Recurrent Severe Subclinical Mastitis and the Risk of HIV Transmission Through Breastfeeding.

Subclinical mastitis (SCM) is an important risk factor of postnatal HIV-1 transmission that is still poorly understood. A longitudinal sub-study of the ANRS12174 trial including 270 breastfeeding mothers in Lusaka, Zambia measured sodium (Na+) and potassium (K+) in archived paired breast milk samples collected at week 14, 26 and 38 postpartum to determine cumulative incidence of SCM and the effects of recurrent severe SCM on HIV-1 shedding in breast milk. A nested retrospective cohort study including 112 mothers was also done to determine longitudinal effects of SCM on four pro-inflammatory cytokines; IL6, IL8, IP10 and RANTES. The cumulative incidence for any SCM (Na + /K + ratio > 0.6) and severe SCM (Na + /K + ratio > 1) were 58.6% (95%CI: 52.7 - 64.5) and 27.8% (95%CI: 22.5 - 33.1), respectively. In majority of affected mothers (51.4%) severe SCM was recurrent. Both breasts were involved in 11.1%, 33.3% and 70% of the mothers with a single episode, 2 and 3 episodes respectively. In affected breasts, an episode of severe SCM resulted in steep upregulation of the four cytokines considered (IL8, IP10, RANTES and IL6) compared to: before and after the episode; contralateral unaffected breasts; and SCM negative control mothers. Recurrent severe SCM significantly increased the odds of shedding cell-free HIV-1 in breast milk (OR: 5.2; 95%CI: 1.7 - 15.6) whereas single episode of severe SCM did not (OR: 1.8; 95%CI: 0.8 - 4.2). A Na+/K+ ratio > 1 indicative of severe SCM is an excellent indicator of breast inflammation characterized by a steep, localized and temporal upregulation of several pro-inflammatory cytokines that favor HIV-1 shedding in mature breast milk and may facilitate postnatal HIV-1 transmission through breastfeeding.

SARS-CoV-2 Poorly Replicates in Cells of the Human Blood-Brain Barrier Without Associated Deleterious Effects.

Various neurological symptoms have been associated to severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection including headache, fever, anosmia, ageusia, but also, encephalitis, Guillain-Barre syndrome and ischemic stroke. Responsible for the current coronavirus disease (COVID-19) pandemic, SARS-CoV-2 may access and affect the central nervous system (CNS) by several pathways such as axonal retrograde transport or through interaction with the blood-brain barrier (BBB) or blood-cerebrospinal fluid (CSF) barrier. Here, we explored the molecular and cellular effects of direct SARS-CoV-2 infection of human BBB cells. We observed low replication of SARS-CoV-2 that was accompanied by very moderate inflammatory response. Using a human in vitro BBB model, we also described low replication levels without strong inflammatory response or modulation of endothelium integrity. Finally, using serum samples from COVID-19 patients, we highlighted strong concentrations of pro-inflammatory factors that did not perturb BBB integrity after short term exposure. Altogether, our results show that the main mechanism of brain access following SARS-CoV-2 infection does not seem to be directed by brain infection through endothelial cells.

Alarmin S100A9 restricts retroviral infection by limiting reverse transcription in human dendritic cells.

Dendritic cells (DC) subsets, like Langerhans cells (LC), are immune cells involved in pathogen sensing. They express specific antimicrobial cellular factors that are able to restrict infection and limit further pathogen transmission. Here, we identify the alarmin S100A9 as a novel intracellular antiretroviral factor expressed in human monocyte-derived and skin-derived LC. The intracellular expression of S100A9 is decreased upon LC maturation and inversely correlates with enhanced susceptibility to HIV-1 infection of LC. Furthermore, silencing of S100A9 in primary human LC relieves HIV-1 restriction while ectopic expression of S100A9 in various cell lines promotes intrinsic resistance to both HIV-1 and MLV infection by acting on reverse transcription. Mechanistically, the intracellular expression of S100A9 alters viral capsid uncoating and reverse transcription. S100A9 also shows potent inhibitory effect against HIV-1 and MMLV reverse transcriptase (RTase) activity in vitro in a divalent cation-dependent manner. Our findings uncover an unexpected intracellular function of the human alarmin S100A9 in regulating antiretroviral immunity in Langerhans cells.

