Year: 2008
Type: dissertation
Abstract: The South African National Antiretroviral Treatment Guidelines recommend the use of \nHIV-1 viral load assays for routine monitoring of HIV-1 positive patients receiving \nhighly active antiretroviral therapy (HAART). This thesis describes the innovative \napproaches to developing more affordable HIV-1 diagnostics and monitoring assays for \nSouth Africa, which take into account the tiered laboratory infrastructure of this country. \nAn in-house HIV-1 viral load assay – the LUX assay, was developed and evaluated with \na view of implementing this more affordable option in high tier laboratories. The LUX \nassay represents quantitative real-time RT-PCR that utilizes the LightCycler® technology \n(Roche) in a novel combination with a LUXÔ primer. The assay showed good analytical \nsensitivity, specificity and reproducibility of its linear dynamic range of 4x102 to 4x106 \nRNA copies/ml. Preliminary clinical evaluation (n = 458) of the LUX assay showed good \nagreement with the COBAS Amplicor assay, and demonstrated its usefulness for long \nterm monitoring of HAART patients. \nELISA based viral load testing approaches were investigated as low cost and less \ntechnically complex alternatives for medium tier laboratories. The HiSens HIV-1 p24 Ag \nUltra (Perkin Elmer) and the ExaVir™ Load Quantitative HIV-RT kits (CAVIDI) were \ncompared with the Roche Amplicor assay. Both assays showed strong association with \nthe Roche Amplicor assay, with R2 = 0.686 and R2 = 0.810, respectively (n = 117). These \nalternative assays seemed most useful in the serial monitoring of patients on HAART. \nMajor drawbacks included the wide variability of both assays, insufficient sensitivity of \nthe p24 antigen assay and low throughput of the RT assay. \nDevelopment of a point-of-care HIV-1 RNA assay could address issues related to early \nand cost effective diagnosis of acute HIV infection. A novel isothermal amplification \ntechnique termed the Reverse Transcription Loop Dependant Amplification (RT-LDA) \nwas developed as one component for a potential point-of-care HIV-1 RNA assay. The \nRT-LDA converted RNA into partially looped ssDNA amplicons, over a wide RNA \nconcentration range (4x103 to 4x108 copies/ml) using a 1 hour incubation at 53ºC. The \nRT-LDA technology is fully compatible with a lateral flow detection system using \ndipsticks and highly suitable for point-of-care testing. \nOverall, this study demonstrates the feasibility of developing novel, more affordable \nHIV-1 testing options that would be appropriate for the tiered laboratory infrastructure \npresent in South Africa. Evaluation of commercially available, less expensive alternative \nHIV viral load assays in local settings facilitates their implementation.
Author: Natela Rekhviashvili
Language: English
Subfield: Infectious Diseases
Field: Medicine
Domain: Health Sciences
Open Access status: green
OpenAlex ID: W2136833369