Low-Cost HIV-1 Diagnosis and Quantification in Dried Blood Spots by Real Time PCR

Nishaki Mehta,Katherine Luzuriaga,Vindu Eulalievyolo,Mohan Somasundaran,Rongheng Lin,Andrea S Foulkes,Sonia Trzmielina,Arthur Ammann,Melissa N Eliot,Bareng A S Nonyane,Kristina Mcneal,John L Sullivan,Lisa F P Ng

doi:10.1371/journal.pone.0005819

Nishaki Mehta, Katherine Luzuriaga + Show 11 more

Open Access

https://doi.org/10.1371/journal.pone.0005819

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Abstract

BackgroundRapid 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 FindingsWe 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.ConclusionsThe 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.

Highlights

It is estimated that 33.2 million people were infected with HIV1 at the end of 2007; 2.5 million were children under 15 years of age, the majority of whom acquired infection through mother-tochild transmission (MTCT; [1])
The accuracy, reliability, genotype inclusivity and affordability, along with the small volumes of blood required for the assay suggest that the real-time LightCycler (rtLC) Dried blood spots (DBS) assay will be useful for early diagnosis and monitoring of pediatric HIV-1 infection in resource-limited settings
We describe a LightCycler-based real time PCR assay to quantify viral loads using RNA extracted from filter paper containing dried blood spots

Summary

Introduction

It is estimated that 33.2 million people were infected with HIV1 at the end of 2007; 2.5 million were children under 15 years of age, the majority of whom acquired infection through mother-tochild transmission (MTCT; [1]). 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. 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

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Conclusion