Abstract
Dried blood spots (DBS) are an easy to collect sample-type that can stabilize biological material at ambient temperature for transport and storage, making them ideal for use in resource-limited settings (RLS). We investigated the effect of storage temperature and duration on ability to detect mixed HIV-1 viral RNA populations, and subsequently viral RNA populations in a background of proviral DNA. Part one of the study used DBS samples of whole blood spiked with specific quantities of HIV-1 subtype-B and -C RNA to study mixed virus population detection. Part two used DBS comprising of HIV-1 subtype-B proviral DNA containing U1 cells combined with HIV-1 subtype-C RNA to mimic HIV-1 infected clinical samples as a model system to study the relative stability of HIV-1 RNA and DNA in DBS. Prepared DBS were stored at -20°C and +30°C for periods of one day, one, two, and four weeks. Samples were genotyped to determine changes in the detection of mixtures in the sample over time. From two weeks onwards, storage at +30°C resulted in gradual, time-related reduction in the detection of mixed virus population at log10 VL 4.0 but not at log10 5.0. Proviral DNA and viral RNA were both stable for at least 52 weeks when stored at -20°C, compared to progressive RNA decay over time at +30°C. DBS storage conditions and duration had a significant effect on HIV-1 RNA amplification. Our results demonstrate that DBS storage at ambient temperature (+30°C) should not exceed two weeks, with long-term storage at -20°C or lower.
Highlights
MethodsSample Material: Viruses, Cell Lines, and Whole BloodCultured HIV-1 subtypes-B (strain BK132; NCBI accession number AY173951) and C (strain ZB18; NCBI accession number AB4856) from the BBI subtype panel (BBI Biotech Research Laboratories Inc., USA) were used to represent circulating virus
The use of dried blood spot samples (DBS) is becoming increasingly popular for HIV-1 drug resistance genotyping and viral load (VL) monitoring in resource limited settings (RLS)
Our results indicate that maintaining the quality of the DBS sample for VL and HIV-1 drug resistance (HIVDR) determination on the one hand and using simple sample collection and shipment procedures on the other can be achieved if reverse transcriptase (RT) storage of DBS does not exceed two weeks, with subsequent long term storage at -20°C or below
Summary
Sample Material: Viruses, Cell Lines, and Whole BloodCultured HIV-1 subtypes-B (strain BK132; NCBI accession number AY173951) and C (strain ZB18; NCBI accession number AB4856) from the BBI subtype panel (BBI Biotech Research Laboratories Inc., USA) were used to represent circulating virus. Sample Material: Viruses, Cell Lines, and Whole Blood. EDTA whole blood (WB) was obtained from the Mini Donor Service (Mini Donor Dienst, MDD) at the University Medical Center Utrecht (UMCU), The Netherlands. The MDD has received positive approval from the Medical Ethics Committee of the UMCU (Medisch Ethische Toetscommissie, METC; www.umcutrecht.nl/metc) for the protocol number 07-125/ C (S1 File). The METC concluded that the MDD protocol is not considered medical research but rather falls under the classification of having to abide by the regulations of Good Clinical. Volunteers of the MDD sign an informed consent document prior to partaking in the MDD (S2 File). All MDD participant information is kept confidential and only MDD personal have access to their particulars. WB that is obtained from the MDD is confirmed at being HIV-1 negative by the MDD service
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