Digital droplet PCR (ddPCR) for the precise quantification of human T-lymphotropic virus 1 proviral loads in peripheral blood and cerebrospinal fluid of HAM/TSP patients and identification of viral mutations

Abstract

An elevated human T cell lymphotropic virus 1 (HTLV)-1 proviral load (PVL) is the main risk factor for developing HTLV-1-associated myelopathy/tropical spastic paraparesis (HAM/TSP) in HTLV-1 infected subjects, and a high cerebrospinal fluid (CSF) to peripheral blood mononuclear cell (PBMC) PVL ratio may be diagnostic of the condition. However, the standard method for quantification of HTLV-1 PVL—real-time PCR—has multiple limitations, including increased inter-assay variability in compartments with low cell numbers, such as CSF. Therefore, in this study, we evaluated a novel technique for HTVL-1 PVL quantification, digital droplet PCR (ddPCR). In ddPCR, PCR samples are partitioned into thousands of nanoliter-sized droplets, amplified on a thermocycler, and queried for fluorescent signal. Due to the high number of independent events (droplets), Poisson algorithms are used to determine absolute copy numbers independently of a standard curve, which enables highly precise quantitation. This assay has low intra-assay variability allowing for reliable PVL measurement in PBMC and CSF compartments of both asymptomatic carriers (AC) and HAM/TSP patients. It is also useful for HTLV-1-related clinical applications, such as longitudinal monitoring of PVL and identification of viral mutations within the region targeted by the primers and probe.

Human T Cell Leukemia Virus Type 1 Infection of the Three Monocyte Subsets Contributes to Viral Burden in Humans

“…The level of HTLV-1 DNA (the viral load [VL]) in PBMCs and in sorted CD4 ϩ cells, CD8 ϩ cells, and monocyte subsets was determined using a QX100 digital droplet PCR (ddPCR) system (Bio-Rad). The assay was performed as described before (59). Briefly, primers specific for HTLV-1 tax and the RNase P protein subunit P30 (RPP30) or the ␤-actin housekeeping gene were used.…”

The Future of Digital Polymerase Chain Reaction in Virology

“…In current virology research, absolute quantification by dPCR has mostly been explored and compared with qPCR for standard viral load testing [12] and low-level detection of viruses [13][14][15][16][17]. Examples of the latter are the quantification of persisting viruses, such as reservoirs of the human immunodeficiency virus (HIV) in HIV-infected individuals receiving antiretroviral therapy [14-16, 18, 19] or low-level detection of GB virus type-C (GBV-C) in cell lines [17], human T-lymphotropic virus (HTLV) in cerebrospinal fluid [13], and covalently closed circular DNA (cccDNA) in hepatitis B virus (HBV)-infected individuals [20].…”

“…Examples of the latter are the quantification of persisting viruses, such as reservoirs of the human immunodeficiency virus (HIV) in HIV-infected individuals receiving antiretroviral therapy [14-16, 18, 19] or low-level detection of GB virus type-C (GBV-C) in cell lines [17], human T-lymphotropic virus (HTLV) in cerebrospinal fluid [13], and covalently closed circular DNA (cccDNA) in hepatitis B virus (HBV)-infected individuals [20]. dPCR was recently described for direct quantification of viral nucleic acids without initial DNA or RNA isolation.…”

HTLV-1 Infection and Neuropathogenesis in the Context of Rag1-/-γc-/- (RAG1-Hu) and BLT Mice

“…Due to the random segregation of DNA fragments into droplets, Poisson algorithm was used to determine absolute copy numbers independently of a standard curve. The PVL was calculated as the percentage of infected cells using the following formula as described earlier (Brunetto et al 2014): $$\text{PVL}=\left[\frac{\text{Quantity}\text{of}\text{HTLV}-1\text{Tax}}{\frac{\text{Quantity}\text{of}\text{housekeeping}\text{gene}}{2}}\right]\times 100$$…”