Mitochondrial DNA Instability Is Common in HIV-Exposed Uninfected Newborns.

Worldwide, one million HIV-exposed uninfected (HEU) children are born yearly, and chronic health impairments have been reported in these children. Mitochondrial DNA (mtDNA) instability and altered mtDNA content have been evidenced in these children, but an exhaustive characterization of altered mitochondrial genomes has never been reported. We applied deep mtDNA sequencing coupled to the deletion identification algorithm eKLIPse to the blood of HEU neonates (n = 32), which was compared with healthy controls (n = 15). Dried blood spots (DBS) from African HEU children were collected seven days after birth between November 2009 and May 2012. DBS from French healthy controls were collected at birth (or <3 days of life) in 2012 and in 2019. In contrast to the absence of mtDNA instability observed at the nucleotide level, we identified significant amounts of heteroplasmic mtDNA deletions in 75% of HEU children and in none of controls. The heteroplasmy rate of the 62 mtDNA deletions identified varied from 0.01% to up to 50%, the highest rates being broadly compatible with bioenergetic defect and clinical expression. mtDNA integrity is commonly affected in HEU neonates. The nature of the deletions suggests a mechanism related to aging or tumor-associated mtDNA instability. This child population may be at risk of additional mtDNA genetic alterations considering that they will be exposed to other mitotoxic drugs including antiretroviral or anti-tuberculosis treatment.

Clinical and Biological Factors Associated With Early Epstein-Barr Virus Infection in Human Immunodeficiency Virus-Exposed Uninfected Infants in Eastern Uganda.

BACKGROUND: Immune control of Epstein-Barr virus (EBV) infection is impaired in individuals with HIV. We explored maternal factors associated with EBV acquisition in HIV-exposed uninfected (HEU) infants and the relationship between EBV infection and serious adverse events (SAEs) during the first year of life. METHODS: 201 HEU infants from Uganda enrolled in the ANRS 12174 trial were tested for antiviral capsid antigen (anti-VCA) antibodies at week 50. Date of infection was estimated by testing EBV DNA at weeks 1, 6, 14, 26, 38, and 50 postpartum on dried blood spots. RESULTS: Eighty-seven (43%) infants tested positive for anti-VCA IgG at week 50. Among the 59 infants positive for EBV DNA, 25% were infected within the first 26 weeks. Almost half (12%) were infected before week 14. Shedding of EBV in breast milk was associated with EBV DNA in maternal plasma (P = .009), HIV RNA detection (P = .039), and lower CD4 count (P = .001) and correlated with plasma EBV DNA levels (P = .002). EBV infant infection at week 50 was associated with shedding of EBV in breast milk (P = .009) and young maternal age (P = .029). Occurrence of a clinical SAE, including malaria and pneumonia, was associated with higher levels of EBV DNA in infants (P = .010). CONCLUSIONS: By assessing EBV infection in HEU infants we observed that infection during the first year is determined by HIV and EBV maternal factors and that EBV DNA levels were higher among infants with clinical SAEs. CLINICAL TRIALS REGISTRATION: NCT00640263.

The CD133+ Stem/Progenitor-Like Cell Subset Is Increased in Human Milk and Peripheral Blood of HIV-Positive Women.

Human milk is a significant source of different CD133+ and/or CD34+ stem/progenitor-like cell subsets in healthy women but their cell distribution and percentages in this compartment of HIV-positive women have not been explored. To date, a decrease of CD34+ hematopoietic stem and progenitor cell frequencies in peripheral blood and bone marrow of HIV-positive patients has been reported. Herein, human milk and peripheral blood samples were collected between day 2-15 post-partum from HIV-positive and HIV-negative women, and cells were stained with stem cell markers and analyzed by flow cytometry. We report that the median percentage of CD45+/highCD34-CD133+ cell subset from milk and blood was significantly higher in HIV-positive than in HIV-negative women. The percentage of CD45dimCD34-CD133+ cell subset from blood was significantly higher in HIV-positive than HIV-negative women. Moreover, percentages of CD45dimCD34+, CD45dimCD34+CD133-, and CD45+highCD34+CD133- cell subsets from blood were significantly lower in HIV-positive than HIV-negative women. The CD133+ stem/progenitor-like cell subsets are increased in early human milk and blood of HIV-positive women and are differentially distributed to CD34+ cell subset frequencies which are decreased in blood.

