Background:  Mother-to-child transmission of HIV-1 via breast milk causes an estimated 350,000 infants to contract HIV each year in developing countries. Heat-treatment of expressed breast milk is one alternative infant feeding option advocated by WHO and several methods have been proposed. However these methods have not been systematically evaluated, in part because present culture-based methods of evaluating the efficacy of inactivating HIV in breast milk are insensitive, slow, technically demanding, and lack precision. This work describes 2 pasteurization methods, flash-heating (FH) and Pretoria pasteurization (PP), and uses a new approach for measuring HIV inactivation based upon the destruction of reverse transcriptase (RT) activity that is sensitive, rapid and precise.

Methods:  Fresh breast milk was obtained from 5 healthy women volunteers and spiked with 1x10⁸ copies/mL of an HIV-1 subtype C isolate. Field conditions were simulated using a 1-qt aluminum pan, 16-oz glass peanut butter jars, and an open flame. Heating methods included:  for FH‑50 mL of breast milk in an uncovered peanut butter jar was placed in 450 mL of water in an aluminum pan over a flame until the water began to boil, then breast milk was immediately removed from the water and heat, and allowed to cool to room temperature; for PP‑450 mL of water was brought to a boil in an aluminum pan, removed from the flame, and a covered peanut butter jar with 50 mL of breast milk was placed in the water for 20 minutes, then removed and allowed to cool to room temperature uncovered. Aliquots of breast milk also served as unheated (No Heat) and unspiked (No Virus) controls. Temperature was tracked using a DuaLogR Thermocouple. One-mL samples of FH, PP, No Heat, and No Virus were quantitatively assessed for RT activity using appropriate dilutions in the ExaVir Quantitative HIV-RT Load assay (Cavidi, Uppsala, Sweden).

Results:   A representative temperature recording of FH and PP is presented at the left. Comparison of RT activity in 5 breast milk samples treated according to the 4 methods is shown at the right.

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Conclusions:  Both the FH and PP methods inactivated 3 logs or more of HIV-1 RT activity. However, the FH method appeared superior to the PP method in eliminating residual RT activity. This approach, utilizing simulated field conditions, accurate and continuous temperature monitoring, and the exquisite biologic sensitivity and range of the RT assay, should allow the direct assessment of HIV-1 inactivation in breast milk from HIV-infected mothers.

Keywords: Breast Milk; Pasteurize; Reverse Transcriptase Assay