671 Quantitation of Human Immunodeficiency Virus Type 1 (HIV-1) in Breast Milk M. Ghosh*1, L. Kuhn2, J. West3, K. Semrau4, D. Decker1, D. Thea4, G. Aldrovandi1 1Univ of Alabama at Birmingham; 2Columbia Univ, New York, NY; 3Univ of Nebraska; and 4Boston Univ, MA
Background: The distribution and stability of HIV-1 in breast milk (BM) components remains largely unknown. Inhibitory effects, if any, of BM on HIV RNA and DNA PCR amplification are also poorly understood. We have addressed these issues by using virus-spiked BM samples from HIV- women.
Methods: BM samples from 10 uninfected women were spiked with HIV-1 virions or cells containing a single integrated copy of HIV DNA (8E5/LAV) and incubated under different conditions. Viral RNA was detected by the Roche Amplicor UltraSensitive assay in the whole milk, skim milk, and lipid fractions. HIV-1 DNA was detected by real time polymerase chain reaction assay. Associations between input and observed viral quantity were described using Spearman rank correlation co-efficients (rho), and Kruskall-Wallis tests were used to describe differences in observed viral quantity under different conditions.
Results: We found excellent correlation between HIV-1 input copy and recovery in whole milk (rho = 0.965, p < 0.0001), skim milk (rho = 0.972, p < 0.0001), and the lipid fraction (rho = 0.905, p < 0.001). Surprisingly, on average, BM lipid contained over 3 times more (9.38% ±3.05% to 11.18% ±2.33%) virus than expected based on the lipid’s relative proportion. PCR inhibition was observed in less than 10% of the virion spiked samples. Similar levels of inhibition were noted in a number of BM samples collected from HIV+ women. Detection of HIV proviral DNA in serially diluted 8E5/LAV spiked BM samples also revealed a linear pattern (correlation between the threshold cycles vs log10 DNA copy number > 0.982). The effects of incubation duration and temperature, as well as repeated freeze-thaw cycles on HIV RNA recovery, were also analyzed. It was observed that HIV RNA levels were stable in whole milk, after 3 freeze-thaw cycles and for up to 30 hrs at room temperature.
Conclusions: HIV RNA and DNA can be accurately detected in breast milk. Using a commercial assay to detect HIV RNA, we observed relatively little (< 10%) PCR inhibition. HIV RNA can be detected in both the skim and lipid fractions of milk. HIV RNA in breast milk is remarkably stable at room temperature and after repeated freeze-thaw cycles. These results should assist investigators in the development of standardized collection, transportation, processing, and storage procedures for studies involving breast milk.
