P10.20 Digital droplet pcr (ddpcr) quantification of human t-lymphotropic virus type-1 clade c in peripheral blood and lung infiltrates of indigenous australian bronchiectasis patients

Abstract

Background Human T-cell Lymphotropic Virus type-1 (HTLV-1) infects an estimated 20 million people worldwide causing a fatal T- cell leukaemia and inflammatory conditions, such as HTLV-1-associated myelopathy, resulting from a dysregulated inflammatory response to the virus. HTLV-1 is highly endemic to remote indigenous communities in Central Australia where infection contributes to racial disparities in morbidity and mortality. HTLV-1 related complications are associated with elevated numbers of T-cells in which the HTLV-1 provirus integrates (HTLV-1 proviral load, PVL). Digital-droplet PCR (ddPCR) is capable of absolute quantification of PVL, offering low variability between assays with few cell numbers. We measured HTLV-1 PVLs from buffy coat cells (BCCs) of infected individuals, and bronchoalveolar lavage (BALs) infiltrates from a bronchiectasis patient to determine the range of HTLV-1 copies/cell relative to illness. Methods All samples were from Alice Springs Hospital HTLV-1 cohort (8 BCCs, 1 BAL) and obtained following ethics approval and patient consent in first language. Genomic DNA was extracted from BCCs for ddPCR PVL quantification. HTLV-1 gag/tax primers and FAM/MGB probes were optimised using temperature gradient amplification. Samples were tested in duplicate relative to an internal reference gene standard, RPP30, or negative threshold controls that used VIC/MGB probe. QuantaSoft software was used for data analysis. Results Of the 8 samples from BCCs, the PVL fell between 320 – 8,040 proviral copies per 10 6 cells. The BAL sample PVL was 1,110 DNA copies per 10 6 cells. The sensitivity (or limit of detection) of our ddPCR assay is 2 copies of proviral DNA per 10 4 cells. Conclusion The ddPCR assay demonstrates extremely high sensitivity, low assay-variability and the capability to reliably quantify HTLV-1 PVL. This technique offers logistic advantages in studying relationships between PVL in HTLV-1 patient’s BCCs and BAL. Disclosure of interest statement No Conflicts of interest. ISSTDR and IUSTI recognise the considerable contribution that industry partners make to professional and research activities. We also recognise the need for transparency of disclosure of potential conflicts of interest by acknowledging these relationships in publications and presentations. No commercial contributions have been received for this research project.