Stability of HIV-1 Nucleic Acids in Dried Blood Spot Samples for HIV-1 Drug Resistance Genotyping

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.

The dried blood spot sampling method in the laboratory medicine

“…Whereas DNA is stable at least one year even, when stored at 37 °C, in most cases RNA is unstable under these conditions. Otherwise, RNA shows a good stability at -70 °C (6-9 months, depending on viral load) (20,21). C-reactive protein (CRP) lev-els decrease quickly in DBS, when stored at ambient temperature, since its stability under these conditions is only 3 days.…”

Participation in adherence clubs and on-time drug pickup among HIV-infected adults in Zambia: A matched-pair cluster randomized trial

“…Viral load suppression at 12 months was a planned secondary outcome but could not be evaluated due to unexpected inaccuracy of test results from dried blood spot (DBS) specimens (collected during study enrollment and exit procedures on all patients) and lack of availability of plasma viral load tests (collected in routine clinical care but available for less than 50% of study participants). Despite use of a validated DBS assay (COBAS AmpliPrep/COBAS Taq-Man, Roche Diagnostics, Indianapolis, Indiana, US) [28][29][30] and detailed laboratory investigation, a clear cause for the assay error could not be determined, as was reported to the institutional review boards at the University of Zambia and University of California, San Francisco. Other secondary outcomes (S1 Text), including costs and cost-effectiveness, are planned for separate publication.…”

Miniaturized devices for point of care molecular detection of HIV

“…For example, blood samples can be preserved by blotting onto cellulose-based filter papers (i.e., dried blood spots (DBS)) for subsequent viral load testing [43], early infant diagnosis [44] and drug resistance genotyping [45]. Pathogens in dried blood spots are deactivated due to the inclusion of appropriate chemicals in the paper [46].…”