Efficient methodologies for sensitive HIV-1 RNA quantitation from plasma and vaginal secretions |
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| Authors: | Tara Randolph Henning Nedra Lacour Angela Martin Amedee |
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| Affiliation: | 1. Division of HIV/AIDS Prevention, Centers for Disease Control and Prevention, United States;2. ICF International, United States;3. Association of Public Health Laboratories, United States;4. Wadsworth Center, New York State Department of Health, United States;5. North Carolina State Laboratory of Public Health, United States;1. Food and Drug Administration, Department of Health, Executive Yuan, Taipei 11561, Taiwan;2. Institute of Biotechnology, National Taiwan University, Taipei 10672, Taiwan;1. Department of Clinical Microbiology 445, Hvidovre Hospital, Kettegård Alle 30, DK-2650 Hvidovre, Denmark;2. Department of Infectious Diseases 144, Hvidovre Hospital, Kettegård Alle 30, DK-2650 Hvidovre, Denmark;3. Department for Clinical Medicine, Faculty of Health and Medical Sciences, University of Copenhagen, Denmark;1. Department of Veterinary Diagnosis and Production Animal Medicine, Iowa State University, Ames, IA, USA;2. Virginia Herd Management Service, P.O. Box 555, Madison, VA, USA;1. Department of Virology, Royal Free London NHS Foundation Trust, London, United Kingdom;2. Institute of Infection & Global Health, University of Liverpool, Liverpool, United Kingdom |
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| Abstract: | BackgroundQuantifying 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.ObjectivesTo 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 designA 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.ResultsThe 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.ConclusionsThe 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. |
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