Zika Virus Infection Promotes Local Inflammation, Cell Adhesion Molecule Upregulation, and Leukocyte Recruitment at the Blood-Brain Barrier.

The blood-brain barrier (BBB) largely prevents toxins and pathogens from accessing the brain. Some viruses have the ability to cross this barrier and replicate in the central nervous system (CNS). Zika virus (ZIKV) was responsible in 2015 to 2016 for a major epidemic in South America and was associated in some cases with neurological impairments. Here, we characterized some of the mechanisms behind its neuroinvasion using an innovative in vitro human BBB model. ZIKV efficiently replicated, was released on the BBB parenchyma side, and triggered subtle modulation of BBB integrity as well as an upregulation of inflammatory and cell adhesion molecules (CAMs), which in turn favored leukocyte recruitment. Finally, we showed that ZIKV-infected mouse models displayed similar CAM upregulation and that soluble CAMs were increased in plasma samples from ZIKV-infected patients. Our observations suggest a complex interplay between ZIKV and the BBB, which may trigger local inflammation, leukocyte recruitment, and possible cerebral vasculature impairment.IMPORTANCE Zika virus (ZIKV) can be associated with neurological impairment in children and adults. To reach the central nervous system, viruses have to cross the blood-brain barrier (BBB), a multicellular system allowing a tight separation between the bloodstream and the brain. Here, we show that ZIKV infects cells of the BBB and triggers a subtle change in its permeability. Moreover, ZIKV infection leads to the production of inflammatory molecules known to modulate BBB integrity and participate in immune cell attraction. The virus also led to the upregulation of cellular adhesion molecules (CAMs), which in turn favored immune cell binding to the BBB and potentially increased infiltration into the brain. These results were also observed in a mouse model of ZIKV infection. Furthermore, plasma samples from ZIKV-infected patients displayed an increase in CAMs, suggesting that this mechanism could be involved in neuroinflammation triggered by ZIKV.

Study of Usutu virus neuropathogenicity in mice and human cellular models.

Usutu virus (USUV), an African mosquito-borne flavivirus closely related to West Nile virus, was first isolated in South Africa in 1959. USUV emerged in Europe two decades ago, causing notably massive mortality in Eurasian blackbirds. USUV is attracting increasing attention due to its potential for emergence and its rapid spread in Europe in recent years. Although mainly asymptomatic or responsible for mild clinical signs, USUV was recently described as being associated with neurological disorders in humans such as encephalitis and meningoencephalitis, highlighting the potential health threat posed by the virus. Despite this, USUV pathogenesis remains largely unexplored. The aim of this study was to evaluate USUV neuropathogenicity using in vivo and in vitro approaches. Our results indicate that USUV efficiently replicates in the murine central nervous system. Replication in the spinal cord and brain is associated with recruitment of inflammatory cells and the release of inflammatory molecules as well as induction of antiviral-responses without major modulation of blood-brain barrier integrity. Endothelial cells integrity is also maintained in a human model of the blood-brain barrier despite USUV replication and release of pro-inflammatory cytokines. Furthermore, USUV-inoculated mice developed major ocular defects associated with inflammation. Moreover, USUV efficiently replicates in human retinal pigment epithelium. Our results will help to better characterize the physiopathology related to USUV infection in order to anticipate the potential threat of USUV emergence.

Mastitis in Autoimmune Diseases: Review of the Literature, Diagnostic Pathway, and Pathophysiological Key Players.

