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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

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