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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 targets of HTLV-1 infection are CD4(+) and CD8(+) T cells. 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 germ line 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 genomic DNA (gDNA) extracted from frozen material (peripheral blood mononuclear cells [PBMCs], bronchoalveolar lavage fluid, 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-1 subtype c (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 and prognostics as well as monitoring therapeutic applications.

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.