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Heat-treated breast milk of HIV-positive mothers has potential to reduce vertical transmission. This study compared the impact of flash-heating (FH) and Pretoria pasteurization (PP) on HIV, nutrients, and antimicrobial properties in human milk.Milk samples were spiked with 1 × 108 copies/mL of clade C HIV-1 and treated with FH and PP. We measured HIV reverse transcriptase (RT) activity before and after heating (n = 5). Heat impact on vitamins A, B6, B12, and C; folate, riboflavin, thiamin, and antimicrobial proteins (lactoferrin and lysozyme) was assessed. Storage safety was evaluated by spiking with Escherichia coli or Staphylococcus aureus.Both methods inactivated ≥3 logs of HIV-1. FH resulted in undetectable RT activity. Neither method caused significant decrease in any vitamin, although reductions in vitamins C and E were noted. Heat decreased immunoreactive lactoferrin (P < 0.05) but not the proportions of lactoferrin and lysozyme surviving digestion. FH seems to retain more antibacterial activity. Both treatments eliminated spiked bacteria.FH may be superior to PP in eliminating all viral activity; both methods retained nutrients and destroyed bacterial contamination. Heat-treated breast milk merits further study as a safe and practical infant feeding option for HIV-positive mothers in developing countries.