Stability of dried blood spots for HIV-1 drug resistance analysis.
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 50microl 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 degrees C) and high humidity (>90%) substantially impaired DBS recovery within two weeks of storage (38%, n=8), whilst storage at -20 degrees C or 4 degrees 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 short-term storage of samples at either 4 degrees C or -20 degrees C is recommended.
Hearps
Anna C
AC
Centre for Virology, Burnet Institute, 85 Commercial Road, Melbourne 3004, Australia. annah@burnet.edu.au
Ryan
Claire E
CE
Morris
Lisa M
LM
Plate
Megan M
MM
Greengrass
Vicki
V
Crowe
Suzanne M
SM
D001769:Blood / Q000821:virology*; D002460:Cell Line; D003890:Desiccation; D024882:Drug Resistance, Viral / Q000235:genetics*; D005838:Genotype; D015497:HIV-1 / Q000235:genetics / Q000502:physiology*; D006403:Hematologic Tests / Q000295:instrumentation / Q000592:standards; D006801:Humans; D006813:Humidity; D010209:Paper / Q000592:standards; D010949:Plasma / Q000821:virology; D015203:Reproducibility of Results; D012680:Sensitivity and Specificity; D013048:Specimen Handling / Q000379:methods* / Q000592:standards; D013696:Temperature; D013997:Time Factors