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The present study compares the ability of distinct immunological assays (chemiluminescence immunoassay-CLIA, western blot-WB and flow cytometry-FC-Simplex and Duplex) to detect anti-HTLV (human T-lymphotropic virus) antibodies in candidates for blood donations at the Amazonas State Blood Center (Brazil) between January 2018 and December 2022. Overall, 257,942 samples from candidates for blood donations were screened using CLIA, which led to 0.15% seropositivity for HTLV (409 samples). A total of 151 candidates for blood donations were enrolled for retesting with CLIA followed by additional testing using WB and FC-Simplex and Duplex analysis. Our results demonstrated that 62% (93/151), 20% (30/151) and 17% (26/151) of the samples presented positive results with retesting using CLIA, WB and FC-Simplex analysis, respectively. Additional analysis of the CLIA, WB and FC-Simplex results revealed an overall agreement of 56% for CLIA and WB (22 co-negative; 30 co-positive samples), 48% for CLIA and FC-Simplex (21 co-negative; 24 co-positive samples) and 80% for WB and FC-Simplex (51 co-negative; 23 co-positive samples). Considering the WB as the reference standard for the diagnosis of infection with HTLV-1/2, we observed that the CLIA results of ≤3.0 RLU and >10.0 RLU in the retest can be used define a negative or positive result, respectively, and could be used as new specific cut-off values. The overall agreement between WB and FC-Duplex for accomplishing the differential diagnosis was evaluated and demonstrated 100% correspondence for the diagnosis of HTLV-1 (15/15) and HTLV-2 (7/7). Our findings demonstrate that gaps in the diagnosis of infection with HTLV-1/2 could be overcome by the simultaneous use of distinct immunological assays during retesting of candidates for blood donations., Competing Interests: The authors have declared that no competing interests exist., (Copyright: © 2024 Santos et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.)

The present study sought to search for the immunodominance related to the N-terminal, Central and C-terminal regions of HTLV-1 Tax using novel, cutting-edge peptide microarray analysis. In addition, in silico predictions were performed to verify the presence of nine amino acid peptides present along Tax restricted to the human leukocyte antigen (HLA)-A2.02*01 haplotype, as well as to verify the ability to induce pro-inflammatory and regulatory cytokines, such as IFN-γ and IL-4, respectively. Our results indicated abundant dose-dependent reactivity for HLA-A*02:01 in all regions (N-terminal, Central and C-terminal), but with specific hotspots. Furthermore, the results of fold-change over the Tax 11-19 reactivity obtained at lower concentrations of HLA-A*02:01 reveal that peptides from the three regions contain sequences that react 100 times more than Tax 11-19 . On the other hand, Tax 11-19 has similar or superior HLA-A*02:01 reactivity at higher concentrations of this haplotype. The in silico analysis showed a higher frequency of IFN-γ-inducing peptides in the N-terminal portion, while the C-terminal portion showed a higher frequency of IL-4 inducers. Taken together, these results shed light on the search for new Tax immunodominant epitopes, in addition to the canonic Tax 11-19 , for the rational design of immunomodulatory strategies for HTLV-1 chronic diseases.

In the present work, we developed and evaluated the performance of a new flow cytometry-based single platform, referred to as "FC-Duplex IgG1 (HTLV-1/2)", for universal and differential serodiagnosis of HTLV-1/2 infection. The proposed technology employs a system for detection of IgG1 antibodies in a single competitive immunofluorescence platform by flow cytometry using fluorescently labeled MT-2/MoT cell line mix coupled to a highly sensitive development system (Biotin/Streptavidin/Phycoerythrin). The stability of fluorescent labeling and the antigenicity of MT-2 and MoT cell lines were confirmed upon storage at -20°C for 2, 6, and 12 months. The anti-HTLV-1/2 IgG1 reactivity, expressed as percentage of positive fluorescent cells (PPFC), was evaluated for each target antigen along the titration curve of test serum samples (1:32 to 1:4,096). Upon selection of target cell line and serum dilutions with higher segregation score between groups, the performance of "FIX" and "FIX & PERM" protocols was evaluated. The "FIX" protocol presented excellent performance indices (Se = 92%/Sp = 94%/AUC = 0.96; Se = 96%/Sp = 100%/AUC = 0.99) for the universal (HTLV-1/2 vs. NI) and differential (HTLV-1 vs. HTLV-2) diagnosis of HTLV-1 infection, respectively. Optimization of the "FIX" protocol using the principle of synchronous and asynchronous pairwise analysis further improved the performance of "FC-Duplex IgG1 (HTLV-1/2)", using the "FIX" protocol for differential diagnosis of HTLV-1 and HTLV-2 infections (Se = 100%/Sp = 100%/AUC = 1.00). In conclusion, the "FC-Duplex IgG1 (HTLV-1/2)" method represents an innovation in the biotechnology segment with the potential to compose a serological kit for differential diagnosis of HTLV-1/2 infection for reference laboratories and blood centers., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2022 Pimenta de Paiva, Coelho-dos-Reis, Trindade, Peruhype-Magalhães, Silva Araújo, Gonçalves, Nogueira, Pereira Martins, Lopes Ribeiro, Starling, Alcântara, Ribeiro, Carneiro-Proietti, Sabino, Alves Bicalho, Teixeira-Carvalho and Martins-Filho.)

