The South African National Antiretroviral Treatment Guidelines recommend the use of
HIV-1 viral load assays for routine monitoring of HIV-1 positive patients receiving
highly active antiretroviral therapy (HAART). This thesis describes the innovative
approaches to developing more affordable HIV-1 diagnostics and monitoring assays for
South Africa, which take into account the tiered laboratory infrastructure of this country.
An in-house HIV-1 viral load assay – the LUX assay, was developed and evaluated with
a view of implementing this more affordable option in high tier laboratories. The LUX
assay represents quantitative real-time RT-PCR that utilizes the LightCycler® technology
(Roche) in a novel combination with a LUXÔ primer. The assay showed good analytical
sensitivity, specificity and reproducibility of its linear dynamic range of 4x102 to 4x106
RNA copies/ml. Preliminary clinical evaluation (n = 458) of the LUX assay showed good
agreement with the COBAS Amplicor assay, and demonstrated its usefulness for long
term monitoring of HAART patients.
ELISA based viral load testing approaches were investigated as low cost and less
technically complex alternatives for medium tier laboratories. The HiSens HIV-1 p24 Ag
Ultra (Perkin Elmer) and the ExaVir™ Load Quantitative HIV-RT kits (CAVIDI) were
compared with the Roche Amplicor assay. Both assays showed strong association with
the Roche Amplicor assay, with R2 = 0.686 and R2 = 0.810, respectively (n = 117). These
alternative assays seemed most useful in the serial monitoring of patients on HAART.
Major drawbacks included the wide variability of both assays, insufficient sensitivity of
the p24 antigen assay and low throughput of the RT assay.
Development of a point-of-care HIV-1 RNA assay could address issues related to early
and cost effective diagnosis of acute HIV infection. A novel isothermal amplification
technique termed the Reverse Transcription Loop Dependant Amplification (RT-LDA)
was developed as one component for a potential point-of-care HIV-1 RNA assay. The
RT-LDA converted RNA into partially looped ssDNA amplicons, over a wide RNA
concentration range (4x103 to 4x108 copies/ml) using a 1 hour incubation at 53ºC. The
RT-LDA technology is fully compatible with a lateral flow detection system using
dipsticks and highly suitable for point-of-care testing.
Overall, this study demonstrates the feasibility of developing novel, more affordable
HIV-1 testing options that would be appropriate for the tiered laboratory infrastructure
present in South Africa. Evaluation of commercially available, less expensive alternative
HIV viral load assays in local settings facilitates their implementation.
| Identifer | oai:union.ndltd.org:netd.ac.za/oai:union.ndltd.org:wits/oai:wiredspace.wits.ac.za:10539/5679 |
| Date | 18 September 2008 |
| Creators | Rekhviashvili, Natela |
| Source Sets | South African National ETD Portal |
| Language | English |
| Detected Language | English |
| Type | Thesis |
| Format | application/pdf, application/pdf |
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