RESUMO
Human T cell lymphotropic virus (HTLV) is the caustive agent of two main conditions i. e., the HTLV-1-associated myelopathy/tropical spastic paraparesis (HAM/TSP) and the adult T-cell leukemia/lymphoma (ATLL). HTLV diagnosis is based on serological and molecular approaches; however, an accurate and validated method is still needed. The objective of this study was to establish a rapid and sensitive molecular test to confirm and discriminate HTLV 1/2 types. The test validation was performed as a multicentric study involving HTLV confirmation centers throughout Brazil. Proviral DNA was extracted from whole blood and the amplification was performed using in-house designed primer and probe sets targeting the pol genomic region. An internal control to validate the extraction and amplification was also included. The limit of detection (LoD) of the assay was four copies/reaction for HTLV-1 and 10.9 copies/reaction for HTLV-2. The diagnostic sensitivity of the platform was 94.6% for HTLV-1, 78.6% for HTLV-2, and the specificity was 100% for both viruses. Cross-reactions of the test with human viruses including HAV, HBV, HCV, HIV-1/2, and parvovirus B19 were not observed. During the multicentric validation, the test was used to screen a total of 692 blood samples obtained from previously confirmed HTLV-positive individuals. From these, 91.1% tested positive being concordant with the previously obtained results. In conclusion, our duoplex-RT-PCR-HTLV1 /2 presented adequate efficiency for HTLV-1/2 differentiation showing high sensitivity and specificity. Therefore, it can be a suitable tool for confirmation of suspected and inconclusive HTLV cases, prenatal and pre-transplant diagnosis, in Brazil and in other countries HTLV-endemic countries.
RESUMO
Human T-lymphotropic virus 1 (HTLV-1) is the etiologic agent of adult cell leukemia/lymphoma (ATL) and HTLV-1 associated myelopathy/tropical spastic paraparesis (HAM/TSP). One of the major questions in HTLV-1 studies is related to the understanding of causes that lead to different clinical manifestations. However, it is well known that the viral genes tax and HTLV-1 basic leucine zipper factor (HBZ) are related to viral infectivity and the development of neurological and hematological diseases. Currently, there is evidence that HTLV-1 infected cells can release small extracellular vesicles (sEVs) involved in the mechanisms of viral particles spreading. Therefore, we evaluated the expression levels of tax and HBZ viral transcripts in serum-derived sEVs from HTLV-1 carriers, as well as the role of these vesicles in the modulation of the immune response. Three HAM/TSP carriers presented detectable levels of tax and HBZ transcripts in sEVs and were positively correlated to the proviral load (PVL) in peripheral blood mononuclear cells (PBMCs). The viral transcripts were only detectable in individuals with a PVL higher than 6,000/105 PBMCs. Additionally, it was observed that HBZ presented a 2-12-folds increase over tax expression units. Gene expression and secretory protein analysis indicated that PBMCs from blood donors and HTLV-1 carriers exposed to increasing doses of tax+ HBZ+ sEVs showed a dose-dependent increase in interferon (IFN)-γ and interleukin (IL)-8 transcripts and proteins. Interestingly, the increase in IL-8 levels was close to those seen in HTLV-1-infected PBMCs with high PVL. Taken together, these findings indicate that the expression of viral transcripts in serum-derived sEVs of HTLV-1 carriers is related to the PVL presented by the infected individual. Additionally, tax+ HBZ+ sEVs can induce the production of inflammatory cytokines in patients with low PVL, which may be related to the development of symptoms in HTLV-1 infection.