Multiplex Droplet Digital PCR Assay for Quantification of Human T-Cell Leukemia Virus Type 1 Subtype c 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 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.

Droplet digital PCR quantification suggests that higher viral load correlates with improved survival in HPV-positive oropharyngeal tumours

“…Previous studies on VL have generally focussed on the amplification of one target and have employed real-time PCR. ddPCR allows absolute quantification of DNA targets and has been used for applied virology where knowledge of load can influence clinical management [20,21]. Around 20,000 data points are generated per sample making it highly accurate and reproducible (2021) and proof principle of this approach when applied to OPC for single HPV target-detection was reported recently [22].…”

Human T-cell leukaemia virus type 1 associated pulmonary disease: clinical and pathological features of an under-recognised complication of HTLV-1 infection

“…The difference between HTLV-1 PVL in respiratory secretions and peripheral blood may be even more pronounced when the HTLV-1 PVL in T-cells is compared [ 93 ]. HTLV-1 PVL in BALF and PBMCs are generally higher in Japanese patients with the inflammatory disorders HAM [ 83 ] and HAU [ 61 ] relative to those who are asymptomatic.…”

Accurate Quantification of T Cells in Copy Number Stable and Unstable DNA Samples Using Multiplex Digital PCR

“…Advantageously, such DNA-based analysis can be performed more rapidly in samples of much lower quantity and quality, but may also reduce various forms of technical and biological measurement variability. 4,5,15,16 For example, when DNA is isolated from a larger proportion of the tumor, spatial differences in T-cell presence can be equaled out and the DNA-based quantification will be a better representation of the whole tumor. Moreover, but in contrast to conventional methods, digital PCR is less dependent on visual inspection and manual thresholding or interpretation.…”

“…2 Previously, digital PCR experiments following the classic model were performed in a duplex configuration: the T-cell marker and reference were measured concurrently on two different channels. 4,5,15,16 The extension within the adjusted model, however, requires the measurement of one extra target (ie, the RC) in the sample of interest. Although this measurement may be performed in another duplex experiment (normalized to the same stable reference) and integrated into the calculation with the Tcell marker afterward, a multiplex analysis would be a more efficient solution.…”