HTLV load testing and genotyping

St. Mary’s hospital London, part of the Imperial NHS Trust, is the national referral centre for HTLV infection. Details of the service can be found at www.imperial.nhs.uk/stmarys/ourservices/htlvservice. MDU provides diagnostic tests in support of this service.

HTLV DNA qPCR

Scope of the test

HTLV-I DNA viral load measurement for diagnosis and monitoring of disease in individual patients infected with human T-lymphotropic virus types 1 & 2.

Test details (back to top)

An in-house method to measure HTLV-1/2 viral load in patient peripheral blood mononuclear cells (PBMC) is used. Primers are specific for HTLV-1 and HTLV-2 Tax gene. HTLV-1 or 2 DNA copy number and β-globin gene copy number of each sample of extracted PBMC DNA are quantified using real-time quantitative PCR monitored by SYBR Green I dye incorporation in a Roche LightCycler 1.5. HTLV-1 copy number is estimated by interpolation from standard curves and is expressed as HTLV-I DNA copies per 100 PBMCs. When extracted DNA has appropriate β-globin gene copy number but no quantifiable HTLV-I DNA is amplified using the RealTime PCR, a PCR is carried out using nested primers specific for HTLV-1 or for HTLV-2 (see typing section below). PCR products are detected on an ethidium bromide stained agarose gel.

HTLV typing by PCR

Scope of the test (back to top)

To discriminate between HTLV-1 and HTLV-2 infection.

Test details (back to top)

PCR amplification of the tax gene sequence of HTLV DNA extracted from PBMCs is performed. PCR is carried out using generic outer primers in the first round and discriminatory inner primers in the second round. PCR products are detected on an ethidium bromide stained agarose gel. HTLV-1 , HTLV-2 and negative controls are run in parallel.

HTLV clonal analysis

Scope of the test (back to top)

To facilitate rapid detection of Adult T-cell Leukaemia/Lymphoma. The method can also be used to monitor therapy response and provide early detection of relapse/remission.

Test details (back to top)

The test is performed to determine whether the dominant integration sites of HTLV-I are monoclonal, oligoclonal or polyclonal, using a vectorette linker and specific proviral primer BIO2. Patients’ DNA is extracted and digested with the restriction enzyme HindIII. The vectorette unit is ligated to the restriction digested DNA fragments and then used as a template in a two-step touchdown PCR to allow amplification of DNA between the known sequence of of HTLV-I and the vectorette unit. After PCR amplification the clonal pattern is analysed by electrophoresis of the amplicons on a 1.5% agarose gel.

Reporting results (back to top)

Reports are routinely made as hard copy but can be supplied as email attachment if required.

Turnaround time (back to top)

We aim to issue reports within two weeks of receiving sample. However turnaround times may be extended if repeat testing is required.

Suitable Samples:

• Whole EDTA blood. CSF to arrive at MDU within 24 hours of venesection/lumbar puncture.
• Cutaneous, lymph node or tissue biopsy – tissue block or histology slide.
• Extracted DNA.

Do not send plasma.

If requesting CSF HTLV-1 DNA viral load send sample EDTA whole blood obtained at the same time point.

Assay failure rate (back to top)

The most common cause of assay failure is old age of sample.

Charges (back to top)

Molecular HTLV tests are provided to UK NHS centres free of charge by the National Centre for Human Retrovirology.

Charges for other uses:

HTLV DNA qPCR	HTLV typing by PCR	HTLV clonal analysis
£80	£70	£130

Complaints (back to top)

If you are unhappy with the service provided by MDU or if you wish to make suggestions on how our service can be improved, please contact the Unit Manager.