Rapid, Point-of-Care Extraction of Human Immunodeficiency Virus Type 1 Proviral DNA from Whole Blood for Detection by Real-Time PCR

Abstract

PCR detection of human immunodeficiency virus type 1 (HIV-1) proviral DNA is the method recommended for use for the diagnosis of HIV-1 infection in infants in limited-resource settings. Currently, testing must be performed in central laboratories, which are usually located some distance from health care facilities. While the collection and transportation of samples, such as dried blood spots, has improved test accessibility, the results are often not returned for several weeks. To enable PCR to be performed at the point of care while the mothers wait, we have developed a vertical filtration method that uses a separation membrane and an absorbent pad to extract cellular DNA from whole blood in less than 2 min. Cells are trapped in the separation membrane as the specimen is collected, and then a lysis buffer is added. The membrane retains the DNA, while the buffer washes away PCR inhibitors, which get wicked into the absorbent blotter pad. The membrane containing the entrapped DNA is then added to the PCR mixture without further purification. The method demonstrates a high degree of reproducibility and analytical sensitivity and allows the quantification of as few as 20 copies of HIV-1 proviral DNA from 100 l of blood. In a blinded study with 182 longitudinal samples from infants (ages, 0 to 72 weeks) obtained from the Women and Infants Transmission Study, our assay demonstrated a sensitivity of 99% and a specificity of 100%.

Point-of-Care Testing

“…This has significant technical challenges, including but not limited to sample preparation. A single-step, filtration-based method for sample preparation has been developed that appears amenable to POC [30], and integrated "lab on a chip" solutions have also been described that include sample preparation, amplification, and detection [37,38]. However these systems have not yet been integrated into devices suitable for clinical and field evaluation.…”

“…Routine diagnosis is currently based on PCR for integrated HIV proviral DNA, and progress has been made toward reduction of the sample preparation step into a POC format [30], although fully integrated nucleic acid POC tests have not yet been validated.…”

Feasibility and Challenges in Providing Antiretroviral Treatment to Children in Sub-Saharan Africa

“…Point-of-care assays are also being developed that are inexpensive and can be performed in most facilities by trained healthcare workers. Several new approaches are based on nanotechnology and microfluidics, and include biobarcode amplification-based assays, microfluidics-based viral detection and quantification, miniaturized PCR chips, miniaturized immunoassays, point-of-care HIV-1 amplification systems and point-of-care DNA extraction systems [29,30].…”

Micro- and Nanotechnology for HIV/AIDS Diagnostics in Resource-Limited Settings

“…Jangam et al presented a solution to the challenges of sample preparation in resource-limited settings when analyzing proviral load with a low-cost extraction technique called filtration isolation of nucleic acids (FINA) [77]. In this technique, whole blood is introduced on a membrane disk and subsequently washed with NaOH resulting in isolation of genomic DNA on the membrane.…”