Mastitis frequently affects women of childbearing age. Of all the pathological breast conditions requiring specific management, autoimmune mastitis is in the third position after infection and breast cancer. The aim of this literature review was to make a comprehensive description of autoimmune diseases targeting the mammary gland. Four main histological patterns of autoimmune mastitis are described: (i) lymphocytic infiltrates; (ii) ductal ectasia; (iii) granulomatous mastitis; and (iv) vasculitis. Our literature search found that all types of autoimmune disease may target the mammary gland: organ-specific diseases (diabetes, thyroiditis); connective tissue diseases (such as systemic erythematosus lupus or Sjögren's syndrome); vasculitides (granulomatosis with polyangiitis, eosinophilic granulomatosis with polyangiitis, giant cell arteritis, polyarteritis nodosa, Behçet's disease); granulomatous diseases (sarcoidosis, Crohn's disease); and IgG4-related disease. Cases of breast-specific autoimmune diseases have also been reported, including idiopathic granulomatous mastitis. These breast-limited inflammatory diseases are sometimes the first symptom of a systemic autoimmune disease. Although autoimmune mastitis is rare, it is probably underdiagnosed or misdiagnosed. Early diagnosis may allow us to detect systemic diseases at an earlier stage, which could help to initiate a prompt, appropriate therapeutic strategy. In case of suspected autoimmune mastitis, we hereby propose a diagnostic pathway and discuss the potential pathophysiological pathways leading to autoimmune breast damage.

Discrepancy of Serological and Molecular Patterns of Circulating Epstein-Barr Virus Reactivation in Primary Sjögren's Syndrome.

Primary Sjögren's syndrome (pSS) is characterized by B cell hyperactivation, production of autoantibodies and increased risk of B cell lymphomas. Serological profile of Epstein-Barr virus (EBV) reactivation and increase EBV DNA levels in exocrine glands are observed in pSS, but whether these abnormalities are accompanied with disturbed systemic EBV control or have any association with pSS activity remains to be investigated. In this observational study, we initially explored anti-EBV antibodies and cell-free DNA in 395 samples from a cross-sectional plasma collection of pSS patients included in ASSESS French national cohort. Results were assessed in relation with disease activity. Further, to assess cell-associated EBV DNA we organized a case-control study including 20 blood samples from pSS patients followed in University Hospital Center of Montpellier. Results were compared with matched controls. Robust response against EBV early antigen (EA) was observed in pSS patients with anti-SSA/B (Sjögren's syndrome A and B) and anti-SSA autoantibodies compared to anti-SSA/B negatives (P < 0.01 and P = 0.01, respectively). Increased beta-2 microglobulin, kappa and lambda light chains, and immunoglobulin G levels were more frequently observed in anti-EA seropositive pSS subjects compared to anti-EA negative subjects (P < 0.001; P = 0.001; P = 0.003, respectively). Beta-2 microglobulin was independently associated with anti-EA positivity in multivariate analysis (P < 0.001). Plasma cell-free EBV DNA and EBV cellular reservoir was not different between pSS patients and controls. We conclude that serological evidence of EBV reactivation was more frequently observed and more strongly associated with anti-SSA/B status and B cell activation markers in pSS. However, serological profile of EBV reactivation was not accompanied by molecular evidence of systemic EBV reactivation. Our data indicated that EBV infection remains efficiently controlled in the blood of pSS patients.

Human papillomavirus (HPV) shedding in breast milk from African women living with HIV.

BACKGROUND: Little is known about human papillomavirus (HPV) shedding in human breast milk. OBJECTIVE: To investigate HPV shedding in mature breast milk specimens collected from breastfeeding African women living with HIV-1 and not receiving antiretroviral treatment. DESIGN: 62 African women enrolled in the ANRS 12174 trial participated in this study. 79 lactoserum specimens obtained from right and/or left breasts from 42 Zambian women as well as lactosera and cell pellets from 40 milk samples collected from right and left breasts among 20 Ugandan women were tested for HPV using the INNO-LiPA HPV Genotyping Extra II assay. RESULTS: HPV DNA was detected in 9 (11.4%) lactoserum specimens collected from 8 (19.0%) Zambian women. Fourteen (17.5%) samples from 5 (25%) Ugandan women were positive for HPV detection. Differences in HPV type identification between the two breasts as well as between lactoserum and cell pellet were oberved. Overall, 13 (21.0%) of the 62 women included in this study had detectable HPV DNA in their breast milk, representing 11 HPV types, including high-risk, probable high-risk and low-risk types. CONCLUSION: This study confirms that HPV can be frequently detected in breast milk in HIV-infected women. Further studies are needed to understand the way by which maternal milk can shed HPV.

The impact of targeting repetitive BamHI-W sequences on the sensitivity and precision of EBV DNA quantification.

