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Optimization of Abbott m2000 RealTime HIV-1 viral load assay on breastmilk, dried blood spots, seminal plasma, and cerebrospinal fluid
Presented by Susan Fiscus (United States).
A. Loftis1, R. Kshatriya1, K. McCall-Culbreath2, S. Fiscus1, J. Nelson1
1University of North Carolina at Chapel Hill, Microbiology and Immunology, Chapel Hill, United States, 2University of North Carolina at Chapel Hill, Medicine, Chapel Hill, United States
Background: The latest technology for measuring HIV-1 RNA in plasma is the real-time PCR platform. Abbott's m2000 RealTime HIV-1 assay is CE-marked and FDA-approved for plasma only, but has not been optimized for off-label use with other sample types. Measurement of HIV-1 RNA levels in other body fluids, including breastmilk (BMK), dried blood spots (DBS), seminal plasma, and cerebrospinal fluid (CSF), is important for understanding pathogenesis and transmission of HIV. More accurate and precise methods for measuring HIV RNA levels in these other sample types are needed.
Methods: Plasma, BMK, whole blood, semen, and CSF were obtained from uninfected donors. 1:10 dilutions of an HIV-1 virus stock was added to plasma, whole BMK, whole blood (for DBS), seminal plasma, and CSF to generate series of samples containing 0-6 log10 copies of HIV-1 per ml of fluid. Each sample type was lysed in different volumes of lysis buffer, with BMK and semen treated with proteinase K prior to extraction. Different m2000sp protocols were used to accommodate different sample volumes after addition of lysis buffer. HIV-1 RNA levels were measured off-label using the RealTime HIV-1 viral load assay in the m2000rt after extraction and the data from the different sample types were compared with the results from the spiked plasma samples.
Results: The results from the dilution series in plasma, BMK, semen, CSF, and DBS had linear results compared to the nominal cp/ml. Comparison of the samples to plasma indicated that HIV-1 RNA recovery after adjusting for sample input and dilution was near 100% from semen, CSF, and DBS, and approximately 50% from BMK.
Conclusions: The Abbott m2000 RealTime HIV-1 assay works well for measuring HIV-1 RNA levels in BMK, semen, CSF, and DBS. More accurate and precise measurements are critical for understanding pathogenesis and transmission, and for clinical trial evaluations.
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