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This Article Full Text Full Text (PDF) 062901.Supplemental Data All Versions of this Article: clinchem.2005.062901v1 52/6/979    most recent Alert me when this article is cited Alert me if a correction is posted Citation Map Services Email this article to a friend Similar articles in this journal Similar articles in Web of Science Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Citing Articles Citing Articles via HighWire Citing Articles via Web of Science (5) Citing Articles via Google Scholar Google Scholar Articles by Timmermans, E. C. Articles by de Baar, M. P. Search for Related Content PubMed PubMed Citation Articles by Timmermans, E. C. Articles by de Baar, M. P. Related Collections Molecular Diagnostics and Genetics Clinical Immunology Drug Monitoring and Toxicology Automation and Analytical Techniques

Real-Time Nucleic Acid Sequence–Based Amplification Assay to Quantify Changes in Mitochondrial DNA Concentrations in Cell Cultures and Blood Cells from HIV-Infected Patients Receiving Antiviral Therapy

¹ Primagen, Amsterdam, The Netherlands.
² University of Pennsylvania, Philadelphia, PA.
³ Laboratory of Genetic Metabolic Diseases and⁴ Department of Human Retrovirology, Academic Medical Centre, Amsterdam, The Netherlands.

^aAddress correspondence to this author at: Primagen, Meibergdreef 59, 1105 BA Amsterdam, The Netherlands. Fax 31-20-566-9081; e-mail M.P.deBaar{at}primagen.com.

Background: To study the clinical relevance of changes in mitochondrial DNA (mtDNA) in peripheral blood mononuclear cells (PBMCs) attributable to HIV infection and/or combination antiretroviral therapy (cART), a high-throughput molecular assay to quantify mtDNA is required.

Methods: We developed a quantitative real-time duplex nucleic acid sequence–based amplification assay in which both mtDNA and nuclear DNA are simultaneously amplified in 1 tube. The assay could accurately quantify mtDNA in a range of 15–1500 copies of mtDNA per 2 genomic copies with an intrarun variation of 11% and an interrun variation of 16%. We compared this real-time assay with the lactate/pyruvate ratios in fibroblasts incubated with glucose and exposed to zalcitabine. Additionally, we studied the effects of platelet contamination and the in vivo effects of cART on mtDNA in PBMCs from a small group of patients.

Results: Decreases in mtDNA preceded the increase in lactate/pyruvate ratios and vice versa when zalcitabine was eliminated from the culture. Platelets affected the mtDNA in PBMCs if >5 platelets per PBMC were present. Within 12 weeks, mtDNA increased and remained increased in PBMCs from patients on continuous treatment with zidovudine/lamivudine/indinavir therapy (P = 0.03), but increased if patients were switched to stavudine/didanosine therapy (P = 0.008).

Conclusion: After drug exposure, the mtDNA assay can detect changes in mtDNA concentrations in cell lines and PBMCs, when properly controlled for platelet effects, earlier than traditional assays.

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