Quantitation of human immunodeficiency virus type 1 in breast milk

Abstract

The distribution and stability of human immunodeficiency virus type 1 (HIV-1) in breast milk (BM) components remain largely unknown. Inhibitory effects, if any, of BM on HIV RNA and DNA PCR amplification are poorly understood. We have addressed these issues by using virus-spiked BM samples from HIV-negative women. BM samples from HIV-negative women were spiked with HIV-1 virions or cells containing a single integrated copy of HIV DNA (8E5/LAV). After incubation under different experimental conditions, viral RNA was detected by the Roche Amplicor UltraSensitive assay in whole-milk, skim milk, and lipid fractions. We found excellent correlation between HIV-1 input copy and recovery in whole milk (r = 0.965, P < 0.0001), skim milk (r = 0.972, P < 0.0001), and the lipid fraction (r = 0.905, P < 0.001). PCR inhibition was observed in less than 10% of the spiked samples. Similar levels of inhibition were noted in BM samples collected from HIV-infected women. HIV proviral DNA was detected in BM samples using real-time PCR (linear correlation between the threshold cycle versus log DNA copy number, >0.982). The effects of incubation duration and temperature and repeated freeze-thaw cycles on HIV RNA recovery were analyzed. HIV RNA levels were remarkably stable in whole milk after three freeze-thaw cycles and for up to 30 h at room temperature. Our findings improve the understanding of the dynamics of HIV detection in BM and the conditions for BM sample collection, storage, and processing.

Figures

FIG. 1.

Aliquots of whole breast milk from 10 women were spiked with serial twofold dilutions of HIV-1 stocks and then assayed using the Roche Amplicor UltraSensitive version 1.0 assay. Next the spiked breast milk was spun, and the skim milk and lipid fractions were analyzed separately. The log10 numbers of HIV-1 RNA copies, measured by the nominal input copy number in the skim milk fraction (A), the lipid fraction (B), and whole milk (C), are shown.

FIG. 2.

Whole breast milk samples were spiked with HIV-1 RNA and then were either frozen immediately or kept at room temperature for 6, 18, or 30 h prior to testing using the Roche Amplicor UltraSensitive version 1.0 assay. The observed log10 numbers of HIV-1 RNA copies per milliliter of milk measured when the input nominal copy number was 50,000 copies/ml (thick lines) or 20,776 copies/ml (thin lines) are shown.

FIG. 3.

Whole breast milk samples from five women were spiked with either 5,000 or 25,000 copies of HIV-1 RNA per ml in duplicate. The Samples were tested immediately for HIV-1 RNA after sample collection and after three freeze-thaw cycles. The HIV-1 RNA quantities in milk spiked with 5,000 copies (thin lines) or 25,000 copies (thick lines) of HIV-1 RNA observed before and after three freeze-thaw cycles are shown.

FIG. 4.

Real-time PCR of HIV-1 standards. A standard curve was obtained by real-time PCR from 10-fold dilutions of 8E5/LAV cells (containing a single integrated HIV proviral copy per cell) into breast milk. The mean Ct is plotted against the logarithmic number of HIV-1 copies detected in the serial dilutions. The uniform slope of the β-globin copies detected demonstrated that an equivalent cellular background was present in each sample.