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.

A pproximately 2 to 3% of human T cell leukemia virus type 1 (HTLV-1)-infected individuals develop adult T-cell leukemia/lymphoma (ATL) and another 2 to 3% develop HTLV-1-associated myelopathy (HAM)/tropical spastic paraparesis (TSP) in their lifetimes (1-4). In addition to HAM/TSP (5, 6), HTLV-1 is also associated with other inflammatory conditions, such as uveitis (6) Sjögren's syndrome (7), bronchoalveolitis and arthritis (8), and polymyositis (9). It is noteworthy that some patients present with more than one of these inflammatory conditions (10). HTLV-1 primarily infects CD4 ϩ and CD8 ϩ effector and memory T cells and regulatory CD4 ϩ CD25 ϩ T cells (11,12). A high viral DNA burden in peripheral blood mononuclear cells (PBMCs) is a risk factor for HAM/TSP (13) and ATL development (14-16), and patients with HAM/TSP have a higher virus level in the cerebrospinal fluid (CSF) than in the peripheral blood (12). The virus level alone is not sufficient to differentiate symptomatic patients from healthy carriers, suggesting the importance of other factors, including the host immune response (16)(17)(18)(19)(20). HAM/TSP patients present diverse immunological alterations, such as increased levels of spontaneous lymphocyte proliferation (21, 22), tax-specific cytotoxic CD8 ϩ T cell expansion, and the production of high levels of inflammatory cytokines (23)(24)(25). Several studies have also suggested that monocytes are involved in immune reg-

The human herpesviruses HHV-6A and HHV-6B have been associated with various neurologic disorders partly due to the detection of elevated viral DNA levels in patients compared to controls. However the reported frequency of these viruses varies widely, likely reflecting differences in PCR methodologies used for detection. Digital droplet PCR (ddPCR) is a third generation PCR technology that enables the absolute quantification of target DNA molecules. Mounting evidence of the biological differences between HHV-6A and HHV-6B has led to their recent reclassification as separate species. As it is now especially relevant to investigate each virus, our objectives were to first design a multiplex HHV-6A and HHV-6B ddPCR assay and then to investigate the incidence of HHV-6A and HHV-6B coinfection in samples from healthy donors and patients with MS, a disease in which HHV-6 is thought to play a role. In our assessment of healthy donors, we observed a heretofore-underappreciated high frequency of coinfection in PBMC and serum, and found that our assay precisely detects both HHV-6A and HHV-6B chromosomally integrated virus, which has important implications in clinical settings. Interestingly, upon comparing the saliva from MS patients and healthy donors, we detected a significantly elevated frequency of coinfection in MS saliva; increased detection of HHV-6A in MS patients is consistent with other studies suggesting that this viral species (thought to be more neurotropic than HHV-6B) is more prevalent among MS patients compared to healthy donors. As the biology and disease associations between these two viral species differ, identifying and quantifying both species of HHV-6 may provide clinically relevant information, as well as enhance our understanding of the roles of each in health and disease.