Characterising HIV-1 transmission in Victoria, Australia: a molecular epidemiological study.

Background: In Australia the incidence of HIV has declined steadily, yet sustained reduction of HIV transmission in this setting requires improved public health responses. As enhanced public health responses and prioritisation of resources may be guided by molecular epidemiological data, here we aimed to assess the applicability of these approaches in Victoria, Australia. Methods: A comprehensive collection of HIV-1 pol sequences from individuals diagnosed with HIV in Victoria, Australia, between January 1st 2000 and December 31st 2020 were deidentified and used as the basis of our assessment. These sequences were subtyped and surveillance drug resistance mutations (SDRMs) identified, before definition of transmission groups was performed using HIV-TRACE (0.4.4). Phylodynamic methods were applied using BEAST (2.6.6), assessing effective reproductive numbers for large groups, and additional demographic data were integrated to provide a high resolution view of HIV transmission in Victoria on a decadal time scale. Findings: Based on standard settings for HIV-TRACE, 70% (2438/3507) of analysed HIV-1 pol sequences were readily assigned to a transmission group. Individuals in transmission groups were more commonly males (aOR 1.50), those born in Australia (aOR 2.13), those with probable place of acquisition as Victoria (aOR 6.73), and/or those reporting injectable drug use (aOR 2.13). SDRMs were identified in 375 patients (10.7%), with sustained transmission of these limited to a subset of smaller groups. Informative patterns of epidemic growth, stabilisation, and decline were observed; many transmission groups showed effective reproductive numbers (R e ) values reaching greater than 4.0, representing considerable epidemic growth, while others maintained low R e values. Interpretation: This study provides a high resolution view of HIV transmission in Victoria, Australia, and highlights the potential of molecular epidemiology to guide and enhance public health responses in this setting. This informs ongoing discussions with community groups on the acceptability and place of molecular epidemiological approaches in Australia. Funding: National Health and Medical Research Council, Australian Research Council.

Surveillance testing using salivary RT-PCR for SARS-CoV-2 in managed quarantine facilities in Australia: A laboratory validation and implementation study.

Background: Regular repeat surveillance testing is a strategy to identify asymptomatic individuals with SARS-CoV-2 infections in high-risk work settings to prevent onward community transmission. Saliva sampling is less invasive compared to nasal/oropharyngeal sampling, thus making it suitable for regular testing. In this multi-centre evaluation, we aimed to validate RT-PCR using salivary swab testing of SARS-CoV-2 for large-scale surveillance testing and assess implementation amongst staff working in the hotel quarantine system in Victoria, Australia. Methods: A multi-centre laboratory evaluation study was conducted to systematically validate the in vitro and clinical performance of salivary swab RT-PCR for implementation of SARS-CoV-2 surveillance testing. Analytical sensitivity for multiple RT-PCR platforms was assessed using a dilution series of known SARS-CoV-2 viral loads, and assay specificity was examined using a panel of viral pathogens other than SARS-CoV-2. In addition, we tested capacity for large-scale saliva testing using a four-sample pooling approach, where positive pools were subsequently decoupled and retested. Regular, frequent self-collected saliva swab RT-PCR testing was implemented for staff across fourteen quarantine hotels. Samples were tested at three diagnostic laboratories validated in this study, and results were provided back to staff in real-time. Findings: The agreement of self-collected saliva swabs for RT-PCR was 84.5% (95% CI 68.6 to 93.8) compared to RT-PCR using nasal/oropharyngeal swab samples collected by a healthcare practitioner, when saliva samples were collected within seven days of symptom onset. Between 7th December 2020 and 17th December 2021, almost 500,000 RT-PCR tests were performed on saliva swabs self-collected by 102 staff working in quarantine hotels in Melbourne. Of these, 20 positive saliva swabs were produced by 13 staff (0.004%). The majority of staff that tested positive occurred during periods of community transmission of the SARS-CoV-2 Delta variant. Interpretation: Salivary RT-PCR had an acceptable level of agreement compared to standard nasal/oropharyngeal swab RT-PCR within early symptom onset. The scalability, tolerability and ease of self-collection highlights utility for frequent or repeated testing in high-risk settings, such as quarantine or healthcare environments where regular monitoring of staff is critical for public health, and protection of vulnerable populations. Funding: This work was funded by the Victorian Department of Health.

Multiply spliced HIV RNA is a predictive measure of virus production ex vivo and in vivo following reversal of HIV latency.

