Background Human T-cell Lymphotropic Virus type-1 (HTLV-1) infects an estimated 20 million people worldwide causing a fatal T- cell leukaemia and inflammatory conditions, such as HTLV-1-associated myelopathy, resulting from a dysregulated inflammatory response to the virus. HTLV-1 is highly endemic to remote indigenous communities in Central Australia where infection contributes to racial disparities in morbidity and mortality. HTLV-1 related complications are associated with elevated numbers of T-cells in which the HTLV-1 provirus integrates (HTLV-1 proviral load, PVL). Digital-droplet PCR (ddPCR) is capable of absolute quantification of PVL, offering low variability between assays with few cell numbers. We measured HTLV-1 PVLs from buffy coat cells (BCCs) of infected individuals, and bronchoalveolar lavage (BALs) infiltrates from a bronchiectasis patient to determine the range of HTLV-1 copies/cell relative to illness.
Methods All samples were from Alice Springs Hospital HTLV-1 cohort (8 BCCs, 1 BAL) and obtained following ethics approval and patient consent in first language. Genomic DNA was extracted from BCCs for ddPCR PVL quantification. HTLV-1 gag/tax primers and FAM/MGB probes were optimised using temperature gradient amplification. Samples were tested in duplicate relative to an internal reference gene standard, RPP30, or negative threshold controls that used VIC/MGB probe. QuantaSoft software was used for data analysis.
Results Of the 8 samples from BCCs, the PVL fell between 320 – 8,040 proviral copies per 106 cells. The BAL sample PVL was 1,110 DNA copies per 106 cells. The sensitivity (or limit of detection) of our ddPCR assay is 2 copies of proviral DNA per 104 cells.
Conclusion The ddPCR assay demonstrates extremely high sensitivity, low assay-variability and the capability to reliably quantify HTLV-1 PVL. This technique offers logistic advantages in studying relationships between PVL in HTLV-1 patient’s BCCs and BAL.
Disclosure of interest statement No Conflicts of interest.
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No commercial contributions have been received for this research project.