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O17.4 Development and validation of a human t-cell lymphotropic virus type-1 proviral load assay
  1. KM Wilson1,
  2. S Dick1,
  3. L Einsiedel2,
  4. S Best1
  1. 1NRL, Melbourne, Australia
  2. 2Flinders Medical Centre, Adelaide, Australia

Abstract

Introduction Human T-cell lymphotropic virus (HTLV-I) infects approximately 20 million people world-wide. Transmission requires cell to cell contact and infection can be acquired through breast milk, exposure to HTLV-1 contaminated blood products or sexual contact with an infected person. The HTLV-I proviral load (PVL) is a strong predictor of the risk of transmission and may also serve as an indicator of those most at risk of acquiring significant complications following infection.

The Australo-Melanesian variant (HTLV-I subtype c) is endemic amongst indigenous communities in Central Australia and demonstrates a highly divergent sequence from other known HTLV-I subtypes. Currently, there are no commercially available HTLV PVL assays and published methods fail to reliably detect HTLV-1c.

Methods We developed and validated a quantitative, real time PCR (qPCR) assay, specific for the current circulating strains of HTLV-I. Primers and probes were designed by sequencing the gag gene from HTLV-1c samples. A highly conserved region of the gag gene which did not cross-react with HTLV-II was chosen.

A dilution series of SP cells which contain 1 copy of the HTLV-I genome, was used for quantification. The standards and specimens were run in parallel throughout the entire extraction and qPCR process, allowing us to eliminate variations due to extraction efficiency, PCR amplification and detection. The albumin gene, was used to determine the number of cells/sample and the PVL expressed as HTLV-I copies/cell.

Results We have now fully validated this assay using both clinical specimens and cultured cell lines. Clinical specimens consisting  of buffy coats, whole blood specimens and dry blood spots have been tested on the HTLV-I PVL assay and demonstrated good concordance with results obtained using gold standard serology assays.

Conclusion We have developed and validated an assay that can reliably quantify the HTLV PVL which may serve as a predictor of the risk of transmission and disease progression.

Disclosure of interest statement The authors and their affiliated organisations have no conflicts of interests. This work has been funded through the NHMRC.

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