Effect of storage conditions of dried plasma and blood spots on HIV-1 RNA quantification and PCR amplification for drug resistance genotyping.
OBJECTIVE
Dried blood spots (DBS) and dried plasma spots (DPS) are easy to collect and store, and have been successfully tested as an alternative to plasma for performing virological analyses. Adequate storage conditions still need to be established and cell-associated proviral DNA in DBS can contribute to the amplified products. We evaluated these two parameters.


METHODS
Residual samples from 34 HIV-1-infected patients [mean viral load (VL) = 3.93 log(10) copies/mL] were used to prepare DPS and DBS, then stored at 20 degrees C and 37 degrees C. HIV-1 nucleic acids were extracted, with or without DNase treatments, to perform HIV-1 VL quantification and nested RT-PCR to amplify the reverse transcriptase gene (798 bp).


RESULTS
For DBS stored for 3 months at 20 degrees C, VL could be measured for all samples and results were comparable to plasma VL. At 37 degrees C, a slight decrease was observed after 2 and 3 months (0.16 and 0.37 log(10) copies/mL mean difference, respectively). For DPS, a significant decrease in VL (0.70 and 1.07 log(10) copies/mL after 1 and 2 months, respectively) was seen at 37 degrees C, but not at 20 degrees C. PCR amplifications from DPS were only successful for 50% of samples with an initial VL >10 000 copies/mL after 1 month at 20 degrees C. From DBS, PCR amplifications are possible until 3 months for samples with plasma VL >5000 copies/mL. VL and PCR results for DBS treated with DNase are close to results obtained for DPS.


CONCLUSIONS
Virological monitoring is still feasible for DBS after 3 months of storage at 37 degrees C when VL is >5000 copies/mL, but DNA contributes largely to the final results.
Monleau
Marjorie
M
UMR 145, Institut de Recherche pour le Développement (IRD) and University of Montpellier 1, Montpellier, France.
Butel
Christelle
C
Delaporte
Eric
E
Boillot
Francois
F
Peeters
Martine
M
D001769:Blood / Q000821:virology*; D003890:Desiccation; D024882:Drug Resistance, Viral; D005838:Genotype; D015497:HIV-1 / Q000235:genetics* / Q000302:isolation & purification; D006801:Humans; D008826:Microbial Sensitivity Tests / Q000379:methods; D010949:Plasma / Q000821:virology*; D016133:Polymerase Chain Reaction / Q000379:methods*; D012367:RNA, Viral / Q000235:genetics / Q000302:isolation & purification*; D013048:Specimen Handling / Q000379:methods*; D013696:Temperature; D013997:Time Factors
D012367:RNA, Viral