Author: Karen Hawke

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The global diversity of circulating HIV-1 subtypes and circulating recombinant forms (CRFs) is growing, including an increasing number of new complex variants. This has serious implications for effective vaccine and treatment strategies worldwide, and may affect sensitivity of diagnostic and viral load assays. In Australia, subtype B has historically been dominant among men who have sex with other men (MSM) but in recent years new non-B infections have appeared, predominantly via heterosexual contact, which follows a global trend. To date, there have been two studies (not including the one resulting from this PhD) published on this change in subtype distribution in Australia, noting a significant increase in the proportion of CRFs and non-B subtypes circulating across Victoria (2012) and Western Australia (2015). To understand epidemics globally it is also important to monitor the transmission of drug resistant strains, known as transmitted drug resistance (TDR), which is rising within the adult population globally. In 2012, UNAIDS assessed global TDR prevalence at 3.1% and likely to increase. TDR can reduce efficacy of antiretroviral therapy and while subtypes display similar drug sensitivity, some may have greater propensity to develop certain mutations. In 2009, an Australian study reported a TDR prevalence of 16% in Victoria. Since 2000, subtype and drug resistance data have been collected as part of routine genotypic resistance testing in South Australia. This PhD examined all newly diagnosed, genotyped HIV cases in South Australia between 2000 and 2013. Initial genotypic drug resistance testing was based on a 1098bp region of the Pol gene, encompassing the protease (PR) and reverse transcriptase (RT) genes. The Stanford calibrated population resistance (CPR) online subtyping tool was used to interpret subtype and drug resistance mutations (Chapter Four and Five), and the 2009 WHO list of surveillance mutations was used to determine TDR. A 530bp region spanning gp41 of the Env gene was then sequenced (Chapter Six), and analysis of the sequenced pol and env data was conducted using Maximum Likelihood. Online subtyping tools (Stanford CPR, jumping profile Hidden Markov Model (jpHMM), REGA, Subtype Classification Using Evolutionary ALgorithms (SCUEAL), National Center for Biotechnology Information (NCBI), and COntext-based Modeling for Expeditious Typing (COMET)) were also used to determine subtype (Chapter Seven). Molecular epidemiological analysis of the HIV pol gene identified an evolving epidemic in South Australia, from predominantly subtype B infection among the MSM community or Australian-born heterosexual population, to an increasing proportion of non-B infections imported from overseas by Australian travelers, or overseas-born people. These non-B infections include an increasing number of complex recombinants that are predominantly acquired in high prevalence countries where multiple strains co-circulate. While the prevalence of TDR decreased over time, the overall rate in the HIV infected population was high, largely due to the forward transmission of K103N amongst MSM infected with subtype B infection. There was also evidence of a moderate but declining rate of Non-Nucleoside Reverse Transcriptase Inhibitor (NNRTI) and Nucleoside Reverse Transcriptase Inhibitor (NRTI) resistance in the non-B cohort who were diagnosed within the last 10 years. This resistance corresponds with the time period NNRTI and NRTI treatments were introduced, then improved, overseas. Phylogeny and rapid online subtyping tool analysis of the pol and env genes found a significantly greater diversity of circulating HIV variants than previously identified using the Stanford CPR tool to assign the pol region alone, including the identification of possible unique recombinant forms. Using two gene regions also identified a larger number of possible transmission events, demonstrating the complexity of this continually evolving virus. Transmission events were predominantly between male/female heterosexual pairings carrying non-B infections acquired overseas, with some evidence of subtype B transmission events occurring between male only pairs and male/female pairs, and a number of larger male subtype B clusters predominantly acquired within Australia. These studies were the first in Australia to link genetic and routine surveillance data to provide an in-depth characterisation of the changing HIV epidemic in Australia over the last 14 years. The findings greatly improve our knowledge of HIV subtype distribution and transmission dynamics in South Australia. They also demonstrate that analysis of multiple gene regions using modern phylogenetic methods provides additional valuable information about the HIV epidemic structure within a region of interest. In addition, we now have an enhanced understanding of treatment failure and treatment adherence rates of current first-line regimens and how these differ between geographic origin and subtype. It is crucial to conduct accurate and detailed surveillance in order to identify factors and molecular mechanisms that affect transmission, replication, and resistance, improve research efforts into strains widely circulating in high prevalence areas, and target subtype-specific prevention and treatment options for at-risk populations. Despite decreasing rates, the number of TDR strains circulating in the MSM population in Australia remain a concern, which highlights the need for continued surveillance, education and early diagnosis. There is also a steady influx of people migrating from high prevalence countries and a growing proportion of these are now being diagnosed with HIV, including some with drug resistant strains. As ART availability continues to increase globally, it is also important to ensure early diagnosis, pre-treatment drug resistance testing, effective treatment regimens and ongoing surveillance are available worldwide. It is imperative that efforts are targeted toward increasing and expanding HIV testing, not only to identify infection early but also to test for transmitted resistance before treatment commences. Resistance patterns in non-B persons infected overseas may influence treatment choice and viral suppression beyond our current understanding of the historical subtype B infection circulating in Australia, and these cases should be monitored closely. In addition to the change in geographic segregation of HIV subtypes and CRFs, the traditional risk-group segregation is also becoming less distinct, with growing evidence of crossover between the MSM, Intravenous Drug Use (IDU) and heterosexual populations. This combined with the increasing genetic diversity including complex and unique variants, highlights the need for careful monitoring of new infections, and the immediate need for non-B subtype research in Australia. Despite evidence that shows social structures have far greater impact on the HIV epidemic than behavioral or treatment strategies, integration and acceptance of social strategies into prevention and control efforts has been very slow, and the prevention focus remains on modifying individual behavior and increasing treatment. Future research should combine genetic and epidemiological data, and use it to create social strategies that identify and counteract issues that impact on individual and societal behavior, such as socioeconomic disadvantage and geographical region, political and cultural systems and gender inequity, which all have considerable effect on the incidence of HIV. In 2014 ethics approval was given for a national project named the Australian Molecular Epidemiology Network to commence. This project is the result of collaboration between South Australia, Western Australia, Victoria, New South Wales and Queensland, and is the first national study in Australia to collect data from all newly diagnosed HIV cases between 2005 and 2018 and phylogenetically analyze sequences to assess subtype distribution, transmission patterns both within each state and across state borders, and to identify the prevalence of unique variant forms of HIV that may be circulating.

Keywords: HIV, subtype, phylogenetic, epidemiology, infection
Subject: Medicine thesis

Thesis type: Doctor of Philosophy
Completed: 2016
School: School of Medicine
Supervisor: Paul Ward