Milk samples
Previous viral infected mothers have been identified by IgG seropositivity towards CMV, in absence of IgM. In 40 out of 57 seropositive mothers (70.2%) the virus reactivated and was excreted in the milk during the six weeks postpartum lactation period. Maternal CMV reactivation was shown by viral shedding in breast milk (Figure 1) concomitantly with positive serum IgG but negative IgM, and negative viruria.
There was not statistically significant difference (p = 0.36) in the values of CMV kinetics, comparing positive samples to total samples examined in the different weeks. The majority of the colostrum samples were CMV DNA positive (31 out of 57, 54.4%) and most milk samples became CMV DNA positive two weeks after delivery (33 out of 52, 63.5%). During the period of the study 109 samples were tested and the highest values of CMV DNA copies, ranging between 104 to 106 copies/mL, were shown from the 4th to the 6th week after delivery. In this period the average DNA load was 61958,8 ± 15818,3 copies/mL. Thereafter, DNA copy number decreased progressively. The 72 h freezing process was capable of decreasing significantly the infectivity, as shown by viral culture, and the viral load (by approximately 75% (Figure 2). However, positive samples remained positive when tested by PCR, since viral DNA is detectable, even in the presence of very few inactivated CMV particles.
Infants
The average gestational age of the newborns was 29 weeks (range 23-34 weeks). The average birth weight was 1158 ± 436.3 g. Among the infants who have been fed with CMV DNA-positive breast milk, only one became infected with CMV. Considering all the checked women, the rate of postnatal infection in exposed newborns was 2.5%: the rate of perinatal infection was 1.7%. The postnatal rate infection (2.5%) has been considered, because the infant became CMV positive 8 weeks after birth and the mother's serological test did not change, showing CMV IgM negative and IgG positive. This results indicate that maternal CMV reactivation was not systemic but occurred only in the breast.
All the infants were followed up to detect long-term sequele as described for congenital and perinatal cytomegalovirus infection: each infant underwent serial monitoring (on 3rd, on 6th, on 12th, on 24th, on 36th month and on 5th of correct gestation age). To date (after at least 3 years from the enrolment), none of them presents health problems correlated with CMV infection, including the symptomatic infants.
Case report
The infant infected by CMV was born by caesarean section, carried out in election because of pre-eclampsia and intrauterine growth retardation, at 28th week and 3 days, having a birth weight of 740 g. He was initially treated for respiratory distress syndrome and hyperbilirubinaemia. During the first day of life, a wide spectrum antibiotic therapy as well as Immunoglobulin IgM-enriched have been administered, because of the increasing value of C-reactive protein in absence of other clinical symptoms. However, all tests performed to detect bacterial infectious were negative: gastric aspiration and tracheobronchial aspiration, including culture test for Mycoplasma and Clamidia, and blood culture. At age 30 days, the newborn developed conjugated hyperbilirubinaemia, and the liver ultrasound showed contracted gallbladder in accordance with relevant parietal thickening. On day 47, the clinical condition of the newborn worsened: elevated serum C-reactive protein levels were found and the infant was again treated with multiple antibiotics because of a suspected sepsis, even if blood and urine cultures were negative. Cholestasis indexes worsened (Bilirubin total 14,99 mg/dL, Bilirubin conjugated 8,31 mg/dL, AST 493 U/L, ALT 183 U/L, GGT 98 U/L). Moreover, the presence of CMV DNA was evidenced in urine, saliva and blood, and of CMV-specific IgM antibodies were positive in serum. The involvement of the digestive system by CMV was confirmed by the presence of a high number of viral DNA copies detected by PCR in the gastric aspirate and stools. Congenital transmission was excluded by negative CMV DNA detection from umbilical cord and Guthrie card, even using highly sensitive real-time PCR. Clinical improvement occurred in accordance with intravenous ganciclovir therapy (7,5 mg/Kg/die twice/day for five days) followed by administration of valganciclovir (25 mg/Kg/die for six months)[5]. The viral load gradually decreased until negativization. Molecular profiles of CMV strain isolated from infants' urine were indistinguishable from that isolated from maternal breast milk, indicating vertical CMV transmission through breast milk.