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Low-cost HIV-1 diagnosis and quantification in dried blood spots by real time PCR
Open Access Articles
  • Nishaki Mehta, University of Massachusetts Medical School
  • Sonia Trzmielina, Texas Tech University
  • Bareng A. S. Nonyane
  • Melissa N. Eliot, University of Massachusetts at Amherst
  • Rongheng Lin, University of Massachusetts at Amherst
  • Andrea S. Foulkes, University of Massachusetts at Amherst
  • Kristina McNeal, University of Massachusetts Medical School
  • Arthur Ammann
  • Vindu Eulalievyolo
  • John L. Sullivan, University of Massachusetts Medical School
  • Katherine Luzuriaga, University of Massachusetts Medical School
  • Mohan Somasundaran, University of Massachusetts Medical School
UMMS Affiliation
Department of Pediatrics; Program in Molecular Medicine
Date
6-5-2009
Document Type
Article
Subjects
AIDS Serodiagnosis; Adolescent; Adult; Anti-Retroviral Agents; Child; Female; Genotype; HIV Infections; HIV-1; Humans; Male; Mothers; Prospective Studies; Reproducibility of Results; Reverse Transcriptase Polymerase Chain Reaction; Time Factors
Abstract

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.

METHODS AND FINDINGS: 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 log10, and %CV <8% up to 4 log10 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.

Comments

Citation: Mehta N, Trzmielina S, Nonyane BAS, Eliot MN, Lin R, et al. (2009) Low-Cost HIV-1 Diagnosis and Quantification in Dried Blood Spots by Real Time PCR. PLoS ONE 4(6): e5819. doi:10.1371/journal.pone.0005819. Link to article on publisher's site

Copyright: © 2009 Mehta et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.

Related Resources
Link to Article in PubMed
PubMed ID
19503790
Citation Information
Nishaki Mehta, Sonia Trzmielina, Bareng A. S. Nonyane, Melissa N. Eliot, et al.. "Low-cost HIV-1 diagnosis and quantification in dried blood spots by real time PCR" Vol. 4 Iss. 6 (2009) ISSN: 1932-6203 (Electronic)
Available at: http://works.bepress.com/andrea_foulkes/5/