Use of Dried Blood Spots to Elucidate Full-Length Transmitted/Founder HIV-1 Genomes.
BACKGROUND
Identification of HIV-1 genomes responsible for establishing clinical infection in newly infected individuals is fundamental to prevention and pathogenesis research. Processing, storage, and transportation of the clinical samples required to perform these virologic assays in resource-limited settings requires challenging venipuncture and cold chain logistics. Here, we validate the use of dried-blood spots (DBS) as a simple and convenient alternative to collecting and storing frozen plasma.


METHODS
We performed parallel nucleic acid extraction, single genome amplification (SGA), next generation sequencing (NGS), and phylogenetic analyses on plasma and DBS.


RESULTS
We demonstrated the capacity to extract viral RNA from DBS and perform SGA to infer the complete nucleotide sequence of the transmitted/founder (TF) HIV-1 envelope gene and full-length genome in two acutely infected individuals. Using both SGA and NGS methodologies, we showed that sequences generated from DBS and plasma display comparable phylogenetic patterns in both acute and chronic infection. SGA was successful on samples with a range of plasma viremia, including samples as low as 1,700 copies/ml and an estimated ∼50 viral copies per blood spot. Further, we demonstrated reproducible efficiency in gp160envsequencing in DBS stored at ambient temperature for up to three weeks or at -20°C for up to five months.


CONCLUSIONS
These findings support the use of DBS as a practical and cost-effective alternative to frozen plasma for clinical trials and translational research conducted in resource-limited settings.
Comprehensive Cancer Center, University of Alabama at Birmingham, Birmingham, AL, USA.
Jesus F
JF
Salazar-Gonzalez
Department of Medicine, University of Alabama at Birmingham, Birmingham, AL, USA.
Maria G
MG
Salazar
Ragon Institute of MHG, MIT, and Harvard, Boston, MA, USA.
Damien C
DC
Tully
Ragon Institute of MHG, MIT, and Harvard, Boston, MA, USA.
Colin B
CB
Ogilvie
Department of Medicine, Perelman School of Medicine, University of Pennsylvania, Philadelphia, PA, USA.
Gerald H
GH
Learn
Ragon Institute of MHG, MIT, and Harvard, Boston, MA, USA.
Todd M
TM
Allen
Department of Medicine, University of Alabama at Birmingham, Birmingham, AL, USA.
Sonya L
SL
Heath
Department of Medicine, University of Alabama at Birmingham, Birmingham, AL, USA.
Paul
P
Goepfert
Department of Medicine, Perelman School of Medicine, University of Pennsylvania, Philadelphia, PA, USA.
Katharine J
KJ
Bar
dried blood spots; resource-limited settings; single genome sequencing; transmitted/founder virus; viral diversity