Stability of Dried Blood Spots for HIV-1 Drug Resistance Analysis

Anna   C.   Hearps; Claire   E.   Ryan; Lisa   M.   Morris; Megan   M.   Plate; Vicki      Greengrass; Suzanne   M.   Crowe

Abstract

The wide scale application of dried blood spots (DBS) as a collection tool for low-cost HIV drug resistance testing requires a greater understanding of the accuracy of DBS for genotype analysis and the stability of DBS under various environmental conditions. Analysis of a 50μl DBS via a single amplicon, nested PCR-based in-house assay (the Burnet genotyping assay) showed an average nucleotide concordance of 98.9% with plasma samples, although only 65% of nucleotide mixtures detected in plasma were also detected within DBS. The analysis of three DBS resulted in the detection of a greater number of nucleotide mixtures (72 and 109 mixtures detected within one and three DBS, respectively, n = 10). Two DBS extraction protocols (silica particle; NucliSENS, bioMerieux and spin column extraction; High Pure, Roche) were assessed and found to be equivalent (79% and 84% recovery success respectively, n = 19). FTA Elute paper (Whatman) was an inferior DBS collection medium compared to Whatman 903 paper. DBS appeared relatively tolerant to multiple freeze/thaw cycles, with 79% of DBS subjected to ten freeze/thaw cycles successfully amplified compared to 93% of DBS defrosted once (n = 14). High temperature (37°C) and high humidity ( > 90%) substantially impaired DBS recovery within two weeks of storage (38%, n = 8), whilst storage at -20°C or 4°C adequately preserved DBS for this period (100% recovery, n = 8). Therefore, whilst DBS are suitable for HIV drug resistance surveillance, the use of multiple DBS may be required to ensure accurate detection of minor HIV quasispecies and shortterm storage of samples at either 4°C or -20°C is recommended.

Keywords: Dried blood spot, HIV drug resistance, HIV genotyping, antiretroviral therapy

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