Breast-feeding mothers frequently transmit cytomegalovirus (CMV) to preterm infants of very low birth weight. Current recommendations for prevention of virus transmission are based on data published 20 y ago in the context of human milk banking. Two recent clinical trials examined storage of breast milk at Ϫ20°C to reduce virus transmission. However, in both studies, CMV transmission occurred. Using sensitive tools like quantitative PCR, CMV pp67 late mRNA assay, and a high-speed, centrifugation-based microculture assay for quantification of CMV infectivity, we reassessed the virological and biochemical characteristics of freeze-storing breast milk at Ϫ20°C, compared it with traditional Holder pasteurization (30 min at 62.5°C), and a new short-term pasteurization (5 s at 72°C) based on the generation of a milk film. Both heat treatment procedures were able to destroy viral infectivity and pp67 RNA completely. Preliminary results showed short-term heat inactivation below 72°C was less harmful in reducing the activity of marker enzymes than Holder pasteurization. Freezing breast milk preserved the biochemical and immunologic quality of the milk; however, late viral RNA and viral infectivity was also preserved. Breast-feeding in premature infants is beneficial (1). However, breast-feeding of a high-risk group of preterm infants (birth weight Ͻ1000 g, gestational age at birth Ͻ30 wk) may be associated with symptomatic CMV infection (2,3). If CMV transmission from breast milk to preterm infants is clinically important, then prevention of virus transmission will be critical (4).Recent recommendations for virus inactivation in human milk by freezing, which destroys the virus (5,6), are still based on three studies that evaluated freezing for viral decontamination of stored milk in the context of human milk banking 20 y ago (7-9). Only two studies analyzed the effect of storage at Ϫ20°C using naturally infected milk specimens (8,9). Storage of milk for 3 d at Ϫ20°C reduced the viral titre by more than 99% (9), and storage of naturally infected milk specimens for 7 d or longer at Ϫ20°C eliminated infectivity (8). On the other hand, CMV survived when the milk sample was frozen at

ObjectivesBreast milk (BM) is the primary source of cytomegalovirus (CMV) transmission to premature infants with potentially harmful consequences. We therefore wanted to evaluate temperature and duration of short-term BM pasteurisation with respect to CMV inactivation, effect on CMV-IgG antibodies and BM enzyme activities.Methods116 artificially CMV-spiked BM and 15 wild-type virus-infected samples were subjected for 5 s to different temperatures (55°C–72°C). CMV-IE-1 expression in fibroblast nuclei was assessed using the milk whey fraction in short-term microculture. BM lipase and alkaline phosphatase (AP) activities and CMV binding using CMV-recomLine immunoblotting and neutralising antibodies using epithelial target cells were analysed before and after heating.ResultsA minimum of 5 s above 60°C was necessary for CMV inactivation in both CMV-AD-169 spiked and wild-type infected BM. Lipase was very heat sensitive (activities of 54% at 55°C, 5% at 60°C and 2% at 65°C). AP showed activities of 77%, 88% and 10%, respectively. CMV-p150 IgG antibodies were mostly preserved at 62°C for 5 s.ConclusionOur results show that short-term pasteurisation of BM at 62°C for 5 s might be efficient for CMV inactivation and reduces loss of enzyme activities, as well as CMV binding, and functional CMV antibodies.