Pasteurization Inactivates SARS-CoV-2–Spiked Breast Milk

Abstract

The current coronavirus disease 2019 pandemic, caused by the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), raises unprecedented questions regarding virus transmission and risks for pregnant or breastfeeding women. We and others described that SARS-CoV-2 RNA is detectable in the breast milk of infected mothers.1–3 In two cases in which the mother continued breastfeeding, the newborns also tested positive for SARS-CoV-2.1,2 However, the origin of the infections of the newborns remained unclear and raised concerns of possible virus transmission via breast milk. The safety and feasibility of breastfeeding is of high importance because breast milk contains nutrients, hormones, and immunoprotective entities that are essential for the development, health, and protection of the neonate. So far, the World Health Organization recommends continued breastfeeding after maternal SARS-CoV-2 infection while taking measures to maintain strict hygiene.4 We here explored the inactivation of SARS-CoV-2 spiked into human milk by Holder pasteurization to reduce the risk of a possible virus transmission while preserving the milk’s beneficial properties.See the Supplemental Information for detailed methods.Viral isolates BetaCoV/France/IDF0372/2020 and BetaCoV/Netherlands/01 were obtained through the European Virus Archive Global and BetaCoV/Germany/Ulm/01/2020, BetaCoV/Germany/Ulm/02/2020, and BetaCoV/UKEssen from patient samples.Milk samples were obtained from 5 healthy human donors after ethical approval by the Ethics Commission of Hanover Medical School and the Ethics Committee of Ulm University. All mothers provided written informed consent for the collection of samples and subsequent analysis. Milk was collected freshly and stored at −80°C until further use as anonymized samples.Tissue culture infectious dose 50 (TCID50) was determined by infection of Cercopithecus aethiops–derived epithelial kidney (Vero E6) or human epithelial colorectal adenocarcinoma (Caco-2) cells. To this end, 20 000 cells were seeded per 96-well plate and, the next day, inoculated with serially diluted samples. For 3 to 6 days, cytopathic effect was monitored, and TCID50 per milliliter was calculated according to the Spearman-Karber method.To test if SARS-CoV-2 retains infectivity in human breast milk and to explore Holder pasteurization as a possible inactivation method, we spiked five different SARS-CoV-2 isolates from Germany, France, and the Netherlands 1:10 into five individual breast milk samples or medium samples and incubated them for 30 minutes at room temperature or 63°C. Residual infectivity was determined as TCID50 after titration on susceptible cells. All five tested SARS-CoV-2 isolates (BetaCoV/UKEssen, BetaCoV/Germany/Ulm/01/2020, BetaCoV/Germany/Ulm/02/2020, BetaCoV/France/IDF0372/2020, and BetaCoV/Netherlands/01) remained infectious in the milk samples that were incubated for 30 minutes at room temperature, with infectious titers of 0.09 to 1.2 × 105 TCID50 per mL (Fig 1). Of note, in each milk sample and independent of the viral strain, we detected a 40.9% to 92.8% decrease of viral titers compared with the medium control. This might indicate an antiviral property of milk potentially due to free fatty acids.5,6 Importantly, after pasteurization, no residual infectivity was detected in any of the samples (Fig 1). Thus, human breast milk potentially containing infectious SARS-CoV-2 can be efficiently inactivated by using standard Holder pasteurization.Holder pasteurization (heating to 63°C for 30 minutes) is a standard procedure to inactivate viral and bacterial agents while at the same time preserving many beneficial and protective effects of human breast milk. Accumulating evidence reveals that SARS-CoV-2 RNA can be shed into the breast milk of infected mothers.1–3 Although no transmission events via breastfeeding have been recorded and no infectious virus has been detected, the possibility needs to be considered, and measures providing safety and supporting continued feeding of breast milk need to be evaluated. Thus, we explored the efficiency of Holder pasteurization against SARS-CoV-2 in five milk samples and found that infectivity of all five tested SARS-CoV-2 isolates is completely eliminated by this treatment. This confirms the findings of earlier studies in which Holder pasteurization of milk spiked with three SARS-CoV-2 isolates prevented viral infection of monkey-derived Vero E6 cells.7–9 We verify the efficiency of Holder pasteurization after inclusion of additional viral strains encompassing the dominant pandemic variant D614G, which reveals increased fitness in cell culture (BetaCoV/Netherlands/01, BetaCoV/UKEssen),10 as well as highly susceptible Caco-2 cells, which express the viral entry factor transmembrane serine protease 2. Thus, despite no infectious virus having been detected in milk so far, Holder pasteurization provides safety for the infant and in milk banks and reassurance for the mother, who might consider discontinuing breastfeeding and substituting for infant formula milk, which lacks many of human milk’s important components.We thank all mothers and all members of the midwife team Luna who contributed to the milk donation. Furthermore, we thank all members of the Department of Molecular and Medical Virology of Ruhr University Bochum for the helpful discussions. We thank Lisa Schwefele, MD (Division of Neonatology, Department of Pediatrics, Ulm University Medical Center), and Frank Reister, MD (Division of Obstetrics, Department of Obstetrics and Gynecology, Ulm University Medical Center), for reviewing the article from a clinical point of view. We thank Karin Steinhart, MD (Administrative District Heidenheim, Public Health Office), for organizing milk samples and Tirza Braun for experimental assistance.

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