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.
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).


CONCLUSIONS
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.
Timmermans
Eveline C
EC
Primagen, Amsterdam, The Netherlands.
Tebas
Pablo
P
Ruiter
Jos P N
JP
Wanders
Ronald J A
RJ
de Ronde
Anthony
A
de Baar
Michel P
MP
D019380:Anti-HIV Agents / Q000627:therapeutic use*; D001483:Base Sequence; D001792:Blood Platelets / Q000737:chemistry; D002467:Cell Nucleus / Q000737:chemistry; D002478:Cells, Cultured; D004272:DNA, Mitochondrial / Q000032:analysis* / Q000097:blood; D016049:Didanosine / Q000627:therapeutic use; D004359:Drug Therapy, Combination; D005240:Feasibility Studies; D005347:Fibroblasts / Q000737:chemistry / Q000187:drug effects; D015658:HIV Infections / Q000097:blood* / Q000188:drug therapy*; D006801:Humans; D019469:Indinavir / Q000627:therapeutic use; D019344:Lactic Acid / Q000032:analysis; D019259:Lamivudine / Q000627:therapeutic use; D007963:Leukocytes, Mononuclear / Q000737:chemistry; D021141:Nucleic Acid Amplification Techniques; D019289:Pyruvic Acid / Q000032:analysis; D018119:Stavudine / Q000627:therapeutic use; D016047:Zalcitabine / Q000494:pharmacology / Q000627:therapeutic use
D019380:Anti-HIV Agents; D004272:DNA, Mitochondrial; D019259:Lamivudine; D019344:Lactic Acid; D019469:Indinavir; D016047:Zalcitabine; D019289:Pyruvic Acid; D018119:Stavudine; D016049:Didanosine