Abstract
Brazil currently has the highest number of individuals infected with human T-lymphotropic virus 1- and 2- (HTLV-1 and HTLV-2) globally. At present, neither molecular protocols nor commercial assays are available for HTLV-1/-2 diagnosis or validated by the Brazilian Ministry of Health regulatory agency (ANVISA). We developed and validated two in-house multiplex quantitative real-time PCR for HTLV-1/-2 (mqPCR_HTLV) assays, targeting the pol and tax genes, for the simultaneous identification of HTLV-1, HTLV-2, and the albumin reference gene. The robustness of the assays was evaluated on two platforms using seven commercial master mix formulations. The reactions employed double plasmids (pHTLV1-Alb and pHTLV2-Alb) for the standard curve’s construction and for expressing the detection limit of the assays. They were able to detect 10 and 10 copies of HTLV-1 and 10 and 70 copies of HTLV-2 for the tax and pol targets, respectively. High efficiency was obtained using both the platforms and all the reagents evaluated and were successfully reproduced by other analysts. DNA samples from HTLV-1/-2-infected and non-infected patients and from HIV/HTLV-coinfected patients were evaluated to determine the feasibility of their use in routine diagnosis. The mqPCR_HTLV (pol and tax) assays demonstrated an overall specificity of 100% and a sensitivity of 97.4% when testing samples from patients without HIV infection, and sensitivities of 77.1% (pol) and 74.6% (tax) in samples from HIV/HTLV-coinfected patients. In addition, they resolved the issue of HTLV western blotting (WB) indeterminate and WB-untyped results in 45.5 and 66.7% of cases, respectively. The developed mqPCR_HTLV (pol and tax) assays indicated their feasibility for efficient and reliable HTLV diagnosis in various core facility laboratories under different conditions and supplies.
Highlights
Human T-lymphotropic virus 1 and 2 (HTLV-1 and HTLV-2) are endemic to Brazil and their distribution varies according to the ethnic background of individuals and geographic regions (CGIST/DDCI/SVS et al, 2020)
Blood samples obtained from patients tested for HTLV-1/2 specific antibodies which were infected and not infected with HIV-1, HBV and HCV were distributed in the following groups: Group 1, 37 patients infected with HTLV-1 alone confirmed by western blotting (WB); Group 2, 152 patients infected with HIV-1 and reactive of HTLV-1/2 infection by screening assays; Group 3, 30 patients not infected with HTLV but infected with HIV-1 and/or HBV and/or HCV
Samples of Group 1 belonged to blood donors from Recife, Pernambuco (Northeast Brazil), tested positive for HTLV-1 during the years 2012–2017, and sent to Instituto Adolfo Lutz (IAL) for HTLV-1 subtype characterization (LTR, env and tax sequencing) using DNA extracted from peripheral blood mononuclear cells (PBMCs) isolated by Ficoll-Paque Plus density gradient centrifugation (GE Healthcare, Uppsala, Sweden)
Summary
Human T-lymphotropic virus 1 and 2 (HTLV-1 and HTLV-2) are endemic to Brazil and their distribution varies according to the ethnic background of individuals and geographic regions (CGIST/DDCI/SVS et al, 2020). Brazil has the highest number of HTLV-1/2-infected individuals in Latin America and worldwide (Gessain and Cassar, 2012; CGIST/DDCI/SVS et al, 2020). The majority of the individuals remain asymptomatic, many people living with HTLV may develop one or more of a range of related diseases (Haziot et al, 2019; Rosadas et al, 2021a). It is important to perform confirmatory and discriminatory diagnosis of HTLV1/2 infections. There is no curative treatment available for these infections, preventive measures, which include correct diagnosis, are the only means to eliminate HTLV transmission (Rosadas et al, 2021b)
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