in breast milk from 62 HIV-infected mothers and proven postnatal MTCT of HIV-1 via breastfeeding. Controls were 62 HIV-positive mothers with HIV-uninfected infants. Results: Median (interquartile range) CMV DNA viral load was significantly higher in cases [88 044 (18 586–233 904)] than in controls [11 167 (3221–31 152)] copies/10 6 breast milk cells (P < 0.001). Breast milk CMV DNA level correlated positively with breast milk HIV-1 RNA level in cases and controls. EBV DNA was detectable in a higher proportion of breast milk samples of cases (37.1%) than controls (16.1%; P ¼ 0.009). HIV-1 MTCT was strongly associated with HIV-1 RNA shedding in breast milk and plasma. In multivariable analysis, every 1 log 10 increase in breast milk CMV DNA was associated with a significant 2.5-fold greater odds of MTCT of HIV-1, independent of breast milk and plasma HIV-1 levels; the nearly three-fold increased risk of HIV-1 MTCT with breast milk EBV DNA detection did not reach significance. Conclusion: We provide the first evidence of an independent association between CMV in breast milk, and postnatal MTCT of HIV-1. This association could fuel persistent shedding of HIV-1 in breast milk in women receiving antiretroviral therapy. EBV DNA detection in breast milk may also be associated with MTCT of HIV-1, but only marginally so. ß 2015 Wolters Kluwer Health, Inc. All rights reserved. AIDS 2015, 29:145–153 Keywords: breast milk, cytomegalovirus, Epstein–Barr virus, postnatal HIV-1 mother-to-child transmission a Africa Centre for Health and Population Studies, University of KwaZulu-Natal, Durban, South Africa, b Inserm U1058, Universite ´ Montpellier 1, c CHRU de Montpellier, De ´partement de Bacte ´riologie-Virologie & Institut de Recherche en Biothe ´rapie and Department of Medical Information, Montpellier, France, d Royal Hospital for Sick Children, Glasgow, UK, e Department of Paediatrics and Child Health, University of KwaZulu-Natal, Durban, South Africa, f Department of Maternal, Newborn, Child and Adolescent Health, World Health Organization, Geneva, Switzerland, and g Faculty of Medicine, University of Southampton, Southampton, UK. Correspondence to Johannes Viljoen, MBChB, FCPath(SA)Viro, University of KwaZulu-Natal, Durban, KwaZulu-Natal, South Africa. E-mail: jviljoen@africacentre.ac.za  Marie-Louise Newell and Philippe van de Perre contributed equally to the writing of this article. Received: 12 June 2014; revised: 16 September 2014; accepted: 28 October 2014. DOI:10.1097/QAD.0000000000000527 ISSN 0269-9370 Copyright Q 2015 Wolters Kluwer Health, Inc. All rights reserved. 145