BACKGROUND: Viral load monitoring and early Epstein-Barr virus (EBV) DNA detection are essential in routine laboratory testing, especially in preemptive management of Post-transplant Lymphoproliferative Disorder. Targeting the repetitive BamHI-W sequence was shown to increase the sensitivity of EBV DNA quantification, but the variability of BamHI-W reiterations was suggested to be a source of quantification bias. We aimed to assess the extent of variability associated with BamHI-W PCR and its impact on the sensitivity of EBV DNA quantification using the 1st WHO international standard, EBV strains and clinical samples. METHODS: Repetitive BamHI-W- and LMP2 single- sequences were amplified by in-house qPCRs and BXLF-1 sequence by a commercial assay (EBV R-gene™, BioMerieux). Linearity and limits of detection of in-house methods were assessed. The impact of repeated versus single target sequences on EBV DNA quantification precision was tested on B95.8 and Raji cell lines, possessing 11 and 7 copies of the BamHI-W sequence, respectively, and on clinical samples. RESULTS: BamHI-W qPCR demonstrated a lower limit of detection compared to LMP2 qPCR (2.33 log10 versus 3.08 log10 IU/mL; P = 0.0002). BamHI-W qPCR underestimated the EBV DNA load on Raji strain which contained fewer BamHI-W copies than the WHO standard derived from the B95.8 EBV strain (mean bias: - 0.21 log10; 95% CI, -0.54 to 0.12). Comparison of BamHI-W qPCR versus LMP2 and BXLF-1 qPCR showed an acceptable variability between EBV DNA levels in clinical samples with the mean bias being within 0.5 log10 IU/mL EBV DNA, whereas a better quantitative concordance was observed between LMP2 and BXLF-1 assays. CONCLUSIONS: Targeting BamHI-W resulted to a higher sensitivity compared to LMP2 but the variable reiterations of BamHI-W segment are associated with higher quantification variability. BamHI-W can be considered for clinical and therapeutic monitoring to detect an early EBV DNA and a dynamic change in viral load.

Increased Epstein-Barr virus in breast milk occurs with subclinical mastitis and HIV shedding.

Epstein-Barr virus (EBV) in breast milk and subclinical mastitis (SCM) are both associated with human immunodeficiency virus (HIV) shedding and possibly with postnatal HIV transmission. The objective of this nested case-control study was to investigate the interplay between SCM and EBV replication in breast milk of HIV-infected mothers.The relationships between EBV deoxyribonucleic acid (DNA) shedding, HIV-1 ribonucleic acid (RNA) level, and SCM were explored in breast milk samples of Zambian mothers participating in the ANRS 12174 trial. Mammary gland inflammation was defined as a breast milk sodium to potassium ratio (Na/K) greater than 0.6 and further subclassified as either "possible SCM" (Na/K ratio 0.6-1.0) or SCM (Na/K ratio ≥ 1.0). Breast milk interleukin 8 (IL-8) was measured as a surrogate marker of mammary gland inflammation.EBV DNA was detected in breast milk samples from 42 out of 83 (51%) participants and was associated with HIV-1 shedding in breast milk (P = 0.006). EBV DNA levels were higher in samples with SCM and "possible SCM" compared to non-SCM breast milk samples (P = 0.06; P = 0.007). An EBV DNA level of >200 copies/mL was independently associated with SCM and "possible SCM" (OR: 2.62; 95%: 1.13-6.10). In patients with SCM, higher EBV replication in the mammary gland was associated with a lower induction of IL-8 (P = 0.013). Resistance to DNase treatment suggests that EBV DNA in lactoserum is encapsidated.SCM and decreased IL-8 responses are associated with an increased EBV shedding in breast milk which may in turn facilitate HIV replication in the mammary gland.

Ebola virus vaccine: benefit and risks of adenovirus-based vectors.

In 2014, an outbreak of Ebola virus spread rapidly in West Africa. The epidemic killed more than 10,000 people and resulted in transmissions outside the endemic countries. WHO hopes for effective vaccines by the end of 2015. Numerous vaccine candidates have been proposed, and several are currently being evaluated in humans. Among the vaccine candidates are vectors derived from adenovirus (Ad). Despite previous encouraging preclinical and Phase I/II trials, Ad vectors used in three Phase II trials targeting HIV were prematurely interrupted because of the lack of demonstrated efficacy. The vaccine was not only ineffective but also led to a higher rate of HIV acquisition. In this context, the authors discuss the potential benefits, risks and impact of using Ad-derived vaccines to control Ebola virus disease.

