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.
| Original language | English |
|---|---|
| Pages (from-to) | 134-140 |
| Number of pages | 7 |
| Journal | Current HIV Research |
| Volume | 8 |
| Issue number | 2 |
| DOIs | |
| Publication status | Published - Mar 2010 |
| Externally published | Yes |
Keywords
- Antiretroviral therapy
- Dried blood spot
- HIV drug resistance
- HIV genotyping
Cite this
}
Stability of dried blood spots for HIV-1 drug resistance analysis. / Hearps, Anna C.; Ryan, Claire E.; Morris, Lisa M; Plate, Megan; Greengrass, Vicki; Crowe, Suzanne M.
In: Current HIV Research, Vol. 8, No. 2, 03.2010, p. 134-140.Research output: Contribution to journal › Article › Research › peer-review
TY - JOUR
T1 - Stability of dried blood spots for HIV-1 drug resistance analysis
AU - Hearps, Anna C.
AU - Ryan, Claire E.
AU - Morris, Lisa M
AU - Plate, Megan
AU - Greengrass, Vicki
AU - Crowe, Suzanne M.
PY - 2010/3
Y1 - 2010/3
N2 - 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.
AB - 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.
KW - Antiretroviral therapy
KW - Dried blood spot
KW - HIV drug resistance
KW - HIV genotyping
UR - http://www.scopus.com/inward/record.url?scp=77949716973&partnerID=8YFLogxK
U2 - 10.2174/157016210790442740
DO - 10.2174/157016210790442740
M3 - Article
C2 - 20163343
AN - SCOPUS:77949716973
VL - 8
SP - 134
EP - 140
JO - Current HIV Research
JF - Current HIV Research
SN - 1570-162X
IS - 2
ER -