Validation of Multiplex Serology for human hepatitis viruses B and C, human T-lymphotropic virus 1 and Toxoplasma gondii.

Multiplex Serology is a high-throughput technology developed to simultaneously measure specific serum antibodies against multiple pathogens in one reaction vessel. Serological assays for hepatitis B (HBV) and C (HCV) viruses, human T-lymphotropic virus 1 (HTLV-1) and the protozoan parasite Toxoplasma gondii (T. gondii) were developed and validated against established reference assays. For each pathogen, between 3 and 5 specific antigens were recombinantly expressed as GST-tag fusion proteins in Escherichia coli and tested in Monoplex Serology, i.e. assays restricted to the antigens from one particular pathogen. For each of the four pathogen-specific Monoplex assays, overall seropositivity was defined using two pathogen-specific antigens. In the case of HBV Monoplex Serology, the detection of past natural HBV infection was validated based on two independent reference panels resulting in sensitivities of 92.3% and 93.0%, and specificities of 100% in both panels. Validation of HCV and HTLV-1 Monoplex Serology resulted in sensitivities of 98.0% and 95.0%, and specificities of 96.2% and 100.0%, respectively. The Monoplex Serology assay for T. gondii was validated with a sensitivity of 91.2% and specificity of 92.0%. The developed Monoplex Serology assays largely retained their characteristics when they were included in a multiplex panel (i.e. Multiplex Serology), containing additional antigens from a broad range of other pathogens. Thus HBV, HCV, HTLV-1 and T. gondii Monoplex Serology assays can efficiently be incorporated into Multiplex Serology panels tailored for application in seroepidemiological studies.

Droplet digital PCR for absolute quantification of proviral load of human T-cell lymphotropic virus (HTLV) types 1 and 2.

BACKGROUND: Human T-lymphotrophic virus (HTLV) types 1 and 2 cause lifelong infection whereby most infected individuals are asymptomatic whilst a minority develop infection-related disease. These latter patients invariably have been found to have high proviral load (PVL). Therefore, infected patients are monitored by determining the proportion of lymphocytes that are infected with HTLV-1/2. An increase in PVL has been shown to represent an increasing risk of developing HTLV-associated diseases. Monitoring of PVL requires a reliable and sensitive method. In this study assays based on droplet digital PCR (ddPCR) were established and evaluated for detection and quantification of HTLV-1/2. OBJECTIVES: To develop two parallel assays to detect the tax genes and determine the PVL of HTLV-1 and -2. STUDY DESIGN: Sixty-seven clinical samples from patients infected with HTLV-1 or HTLV-2 were analysed. The samples had previously been analysed with a qPCR and a comparison between ddPCR and qPCR was performed. The specificity of the assays were determined by analyzing samples from 20 healthy blood donors. RESULTS: The ddPCR was a stable and sensitive method for detection and quantification of HTLV-1 and -2. When comparing the qPCR and ddPCR the correlation was high (Pearsons correlation coefficient 0.96). The variability of the ddPCR was very low with intra-assay coefficient of variation (CV) of 0.97-3.3% (HTLV-1) and 1.7-8.2% (HTLV-2) and inter-assay CV of 1.8-6.1% (HTLV-1) and 1.2-12.9% (HTLV-2). CONCLUSIONS: The ddPCR reliably quantified HTLV DNA in clinical samples and could be a useful tool for monitoring of PVLs in HTLV-infected individuals.

Long-term clinical remission maintained after cessation of zidovudine and interferon-α therapy in chronic adult T-cell leukemia/lymphoma.

Globally, > 5-10 million people are estimated to be infected with Human T-lymphotropic virus type 1 (HTLV-1), of whom ~ 5% develop adult T-cell leukemia/lymphoma (ATL). Despite advances in chemotherapy, overall survival (OS) has not improved in the 35 years since HTLV-1 was first described. In Europe/USA, combination treatment with zidovudine and interferon-α (ZDV/IFN-α) has substantially changed the management of patients with the leukemic subtypes of ATL (acute or unfavorable chronic ATL) and is under clinical trial evaluation in Japan. However, there is only a single published report of long-term clinical remission on discontinuing ZDV/IFN-α therapy and the optimal duration of treatment is unknown. Anecdotal cases where therapy is discontinued due to side effects or compliance have been associated with rapid disease relapse, and it has been widely accepted that the majority of patients will require life-long therapy. The development of molecular methods to quantify minimal residual disease is essential to potentially guide therapy for individual patients. Here, for the first time, we report molecular evidence that supports long-term clinical remission in a patient who was previously treated with ZDV/IFN-α for 5 years, and who has now been off all therapy for over 6 years.

Risk stratification of adult T-cell leukemia/lymphoma using immunophenotyping.

