A Multiplex Droplet Digital PCR Assay for Quantification of HTLV-1c DNA Proviral Load and T-Cells from Blood and Respiratory Exudates Sampled in a Remote Setting

Abstract

During human T-cell leukemia virus type-1 (HTLV-1) infection the frequency of cells harboring an integrated copy of viral cDNA, the proviral load (PVL), is the main risk factor for progression of HTLV-1-associated diseases. Accurate quantification of provirus by droplet digital PCR (ddPCR) is a powerful diagnostic tool with emerging uses for monitoring viral expression. Current ddPCR techniques quantify HTLV-1 PVL in terms of whole genomic cellular material, while the main target of HTLV-1 infection is the CD4+ and CD8+ T-cell. Our understanding of HTLV-1 proliferation and the amount of viral burden present in different compartments is limited. Recently a sensitive ddPCR assay was applied to quantifying T-cells by measuring loss of germline T-cell receptor genes as method of distinguishing non-T-cell from recombined T-cell DNA. In this study, we demonstrated and validated novel applications of the duplex ddPCR assay to quantify T-cells from various sources of human gDNA extracted from frozen material (PBMCs, bronchoalveolar lavage, and induced sputum) from a cohort of remote Indigenous Australians and then compared the T-cell measurements by ddPCR to the prevailing standard method of flow cytometry. The HTLV-1c PVL was then calculated in terms of extracted T-cell gDNA from various compartments. Because HTLV-1c preferentially infects CD4+ T-cells, and the amount of viral burden correlates with HTLV-1c disease pathogenesis, application of this ddPCR assay to accurately measure HTLV-1c-infected T-cells can be of greater importance for clinical diagnostics, prognostics as well as monitoring therapeutic applications.