Subjects and serum specimens
In collaborative studies on the prevention of mother-to-child transmission of hepatitis B virus conducted in 5 hospitals in Jiangsu Province, China, between April 2012 and March 2015, we prospectively recruited 478 pregnant women with positive hepatitis B surface antigen [17, 18]. Serum samples from these women during pregnancy and/or at delivery were collected and kept at –30ºC. The data about the pregnancy, delivery, and neonatal outcomes were prospectively collected and recorded in a computerized database by the obstetricians at each participating hospital. The umbilical blood samples from newborn infants were also collected and kept at –30ºC. All the neonates received recommended combined immunoprophylaxis against hepatitis B. In the follow-up study, serum samples from those women and their 418 children (4 pairs of twins) after 7 months of age were further prospectively collected. In the present study, we aimed to conduct a secondary analysis of data from the paired mothers and children to investigate the influence of breastfeeding and vaginal delivery on postnatal infection of CMV, and further observe the infant’s outcomes with postnatal CMV infection. Therefore, by further measuring CMV IgG and IgM of serum samples collected in the previous study [17, 18], we included 380 mothers who were CMV IgG positive/CMV IgM negative during pregnancy and their 384 (4 pairs of twins) children in whom umbilical blood was CMV IgM negative in the present study. Those 19 women with both negative CMV IgG and IgM were not included. Fifteen paired mothers and infants were also excluded from analysis because there was insufficient serum volume. In the present study, perinatal information, including maternal gestational age, delivery mode, birth weight and height, and neonatal complications were extracted retrospectively from the originally computerized database collected during 2012–2015 [17, 18]. Furthermore, in this study, we also retrospectively retrieved the relevant data recorded in the previous follow-up study [17, 18], including feeding mode, infant’s age, height and weight, alanine aminotransferase (ALT) levels, and health condition. Formula-fed infants were fed exclusively with formula, while breastfed infants were defined as those who received exclusive breastfeeding or mixed feeding.
This study was approved by the ethics committee of each participating hospital. The study was performed in accordance with the ethical standards in the Declaration of Helsinki. All the pregnant women provided written informed consent and consented to follow up of their infants; each infant’s informed consent was assigned by his/her mother in the previous study conducted in 2012–2015 [17, 18]. Therefore, relevant data and serum samples of the mothers and their children were used in the current study via an exemption approved by the institutional review board of each participating hospital.
Quantification of CMV IgG and IgM
Serum samples were quantitatively tested for CMV IgG using a commercial enzyme-linked immunosorbent assay kit (Dia.Pro Diagnostic Bioprobes, Milano, Italy) as previously reported . The kit contains human plasma derived calibrators with CMV IgG at concentrations of 0, 0.5, 1, 2, 4, and 8 IU/ml. In the measurement, paired maternal and neonatal blood, diluted 1:101 with diluent (2% casein, 10 mM Tris-citrate buffer and 0.1% Tween 20), were tested in parallel, and positive and negative controls provided in the kit were also included. Calibration curve was established for each test, and the IgG level of serum samples was further quantified. Based on the manufacturer’s instruction, the diluted sample with a concentration > 0.5 IU/ml was considered positive for CMV IgG, thus, the sample with a concentration ≤ 0.5 IU/ml was considered negative. When the IgG concentration was beyond the upper detection limit (8 IU/ml), the sera were retested by further dilution.
CMV IgM was measured by the CMV IgM capture immunoassay (Dia.Pro Diagnostic Bioprobes). In each measurement, serum samples were diluted same as in detection of CMV IgG, and positive and negative controls were included as previously reported . As recommended by the manufacturer, the cut-off value was calculated as follows: cut-off = OD450 for negative control + 0.250. The test result was interpreted as a ratio of the sample OD450 and the cut-off value (S/Co). The sample was considered positive if S/Co value was > 1.2, indeterminate if it was 1.0–1.2, and negative if it was < 1.0. The indeterminate sample was retested; the sample was considered positive if S/Co value was 1.0–1.2 or > 1.2, and negative if it was < 1.0.
Data were analyzed with the SPSS software (SPSS Standard version 11.0, SPSS Inc., Chicago, IL). Continuous variables normally distributed were expressed as mean ± standard deviation and compared by two-sample or paired t-test. Quantitative data non-normally distributed are presented as median and range, and compared by Man-Whitney U test. Categorical variables were reported as number and percentage, and compared by χ2 analysis or Fisher’s exact test where appropriate. Logistic regression analyses were further performed to determine the independent role of the feeding and delivery mode in postnatal CMV infection of the infants; the results were expressed by the adjusted odds ratios (OR) with 95% confidence intervals (CI). A two-sided P value fewer than 0.05 was considered statistically significant.