Early infant feeding patterns and HIV-free survival: findings from the Kesho-Bora trial (Burkina Faso, Kenya, South Africa).

OBJECTIVE: To investigate the association between feeding patterns and HIV-free survival in children born to HIV-infected mothers and to clarify whether antiretroviral (ARV) prophylaxis modifies the association. METHODS: From June 2005 to August 2008, HIV-infected pregnant women were counseled regarding infant feeding options, and randomly assigned to triple-ARV prophylaxis (triple ARV) until breastfeeding cessation (BFC) before age 6 months or antenatal zidovudine with single-dose nevirapine (short-course ARV). Eighteen-month HIV-free survival of infants HIV-negative at 2 weeks of age was assessed by feeding patterns (replacement feeding from birth, BFC <3 months, BFC ≥3 months). RESULTS: Of the 753 infants alive and HIV-negative at 2 weeks, 28 acquired infection and 47 died by 18 months. Overall HIV-free survival at 18 months was 0.91 [95% confidence interval (CI): 0.88-0.93]. In the short-course ARV arm, HIV-free survival (0.88; CI: 0.84-0.91) did not differ by feeding patterns. In the triple ARV arm, overall HIV-free survival was 0.93 (CI: 0.90-0.95) and BFC <3 months was associated with lower HIV-free survival than BFC ≥3 months (adjusted hazard ratio: 0.36; CI: 0.15-0.83) and replacement feeding (adjusted hazard ratio: 0.20; CI: 0.04-0.94). In the triple ARV arm, 4 of 9 transmissions occurred after reported BFC (and 5 of 19 in the short-course arm), indicating that some women continued breastfeeding after interruption of ARV prophylaxis. CONCLUSIONS: In resource-constrained settings, early weaning has previously been associated with higher infant mortality. We show that, even with maternal triple-ARV prophylaxis during breastfeeding, early weaning remains associated with lower HIV-free survival, driven in particular by increased mortality.

Performances of fourth generation HIV antigen/antibody assays on filter paper for detection of early HIV infections.

BACKGROUND: Point-of-care testing and diagnosis of HIV acute infections play important roles in preventing transmission, but HIV rapid diagnosis tests have poor capacity to detect early infections. Filter paper can be used for capillary blood collection and HIV testing using 4th generation immunoassays. OBJECTIVES: Antigen/antibody combined immunoassays were evaluated for their capacity to identify early HIV infections using filter paper in comparison with rapid test. STUDY DESIGN: Thirty nine serum samples collected from HIV seroconverters were spotted onto filter paper and tested by the Roche Elecsys(®) HIV Combi PT test and the DiaSorin Liaison XL Murex HIV Ab/Ag assay. RESULTS: Fourth generation immunoassays identified 34 out of 39 HIV early infections using dried serum spot, whereas the Determine™ HIV-1/2 rapid test detected 24 out of 39 HIV positive serum (87.2% vs 61.5% respectively, p = 0.009). p24 antigen was detected by the Liaison XL in 19 dried serum samples (48.7%). In the group characterized by a negative western blot, 7 out of 8 (87.5%) and 6 out of 8 (75.0%) samples were found positive for HIV using the Elecsys and the Liaison XL, respectively. None of these eight samples classified in this group of early acute infections were found positive by the rapid test. CONCLUSION: Fourth generation Ag/Ab immunoassays performed on dried serum spot had good performance for HIV testing during the early phases of HIV infection. This method may be useful to detect HIV early infections in hard-to-reach populations and individuals living in remote areas before rapid tests become positive.

Cytomegalovirus, and possibly Epstein-Barr virus, shedding in breast milk is associated with HIV-1 transmission by breastfeeding.

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 compartment. 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 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 = 0.009). HIV-1 MTCT was strongly associated with HIV-1 RNA shedding in breast milk and plasma. In multivariable analysis, every 1 log10 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.