Gen tax evidence of HTLV-1 co-infections in HIV-positive patients in a hospital in Bilbao, Spain
Abstract number: p766
Ortiz N., Basaras M., Sota M., Cisterna R.
Human T-cell leukaemia virus type I (HTLV-I), is the pathogenic agent of adult T-cell leukaemia-lymphoma (ATLL) and HTLV-1 associated myelopath/tropicalspastic paraparesia (HAM/TSP). In HIV infected patients have been shown to be frequent, probably in consequence of their similar modes of transmission. The gen tax of a human T-cell leukaemia virus type 1 (HTLV-1) induces the expression of several cellular genes that are involved in T cell activation and proliferation. The aim of this study was to analyse HTLV-I proviral DNA presence in HIV infected patients of Bilbao using primers of the region tax of the HTLV-1.
Materials and methods:
We analysed peripheral blood mononuclear cells (PBMCs) from 100 patients samples HIV infected patients (EIA, and confirmed with Western blot). These patients were treated with antiretroviral therapy. A conventional PCR and a real time PCR assay using SYBR Green were established. Both assays were designed using primers from tax gene (156 bp) of HTLV-I. The real-time PCR assay reliably detected a single copy of HTLV-I proviral genome in DNA from 1 × 106 PBMCs. For conventional PCR we also included in each sample the HLA DQ alpha gene (242 bp) to check the presence of PCR inhibitors and the quality of DNA extraction. As positive control we used HTLV-I infected MT-2 cell line, which had integrated stable tax gene. Cells were incubated at 37 °C in a 5% CO2 atmosphere in RPMI 1640 medium supplemented with 10% heat inactivated Bovine foetal serum (FBS), penicillin and streptomycin.
All the samples analysed had presented HLA DQ alpha gene amplicon indicating that PCR had been carried out under good conditions. In five of the HIV infected patients the band corresponding to the gene tax (156 bp) of the HTLV-1 was detected. In the real time PCR's the MT-2 cells were in use for obtaining a standard melting curve with a melting temperature of 88 C (±1 C). The melting temperature of nine PCR samples from HIV infected patients was similar to the one obtained in the standard melting curve but only five of the samples had been reported as positive when conventional PCR was performed.
The results of this study suggest that both assays could be used to diagnostic HTLV-1 infection in samples of HIV infected patients. Besides, the results also suggest that real time PCR is faster and has more sensitivity fact the conventional PCR but further studies are needed.
|Session name:||XXIst ISTH Congress|
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