Adult T-cell leukemia/lymphoma (ATL), a human T-lymphotropic virus type 1 (HTLV-1)-associated disease, has a highly variable clinical course and four subtypes with therapeutic and prognostic implications. However, there are overlapping features between ATL subtypes and between ATL and nonmalignant (non-ATL) HTLV-1 infection complicating diagnosis and prognostication. To further refine the diagnosis and prognosis of ATL, we characterized the immunophenotype of HTLV-1-infected cells in ATL and non-ATL. A retrospective study of peripheral blood samples from 10 HTLV-1-uninfected subjects (UI), 54 HTLV-1-infected patients with non-ATL, and 22 with ATL was performed using flow cytometry. All patients with ATL had CD4+  CCR4+  CD26- immunophenotype and the frequency of CD4+  CCR4+  CD26- T cells correlated highly significantly with the proviral load in non-ATL suggesting CD4+  CCR4+  CD26- as a marker of HTLV-1-infected cells. Further immunophenotyping of CD4+  CCR4+   CD26- cells revealed that 95% patients with ATL had a CD7- (≤30% CD7+ cells), whereas 95% HTLV+ non-ATL had CD7+ (>30% CD7+ cells) immunophenotype. All patients with aggressive ATL had a CCR7+ (≥30%), whereas 92% with indolent ATL and 100% non-ATL had a CCR7- (<30%) immunophenotype. Patients with nonprogressing indolent ATL were CD127+ but those with progressive lymphocytosis requiring systemic therapy had a CD127- (≤30%) immunophenotype. In summary, HTLV-1-infected cells have a CD4+  CCR4+  CD26- immunophenotype. Within this population, CD7- phenotype suggests a diagnosis of ATL, CCR7+ phenotype identifies aggressive ATL, while CCR7- CD127- phenotype identifies progressive indolent ATL.

Human T cell lymphotropic virus type 1 viral load variability and long-term trends in asymptomatic carriers and in patients with human T cell lymphotropic virus type 1-related diseases.

Human T cell lymphotropic virus type 1 (HTLV-1) proviral load (PVL) in peripheral blood mononuclear cells (PBMCs) is high in patients with adult T cell leukemia/lymphoma or HTLV-1-associated myelopathy/tropical spastic paraparesis (HAM/TSP) and in some asymptomatic carriers, but fluctuates. Our objectives were to document ranges of HTLV PVL across a broader spectrum of diseases and tissues, to quantify the normal range of intrapatient PVL variability, and to identify which PVL values and changes deserve further investigation. PVL was measured in 191 patients with HTLV-1-associated diseases and in 211 asymptomatic carriers, using real-time quantitative PCR. The intraassay variability increases as viral load decreases: 8% at high load, 17% at medium load, and 33% at low load. The interassay variability is not different from the intraassay. Mean intrapatient CV is 65% (SD 21) in asymptomatic carriers and 59% (SD 22) in HAM/TSP. PVL values varied widely between individuals, but were relatively constant within individuals. High PVL in cerebrospinal fluid (CSF) and lymph nodes (LN) was associated with disease but 57% of asymptomatic carriers had a PVL greater than 1% in PBMCs. Our results suggest that (1) PVL changes falling outside a coefficient of variation of 100% require more detailed assessment, (2) asymptomatic carriers with PVL higher than 10% should undergo more frequent clinical and laboratory monitoring, and (3) HTLV-1 PVL in blood and tissue is helpful in the diagnosis and monitoring of HTLV-1 infection.

Ciclosporin A proof of concept study in patients with active, progressive HTLV-1 associated myelopathy/tropical spastic paraparesis.

INTRODUCTION: Patients with HTLV-1-associated myelopathy/tropical spastic paraparesis (HAM/TSP) become progressively impaired, with chronic pain, immobility and bladder, bowel and sexual dysfunction. Tested antiretroviral therapies have not been effective and most patients are offered a short course of corticosteroids or interferon-α, physiotherapy and symptomatic management. Pathogenesis studies implicate activated T-lymphocytes and cytokines in tissue damage. We therefore tested the hypothesis that inhibition of T-cell activation with ciclosporin A would be safe and clinically beneficial in patients with early and/or clinically progressing HAM/TSP. MATERIALS AND METHODS: Open label, proof of concept, pilot study of 48 weeks therapy with the calcineurin antagonist, ciclosporin A (CsA), in seven patients with 'early' (50% deterioration in timed walk during the preceding three months) HAM/TSP. Primary outcomes were incidence of clinical failure at 48 weeks and time to clinical failure. RESULTS: All patients completed 72 weeks study participation and five showed objective evidence of clinical improvement after 3 months treatment with CsA. Two patients exhibited clinical failure over 6.4 person-years of follow-up to week 48. One patient had a >2 point deterioration in IPEC (Insituto de Pesquisa Clinica Evandro Chagas) disability score at weeks 8 and 12, and then stopped treatment. The other stopped treatment at week 4 because of headache and tremor and deterioration in timed walk, which occurred at week 45. Overall pain, mobility, spasticity and bladder function improved by 48 weeks. Two patients recommenced CsA during follow-up due to relapse. CONCLUSIONS: These data provide initial evidence that treatment with CsA is safe and may partially reverse the clinical deterioration seen in patients with early/progressive HAM/TSP. This trial supports further investigation of this agent's safety and effectiveness in larger, randomised controlled studies in carefully selected patients with disease progression.