Low-cost HIV-1 diagnosis and quantification in dried blood spots by real time PCR.
BACKGROUND
Rapid and cost-effective methods for HIV-1 diagnosis and viral load monitoring would greatly enhance the clinical management of HIV-1 infected adults and children in limited-resource settings. Recent recommendations to treat perinatally infected infants within the first year of life are feasible only if early diagnosis is routinely available. Dried blood spots (DBS) on filter paper are an easy and convenient way to collect and transport blood samples. A rapid and cost effective method to diagnose and quantify HIV-1 from DBS is urgently needed to facilitate early diagnosis of HIV-1 infection and monitoring of antiretroviral therapy.


RESULTS
We have developed a real-time LightCycler (rtLC) PCR assay to detect and quantify HIV-1 from DBS. HIV-1 RNA extracted from DBS was amplified in a one-step, single-tube system using primers specific for long-terminal repeat sequences that are conserved across all HIV-1 clades. SYBR Green dye was used to quantify PCR amplicons and HIV-1 RNA copy numbers were determined from a standard curve generated using serially diluted known copies of HIV-1 RNA. This assay detected samples across clades, has a dynamic range of 5 log(10), and %CV <8% up to 4 log(10) dilution. Plasma HIV-1 RNA copy numbers obtained using this method correlated well with the Roche Ultrasensitive (r = 0.91) and branched DNA (r = 0.89) assays. The lower limit of detection (95%) was estimated to be 136 copies. The rtLC DBS assay was 2.5 fold rapid as well as 40-fold cheaper when compared to commercial assays. Adaptation of the assay into other real-time systems demonstrated similar performance.


CONCLUSIONS
The accuracy, reliability, genotype inclusivity and affordability, along with the small volumes of blood required for the assay suggest that the rtLC DBS assay will be useful for early diagnosis and monitoring of pediatric HIV-1 infection in resource-limited settings.
Mehta
Nishaki
N
Department of Pediatrics and Program in Molecular Medicine, University of Massachusetts Medical School, Worcester, Massachusetts, United States of America.
Trzmielina
Sonia
S
Nonyane
Bareng A S
BA
Eliot
Melissa N
MN
Lin
Rongheng
R
Foulkes
Andrea S
AS
McNeal
Kristina
K
Ammann
Arthur
A
Eulalievyolo
Vindu
V
Sullivan
John L
JL
Luzuriaga
Katherine
K
Somasundaran
Mohan
M
D015492:AIDS Serodiagnosis / Q000191:economics* / Q000379:methods*; D000293:Adolescent; D000328:Adult; D044966:Anti-Retroviral Agents / Q000494:pharmacology; D002648:Child; D005260:Female; D005838:Genotype; D015658:HIV Infections / Q000097:blood* / Q000175:diagnosis*; D015497:HIV-1 / Q000378:metabolism*; D006801:Humans; D008297:Male; D009035:Mothers; D011446:Prospective Studies; D015203:Reproducibility of Results; D020133:Reverse Transcriptase Polymerase Chain Reaction / Q000379:methods*; D013997:Time Factors
D044966:Anti-Retroviral Agents