Validation of a Real-Time PCR Assay for Fully Automated Detection of Bacillus cereus in Donor Human Milk

Abstract

Donor human milk (DHM) can harbor microbial contaminants that cause serious infections in premature infants. Bacillus cereus is a pathogen frequently found in DHM, capable of forming spores that can resist Holder pasteurization (62.5 °C, 30 min). Since no microbial growth is acceptable in post-pasteurized DHM, microbiological testing of pre-pasteurized DHM provides information about its contamination level to determine if it should be accepted for pasteurization. Culture is the gold standard in microbiological control but it requires 24-48 h to provide results. In this study we developed and validated a non-commercial real-time PCR assay for the detection of Bacillus cereus (BC test) in DHM specimens on a fully automated high-throughput platform, the cobas® 6800 system. The BC test showed excellent sensitivity and specificity, repeatability and linearity over an 8-log range and a low limit of detection in milk specimens, as well as good agreement with selective culture methods. BC test was then used to systematically control all milk donations (3439) over a 24-month period. Bacillus cereus was detected in 14.2% of DHM, with monthly rates ranging from 6 to 29% and a significantly higher incidence in warmer months. Incorporating this assay into our laboratory workflow improved efficiency and reduced turnaround time.