Dann, Louise Claire; (2002) Nucleic acid sequence-based amplification: Relative performance and applications in HIV-1 disease monitoring and patient management. Doctoral thesis (Ph.D), UCL (University College London).

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Abstract

In recent years there have been significant advances in the understanding of the pathogenesis of HIV-1 infection. Central to this progress has been the development of accurate and reproducible methods of measuring HIV-1 RNA in plasma. This has been pivotal in studies of viral dynamics, disease progression and antiretroviral drug efficacy. This thesis describes evaluations of the performance of Nucleic Acid Sequence-Based Amplification (NASBA) technologies in quantifying HIV-1 RNA, relative to other commercial systems, investigating reproducibility, sensitivity and reliability across subtypes using a number of panels designed with either plasma dilutions of laboratory strains of HIV-1, or by selected clinical viruses of interest. Quantification of HIV-1 B and non B subtypes was investigated to study the impact of increasing diversity of HIV-1 subtypes in our clinic population. The evaluations described here involve close collaborations with assay manufacturers (as an alpha testing site) and clinical users, and have encouraged the continual improvement of viral load quantification systems, supporting the development of new versions of RT-PCR and NASBA assays. Further, attempts were made to develop an economic in house viral quantification system based on immunocapture and RT-PCR using an internal biological standard. To investigate the versatility of viral load measurement in different clinical settings, studies were performed in primary HIV-1 infection and established HIV-1 infection. A well characterised cohort of patients undergoing primary HIV-1 infection (n = 47), were evaluated for the relative performance of NASBA quantification during the initial stages of infection and followed for up to 3 years. In addition, this enabled a comparison of virological responses to therapy in early HIV-1 disease and analysis for antiretroviral resistance mutations to investigate the prevalence of resistance transmission in this recently infected group. A clinical trial of patients with established infection was undertaken to determine the practical significance of virological measures relative to immunological responses and therapeutic drug monitoring. The performance of NASBA in quantifying viral load in a wide range of studies was shown to be less sensitive than the bDNA and RT-PCR assays and also less able to amplify viruses of more diverse subtypes. In a number of studies the quantification of samples with a low viral load (>5,000 copies/ml) by NASBA was shown have greater variance. However, at higher viral copy numbers (>5,000 copies/ml), the reproducibility of results was equivalent to alternative viral load systems and other markers of disease monitoring and drug efficacy. NASBA performed adequately in characterising patients undergoing primary HIV-1 infection relative to other disease markers such as p24 antigen, anti-HIV-1 serology, and the conventional understanding of virological events during this early infection period. Further, NASBA viral load measures demonstrated that although antiretroviral therapy was very effective during this period, it was not significantly more effective than therapy initiated later. It was noted in this cohort that the frequency of resistance transmission was low for nucleoside (15%) and non-nucleoside (6%) inhibitor resistance, and absent for protease inhibitors. The investigations into an in house system showed that a modified immunocapture using latex microparticle produced inconsistent results, and a variable high background in the detection system precluded further evaluation. By comparison, the reverse transcription and amplification step was modified successfully and further optimisation of this method was undertaken within the department to allow routine use. Throughout this study, the objective has been to evaluate the current and future use of viral load measurement. This thesis has gone some way towards validating the expanding use of viral load since the identification of HIV-1 RNA as a disease marker. As long as the reliability of viral quantification systems supports the prevailing clinical environment and is evaluated in studies such as this, it may continue to act as a principal tool in HIV-1 clinical research and patient care.

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