BACKGROUND: One strategy being pursued to clear latently infected cells that persist in people living with HIV (PLWH) on antiretroviral therapy (ART) is to activate latent HIV infection with a latency reversing agent (LRA). Surrogate markers that accurately measure virus production following an LRA are needed. METHODS: We quantified cell-associated unspliced (US), multiply spliced (MS) and supernatant (SN) HIV RNA by qPCR from total and resting CD4+ T cells isolated from seven PLWH on ART before and after treatment ex vivo with different LRAs, including histone deacetylase inhibitors (HDACi). MS and plasma HIV RNA were also quantified from PLWH on ART (n-11) who received the HDACi panobinostat. FINDINGS: In total and resting CD4+ T cells from PLWH on ART, detection of US RNA was common while detection of MS RNA was infrequent. Primers used to detect MS RNA, in contrast to US RNA, bound sites of the viral genome that are commonly mutated or deleted in PLWH on ART. Following ex vivo stimulation with LRAs, we identified a strong correlation between the fold change increase in SN and MS RNA, but not the fold change increase in SN and US RNA. In PLWH on ART who received panobinostat, MS RNA was significantly higher in samples with detectable compared to non0detectable plasma HIV RNA. INTERPRETATION: Following administration of an LRA, quantification of MS RNA is more likely to reflect an increase in virion production and is therefore a better indicator of meaningful latency reversal. FUNDING: NHMRC, NIH DARE collaboratory.

Virological fitness of HIV in patients with resistance to enfuvirtide.

Resistance to the HIV fusion inhibitor enfuvirtide is associated with mutations in the first heptad repeat region of gp41, but little is known of their impact on replicative fitness in vivo. We followed seven patients undergoing salvage therapy that included enfuvirtide in order to document the temporal generation of genotypic and phenotypic resistance in parallel with replicative fitness. Resistance to enfuvirtide was not associated with decreased replicative fitness of HIV strains infecting these patients.

Analysis of transmitted HIV drug resistance from 2005 to 2015 in Victoria, Australia: a comparison of the old and the new.

Background Baseline genotyping is part of standard-of-care treatment. It reveals that transmitted drug resistance (TDR) continues to be important for the management of HIV infection. Attention is typically focused on determining whether resistance to the protease inhibitors (PI) and reverse transcriptase inhibitors (RTI) occurs. However, the increasing use of integrase inhibitors (INIs) raises a concern that TDR to this class of antiretroviral drug may also occur. METHODS: PI and RTI drug resistance genotyping was performed on blood samples collected between 2005 and 2015 from 772 treatment-naïve Victorian patients infected with HIV within the previous 12 months. Integrase genotyping was performed on 461 of the 485 patient samples collected between 2010 and 2015. RESULTS: In the period 2005-10, 39 of 343 patients (11.4%) had at least one PI- or RTI-associated mutation, compared with 34 of 429 (7.9%) during the period 2011-15. Compared with 2005-10, during 2011-15 there was a significant decline in the prevalence of the non-nucleoside-associated mutation K103N and the nucleoside-associated mutations at codons M41 and T215. One patient was detected with a major INI resistance mutation, namely G118R. However, this mutation is rare and its effect on susceptibility is unclear. A small number of patients (n=12) was infected with HIV containing accessory resistance mutations in the integrase gene. CONCLUSIONS: The lack of transmitted resistance to INIs is consistent with a low level of resistance to this class of drugs in the treated population. However, continued surveillance in the newly infected population is warranted as the use of INIs increases.

Prevalence of transmitted HIV drug resistance since the availability of highly active antiretroviral therapy.

We investigated the prevalence of HIV-1-associated transmitted drug resistance (TDR) in Victoria from the time of first availability of highly active antiretroviral therapy. Drug resistance genotyping was performed on virus present in blood samples collected from individuals with serologically confirmed primary infection, between 1996 and 2007. The significance of any mutations detected was interpreted according to a standardised list of drug resistance mutations. The main outcomes measured were the prevalence by year of TDR to any antiretroviral drug class, the numbers of infected individuals with TDR involving multiple drug classes, and the resistance mutations implicated in all cases. There was an average annual prevalence of TDR of 16%, predominantly associated with nucleoside and non-nucleoside reverse transcriptase (RT) inhibitors and most commonly occurring at codons 41, 103 and 215 in the RT. The prevalence of thymidine-associated mutations remained high throughout the period of study. While mutations known to cause resistance to protease inhibitors were uncommon, they were present in several individuals infected with virus resistant to multiple drug classes. The prevalence of TDR in Victoria is similar to geographical locations outside Australia where HIV-specific drug treatment is widely available. Primary infection with drug resistant HIV is a future treatment issue for the individual patient and for the wider population at risk of infection. At this time TDR shows no sign of waning and our data support recent treatment guidelines recommending baseline testing for TDR before therapy is initiated.