Inactivation of Cytomegalovirus in Breast Milk Using Ultraviolet-C Irradiation: Opportunities for a New Treatment Option in Breast Milk Banking

Abstract

Pasteurized donor human milk is provided by milk banks to very preterm babies where their maternal supply is insufficient or unavailable. Donor milk is currently processed by Holder pasteurization, producing a microbiologically safe product but significantly reducing immunoprotective components. Ultraviolet-C (UV-C) irradiation at 254 nm is being investigated as an alternative treatment method and has been shown to preserve components such as lactoferrin, lysozyme and secretory IgA considerably better than Holder pasteurization. We describe the inactivation of cytomegalovirus, a virus commonly excreted into breast milk, using UV-C irradiation. Full replication was ablated by various treatment doses. However, evidence of viral immediate early proteins within the cells was never completely eliminated indicating that some viral gene transcription was still occurring. In conclusion, UV-C may be a safe alternative to pasteurisation for the treatment of human donor milk that preserves the bioactivity. However, our data suggests that CMV inactivation will have to be carefully evaluated for each device designed to treat breast milk using UV-C irradiation.

Fig 1. The effect of low temperatures on HCMV viability.

Pasteurized breast milk was spiked with AD169 stock 1:5 and 0.5 mL aliquots were prepared and either stored at 4°C or frozen at -20°C. After 24, 48, 72 and 96 hours at 4°C or -20°C, samples were removed/thawed and analysed by TCID₅₀ on human foreskin fibroblasts (HFF). One sample was stored at -20°C for 21 days (504 hours). The presence or absence of CPE was determined for each well and calculated using the Reed and Muench Calculator. Results are presented as TCID₅₀/mL. Superscripts (1–4) underneath multiple freeze/thaw samples designate the number of times the sample was thawed.

Fig 2. UV exposed AD169 spiked milk and media.

Pasteurized breast milk or MEM culture medium were spiked with AD169 culture 1:5. 150 μL samples were irradiated for 10 seconds at 1, 2, 3 or 4 cm from the UV light source (estimated UV intensity 400, 100, 25 and 4 W/cm2 respectively). The irradiated samples were inoculated on to human foreskin fibroblasts (HFF). The presence or absence of CPE was determined for each well and calculated using the Reed and Muench Calculator. Results are presented as TCID₅₀/mL. Breast milk samples with values below the limit of detection are designated (*).

Fig 3. UV-C inactivation of HCMV.

A—C. UV-C exposure of spiked breast milk for 10 seconds from 1 to 5 cm from the UV light source. Pasteurized breast milk was spiked with AD169 culture 1:5. The irradiated samples were diluted tenfold and inoculated on to human HFF and the TCID₅₀ calculated using the method described by Reed and Muench [15]. A. Results are presented as TCID₅₀/mL. Breast milk samples with values below the limit of detection are designated (*). B. Irradiated samples were inoculated onto HFF, centrifugally enhanced and cells were analysed by immunofluorescence at 7 days post infection. Results are presented as % infected cells. C: UV dose delivered. D—F. UV-C exposure of spiked breast milk at 5 cm from the UV light source for different durations. D: Pasteurized breast milk was spiked with AD169 and samples were irradiated at 5 cm from the UV light source for 10 to 50 seconds. The irradiated samples were assessed by TCID₅₀. Breast milk samples with values below the limit of detection are designated (*). E. Irradiated samples were inoculated onto HFF, centrifugally enhanced and cells were analysed by immunofluorescence at 7 days post infection. Results are presented as % immunofluorescently labelled cells. * <1% of cells fluorescently labelled. F: Inset shows results for 30, 40 and 50 seconds exposure. G: UV dose delivered. Where error bars are present, images show mean ± standard deviation.

Fig 4. CMV infected HFF cells stained using DAPI and MABX.

A Uninfected HFF cells (DAPI), B: Uninfected cells (MABX) C, D: Cells infected with AD169, no UV-C treatment, C: DAPI, D: MABX. E, F: Cells infected with AD169, 10 seconds UV-C treatment (5 cm), E. DAPI, F: MABX, G, H: Cells infected with AD169, 50 seconds UV-C treatment (5 cm) G: DAPI, H: MABX.