Enhancing Access and Utility of Assays for ART Initiation and Monitoring in HIV Patients in Resource-Limited Settings

Background: Strategies to expand access to routine HIV monitoring assays in resource limited settings (RLS) include the use of point-of-care (POC) tests and dried blood spots (DBS) collected at remote settings and shipped to central laboratories for testing for viral load (VL) testing. Currently access to CD4 testing is gradually expanding due to the emergence of relatively rapid, instrument-based POC tests. There are also instrument-free CD4 tests in the pipeline among which is VISITECT CD4 test, a semi-quantitative lateral-flow based technology. Though few near POC VL tests are commercially available currently and a few in the pipeline, these have not yet had much impact on the total level of VL monitoring in resource-poor settings they. As a result, the use of DBS is suggested as interim option for expanding access to VL testing in RLS. However, due to inclusion of HIV nucleic acids from white blood cells rather than only plasma HIV RNA, many studies reported its lack of accuracy to diagnose anti-retroviral therapy (ART) failure when used in most RT-PCR platforms
Aims: The broad aim of this thesis was to enhance access to HIV monitoring assays in RLS by improving performance and utility of assay technologies/methods. Specific aims included; studying the modified use of the standard VISITECT CD4 test for TB/HIV co-infected patients who need a different cut-off for timing of ART initiation, and development of a filter paper-based method of plasma separation (referred to as Filtered and Dried Plasma, FDP) from whole blood to enhance accurate quantification of HIV VL in RLS.
Methods: Experiments were conducted to investigate the potential use of test/reference ratio values generated by AX-2 reader of VISITECT CD4 test for TB/HIV co-infected
patients ART initiation (Chapter 2). In Chapter 4, different filtration materials (filter papers and biological reagents) and approaches were examined to develop a novel plasma separation method, known as Filtered and Dried Plasma (FDP). Proof-of-concept experiments to evaluate the new method efficiency in removing blood cells and generating cell-free plasma were also conducted. A prospective study to evaluate the performance of FDP in HIV VL quantification was performed using clinical samples (Chapter 5). Furthermore, long term storage stability of FDP was studied in laboratory conditions (Chapter 6).
Results: Only preliminary experiments that pinpoint a potential approach where modification of VISITECT CD4 test can be made were conducted and presented in Chapter 2. A preferred method (FDP) to DBS has been developed using filter papers that demonstrated effective elimination of RBCs and 93% of WBCs (Chapter 4). The prospective study (Chapter 5) demonstrated its accurate detection of treatment failure at the current cut-off (1000 cps/ml). Storage stability studies demonstrated adequate stability of FDP both in the pre and post- sample application storage (Chapter 6).
Conclusion: This thesis highlighted the diagnostic challenges in HIV/AIDS management, and strategies to enhance access and utility of diagnostics in RLS. Limited work were done and presented under the aim to investigate the modified use of VISITECT CD4 test (Chapter 2) due to delays in the final manufacturing product. The prototype plasma separation method, FDP, demonstrated efficient performance in HIV VL quantification and stability giving the promise to be used as the preferred dried sample for VL testing in RLS.