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Assessment of circulating mitochondrial cell-free DNA dynamics in patients with tuberculosis: pilot study

L Freimane(1) A Viksna(2) V Ulanova(1) A Kivrane(1) D Sadovska(1) R Ranka(1)

1:Latvian Biomedical Research and Study Centre, Riga, LV-1067, Latvia; 2:Centre of Tuberculosis and Lung Diseases, Riga East University Hospital, Riga, LV – 2118, Latvia

Circulating cell-free (ccf) mtDNA copy number (CN) variations can indicate physical injury, inflammation, innate immunity system activity, and predict mortality and treatment outcome in critically ill individuals. Also, pathogen-derived ccf DNA, including M. tuberculosis (Mtb), has been detected in humans, which could directly show infection load and serve as biomarker for disease monitoring.

In this study, blood samples were collected from ten newly diagnosed patients with Mtb infection before treatment and after every two weeks for two months. For ccf-mtDNA and ccf-Mtb CN evaluation DNA was extracted respectively from (a) 200 μL freshly frozen blood plasma, and (b) 500 μL whole blood samples. Absolute CN (CN/μL of plasma/blood) was quantified using the QX200 droplet digital ddPCR System (Bio-Rad) via two separate multiplex amplifications within ND1 and CYB (mtDNA), and IS16110 and CFP-10 (Mtb) gene regions. QuantaSoft software (1.0.596) was used to analyse the data. Statistical analysis was performed using GraphPad Prism 5.0 software.

Almost all patients showed extremely high ccf-mtDNA plasma concentrations (maximum of 28163 CN/μL before therapy); we could detect ccf-mtDNA CN fluctuations in time which were statistically significant in those patients which showed CN decrease in the first two weeks of the therapy (p=0.03). We detected ccf-Mtb only in one patient before treatment with concentration of 0.023 CN/μL.

In conclusion, ccf-Mtb CN wasn’t informative in our settings due to extremely low concentrations. Ccf-mtDNA may serve as a useful biomarker for Mtb infection monitoring, but further studies are necessary. This study was supported by ERDF grant No.

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