Increased Cytomegalovirus Secretion and Risks of Infant Infection by Breastfeeding Duration From Maternal Human Immunodeficiency Virus Positive Compared to Negative Mothers in Sub-Saharan Africa

Abstract

Background: Breastfeeding imparts beneficial immune protection and nutrition to infants for healthy growth, but it is also a route for human immunodeficiency virus (HIV) and human cytomegalovirus (HCMV) infection. In previous studies, we showed that HCMV adversely affects infant development in Africa, particularly with maternal HIV exposure. In this study, we analyzed infants risks for acquisition of HCMV infection from breastfeeding and compared HIV-positive and HIV-negative mothers.

Methods: Two cohorts were studied in Zambia. (1) Two hundred sixty-one HIV-infected and HIV-uninfected mothers were compared for HCMV deoxyribonucleic acid (DNA) loads and genotypes (glycoprotein gO) in milk from birth to 4 months postpartum. (2) Maternally HIV-exposed and HIV-unexposed infants were compared for HCMV infection risk factors. The second cohort of 460 infants, from a trial of micronutrient-fortified complementary-food to breastfeeding, were studied between 6 and 18 months of age. Human cytomegalovirus seroprevalence was assayed, and logistic regression was used to calculate risk factors for HCMV infection, including maternal HIV exposure and breastfeeding duration.

Results: Human cytomegalovirus was detected in breast milk from 3 days to 4 months postpartum, with significantly raised levels in HIV-positive women and independent of genotype. In infants, HCMV antibody seroprevalence was 83% by 18 months age. Longer breastfeeding duration increased infection risk in maternally HIV-unexposed (odds ratio [OR] = 2.69 for 18 months vs <12 months; 95% confidence interval [CI], 0.84-8.59; P = .03) and HIV-exposed infants (OR = 20.37 for >6 months vs never; 95% CI, 3.71-111.70; P < .001).

Conclusions: Prolonged breastfeeding, which is common in Africa, increased risk of HCMV infection in infants. Both HIV-positive and HIV-negative women had extended milk HCMV secretion. Women who were HIV-positive secreted higher HCMV levels, and for longer duration, with their children at increased infection risk. Human cytomegalovirus control is required to maintain health benefits of breastfeeding.

Keywords: HCMV; HIV-exposed infants; breast milk; infant HIV; maternal HIV.

Figure 1.

Human cytomegalovirus (HCMV) shedding kinetics in milk of human immunodeficiency virus (HIV)-positive and HIV-negative women. Comparison of milk HCMV deoxyribonucleic acid (DNA) kinetics between HIV-positive (black lines) and negative women (gray lines) over the first 16 postpartum weeks (n = 40, 20 in each group). Human cytomegalovirus DNA load, log copies/mL milk, increased from comparable levels in the 2 groups from day 3 (week 1 [W1]) to peak levels by week 4 (W4) postpartum. Sensitivity cut-off is indicated by the lower dotted line. Milk DNA loads from HIV-positive women were raised compared with HIV-negative women from W4. Box plots show the median and interquartile range using a Mann–Whitney U test, **P = .026 and ***P < .001 indicate significant differences from W4 and week 16 (W16), respectively.

Figure 2.

Human cytomegalovirus (HCMV) deoxyribonucleic acid (DNA) loads in week 16 milk samples, stratified by human immunodeficiency virus (HIV) serostatus. Scatter plot showing HCMV DNA levels in all available milk samples, at week 16 (W16) (92 HIV-positive and 113 HIV-negative). Human immunodeficiency virus-positive women had a significantly higher mean milk HCMV DNA load, approaching 1 log higher, compared with their HIV-negative counterparts. Furthermore, a higher proportion of milk samples from the HIV-positive group remained with detectable HCMV at this late time point (88.0% [81 of 92] vs 59.3% [67 of 113]; P < .001), with correspondingly decreased proportions below the limit of detection, indicated by the dashed line, in the HIV-positive compared with HIV-negative group (12% [11 of 92] vs 40.7% [46 of 113]). Means were 7.9 × 10⁴ and 1.1 × 10⁴ copies/mL in HIV-positive and HIV-negative women, respectively (P < .001, 2 sample, 2 tailed Student's t test with 95% confidence interval indicated by error bars), with similar differences in the W16 values for the subset with the complete kinetics from Figure 1 as indicated by black circles, mean 8.2 × 10⁴ and 0.5 × 10⁴ copies/mL for HIV-positive compared with HIV-negative women.

Figure 3.

Phylogenetic analyses glycoprotein O (gO) genotypes. Representatives of genotype groups defined here were analyzed in comparison to reference strains, as described previously [32, 33]. Multiple alignments were performed using CLUSTAL in MEGA6 [34], followed by phylogenetic constructions inferred using the Maximum Likelihood method based on the JTT matrix-based model. The analysis involved 39 amino acid sequences and 154 positions in the final dataset. Reference strains for gO genotypes are indicated. Bootstrapping analyses indicate that major nodes are well supported.

Figure 4.

Genotype-independent increases in human cytomegalovirus (HCMV) load in milk from human immunodeficiency virus (HIV)-positive women. Viral loads per genotype were examined for HIV-positive and HIV-negative women. The main 3 glycoprotein O (gO) genotypes (gO1a, gO1b, and gO5) were plotted, and all the remaining genotypes were grouped together. All genotypes appear increased in HIV-positive women compared with HIV-negative women. Genotype gO1a (black circle), gO1b (gray circle), gO5 (white circle), other gO genotypes (crossed circle) are shown. Mean values are as follows: 2.6 × 10⁵ and 5.9 × 10⁴ for HIV-positive and HIV-negative women at both maximal and minimal secreted levels, at 4 and 16 weeks postpartum. Abbreviation: DNA, deoxyribonucleic acid.