The impact of HIV co-infection on the plasma immunological biomarker profile of HTLV-1 infected patients was evaluated. The plasma levels of leukotrienes and chemokines/cytokines were quantified by ELISA and Cytometric Bead Array. A total of 138 volunteers were enrolled and divided into two subgroups ("HTLV-1(+)HIV(-)" and "HTLV-1(+)(HIV(+)"), which were categorized according to the HTLV-1-associated neurological disease (AS, pHAM and HAM). Reference controls were BD and HIV mono-infected patients. HAM(+) exhibited higher CD4 + T-cell counts as compared to HIV+ mono-infected patients and lower HTLV-1 proviral load as compared to mono-infected HAM(-) patients. AS(+) exhibited higher levels of CysLT, CXCL8/IL-8 and lower levels of CCL5/RANTES as compared to AS(-). Increased levels of IL-6 and TNF with reduced levels of CXCL10/IP10 and CCL5/RANTES were observed in co-infected pHAM(+) as compared to mono-infected pHAM(-). HAM(+) patients revealed an increase in CXCL8/IL-8, CCL2/MCP-1, CXCL-10/IP-10, TNF and a decrease in IL-2 as compared to HAM(-) subgroup., (Copyright © 2021 European Federation of Immunological Societies. Published by Elsevier B.V. All rights reserved.)

In the present study, the frequency, the activation and the cytokine and chemokine profile of HTLV-1 carriers with or without dermatological lesions were thoroughly described and compared. The results indicated that HTLV-1-infected patients with dermatological lesions have distinct frequency and activation status when compared to asymptomatic carriers. Alterations in the CD4(+)HLA-DR(+), CD8(+) T cell, macrophage-like and NKT subsets as well as in the serum chemokines CCL5, CXCL8, CXCL9 and CXCL10 were observed in the HTLV-1-infected group with skin lesions. Additionally, HTLV-1 carriers with dermatological skin lesions showed more frequently high proviral load as compared to asymptomatic carriers. The elevated proviral load in HTLV-1 patients with infectious skin lesions correlated significantly with TNF-α/IL-10 ratio, while the same significant correlation was found for the IL-12/IL-10 ratio and the high proviral load in HTLV-1-infected patients with autoimmune skin lesions. All in all, these results suggest a distinct and unique immunological profile in the peripheral blood of HTLV-1-infected patients with skin disorders, and the different nature of skin lesion observed in these patients may be an outcome of a distinct unbalance of the systemic inflammatory response upon HTLV-1 infection.

Background: HTLV-1 infects millions of people around the world and induces myelopathy (HAM/TSP), adult T-cell leukemia (ATL) or other inflammatory or rheumatologic diseases. The host-virus interaction causes asymptomatic carriers to develop HAM/TSP. Biomarkers are needed to predict patients who are at risk for HAM/TSP. Tax is highly immunogenic and is a major target protein recognized by cytotoxic T lymphocytes. Anti-Tax antibodies are involved in HAM/TSP pathogenesis., Objectives: To assess anti-Tax IgG reactivity with a flow cytometry assay (FCA) using an infection/transfection system with Vaccinia virus and pLW44/Tax-expressing Tax and to correlate the anti-Tax response and the HTLV-1 proviral load., Study Design: : We enrolled 81 individuals: 9 HTLV-1 seronegative (NP) and 72 HTLV-1 positive (23 HTLV-1 asymptomatic carriers (AC), 12 oligosymptomatic patients (OL), 7 with rheumatologic diseases (DR) and 30 with HAM/TSP (HT)). Anti-Tax reactivity was assessed by FCA, and HTLV-1 proviral load was measured with real time PCR., Results: The HT and DR groups showed greater anti-Tax IgG reactivity (p<0.001 and p<0.05 comparing HT to the OL and AC group, respectively; p<0.05 comparing DR to the OL group), and the reactivity in the DR+HT group was significantly different when compared to the AC group (p<0.05) and to the OL group (p<0.001). The proviral load was higher in the HT group compared to the OL (p<0.001) and in the HT+DR group compared to OL (p<0.001). There was no correlation between anti-Tax IgG reactivity and proviral load in any of the HTLV-1-infected groups., Conclusion: These findings suggest that although anti-Tax IgG reactivity and the HTLV-1 proviral load are important markers of the development of HTLV-1-associated diseases, their levels are not correlated., (Copyright © 2010 Elsevier B.V. All rights reserved.)

In this study, the performance of IgG and IgG1 anti-HTLV-1 reactivity obtained by a flow cytometric assay was evaluated to verify its applicability for the diagnosis of persons infected with HTLV-1, including asymptomatic carriers and patients with myelopathy. The ability to identify patients with myelopathy among persons infected with HTLV-1 was also examined. Western blot assays were performed to assess the reactivity profiles of sera from asymptomatic carriers and patients with myelopathy against viral proteins. The data showed that IgG1 detected by flow cytometric assay is effective for the diagnosis of persons infected with HTLV-1 with 97% sensitivity and 100% specificity. IgG and IgG1 exhibited high performance in distinguishing patients with myelopathy from asymptomatic carriers. Using serum dilutions and cut-off points established previously a second HTLV-1 carrier group was tested using flow cytometric assay to detect IgG and IgG1. The data demonstrated sensitivity of 93% and 98%, respectively, confirming the high reactivity of persons infected with HTLV-1 detected by this method. Western blot assays confirmed the high specificity of MT-2 cells as a reliable source of viral antigen since only sera from persons infected with HTLV-1 recognised MT-2 proteins. Furthermore, a high reactivity to Gag and Env proteins was observed, especially among patients with myelopathy. These data suggest that flow cytometric detection of IgG1 is a valuable, non-conventional serological method to diagnose HTLV-1 infection and for research purposes.