Efficient methodologies for sensitive HIV-1 RNA quantitation from plasma and vaginal secretions

Abstract 

Background

Quantifying HIV levels in mucosal secretions is essential to study compartmentalized expression of HIV and facilitate development of intervention strategies to prevent disease progression and transmission.

Objectives

To develop a sensitive, reliable, and cost-effective technique to quantify HIV from blood and vaginal secretions that is compatible with efficient implementation in clinical research environments.

Study design

A sensitive, reliable, internally-controlled real-time reverse transcriptase (RT) PCR assay, which uses the HIV-1 pol gene as a target (Hpol assay) was developed to quantify HIV levels in plasma and genital secretions, and compared to the widely used Roche Amplicor™ HIV-1 Monitor assay. In addition, a simplified method of sample collection and processing of genital secretions (self-collection and use of RNAlater with batch processing) was compared to provider collection of samples and immediate processing.

Results

The sensitivity and reliability of HIV levels detected by the assay described herein correlate well with measurements from Roche Amplicor™ HIV-1 Monitor assay for both plasma and vaginal secretions (R²=0.9179 and R²=0.942, respectively). The Hpol assay reproducibly quantifies a lower limit of 5 HIV-1 RNA copies per reaction, with low-levels of inter-assay and intra-assay variation. Additionally, vaginal viral levels and detection frequency did not differ significantly between the two the collection and processing methods.

Conclusions

The methodologies developed here provide sensitive, reliable, and cost-effective quantification of HIV levels in plasma and mucosal secretions, and are compatible with efficient use in clinical research studies.

Abbreviations: HIV/HIV-1, human immunodeficiency virus/human immunodeficiency virus type 1, PCR, polymerase chain reaction, RT, reverse transcriptase, RNA, ribonucleic acid, DNA, deoxyribonucleic acid, EDTA, ethylenediaminetetraacetic acid, PBS, phosphate buffered solution, BMV, brome mosaic virus

Keywords: Human immunodeficiency virus, Real-time PCR, Genital, Plasma, RNAlater