Detection and genotyping of enteroviruses in cerebrospinal fluid in patients in Victoria, Australia, 2007-2013.

Genotyping by VP1 fragment polymerase chain reaction (PCR) and nucleic acid sequencing to detect enterovirus (EV) genotypes was performed directly on 729 EV PCR positive cerebrospinal fluid (CSF) samples collected between 2007 and 2012 from Victorian hospital inpatients. The overall genotype identification rate from CSF-positive material was 43%. The four most common genotypes identified were Echovirus 6 (24%), Echovirus 30 (17%), Echovirus 25 (10%), and Coxsackievirus A9 (10%), together comprising 61% of all EVs typed. The seasonal distribution of all EVs identified followed the recognized pattern of mainly summer epidemics. Three of the four predominant genotypes were present in each of the 6 years in which the study was conducted, with 20 other EV genotypes also detected, often in only a single year. Genotyping of EVs directly in CSF is faster, simpler and more sensitive than traditional virus neutralization assays performed on EV positive samples.

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.

Laboratory diagnosis of HIV: a contemporary overview in the Australian context.

Over the past decade there have been technical advances in human immunodeficiency virus (HIV) assays and updates to testing regulations that have substantially changed the landscape of laboratory testing for HIV. In addition, there have been significant changes in the epidemiology of HIV in Australia in the context of highly effective contemporary biomedical treatment and prevention strategies. Here, we provide an update on contemporary issues for the laboratory detection and confirmation of HIV in Australia. These include (1) the impact of early treatment and biological prevention strategies on the serological and virological detection of HIV; (2) the updated national HIV laboratory case definition and its interaction with testing regulations, public health and clinical guidelines; and (3) novel strategies for the laboratory detection of HIV, including the incorporation of HIV nucleic acid amplification tests (NAATs) into testing algorithms. These developments present an opportunity to develop a nationally consistent contemporary HIV testing algorithm that would result in optimisation and standardisation of HIV testing in Australia.

Adaptation of Droplet Digital PCR-Based HIV Transcription Profiling to Digital PCR and Association of HIV Transcription and Total or Intact HIV DNA.

In most people living with HIV (PLWH) on effective antiretroviral therapy (ART), cell-associated viral transcripts are readily detectable in CD4+ T cells despite the absence of viremia. Quantification of HIV RNA species provides insights into the transcriptional activity of proviruses that persist in cells and tissues throughout the body during ART ('HIV reservoir'). One such technique for HIV RNA quantitation, 'HIV transcription profiling', developed in the Yukl laboratory, measures a series of HIV RNA species using droplet digital PCR. To take advantage of advances in digital (d)PCR, we adapted the 'HIV transcription profiling' technique to Qiagen's dPCR platform (QIAcuity) and compared its performance to droplet digital (dd)PCR (Bio-Rad QX200 system). Using RNA standards, the two technologies were tested in parallel and assessed for multiple parameters including sensitivity, specificity, linearity, and intra- and inter-assay variability. The newly validated dPCR assays were then applied to samples from PLWH to determine HIV transcriptional activity relative to HIV reservoir size. We report that HIV transcriptional profiling was readily adapted to dPCR and assays performed similarly to ddPCR, with no differences in assay characteristics. We applied these assays in a cohort of 23 PLWH and found that HIV reservoir size, based on genetically intact proviral DNA, does not predict HIV transcriptional activity. In contrast, levels of total DNA correlated with levels of most HIV transcripts (initiated, proximally and distally elongated, unspliced, and completed, but not multiply spliced), suggesting that a considerable proportion of HIV transcripts likely originate from defective proviruses. These findings may have implications for measuring and assessing curative strategies and clinical trial outcomes.

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.

Phylogenetic clustering networks among heterosexual migrants with new HIV diagnoses post-migration in Australia.

BACKGROUND: It is estimated that approximately half of new HIV diagnoses among heterosexual migrants in Victoria, Australia, were acquired post-migration. We investigated the characteristics of phylogenetic clusters in notified cases of HIV among heterosexual migrants. METHODS: Partial HIV pol sequences obtained from routine clinical genotype tests were linked to Victorian HIV notifications with the following exposures listed on the notification form: heterosexual sexual contact, injecting drug use, bisexual sexual contact, male-to male sexual contact or heterosexual sexual contact in combination with injecting drug use, unknown exposure. Those with heterosexual sexual contact as the only exposure were the focus of this study, with the other exposures included to better understand transmission networks. Additional reference sequences were extracted from the Los Alamos database. Maximum likelihood methods were used to infer the phylogeny and the robustness of the resulting tree was assessed using bootstrap analysis. Phylogenetic clusters were defined on the basis of bootstrap and genetic distance. RESULTS: HIV pol sequences were available for 332 of 445 HIV notifications attributed to only heterosexual sexual contact in Victoria from 2005-2014. Forty-three phylogenetic clusters containing at least one heterosexual migrant were detected, 30 (70%) of which were pairs. The characteristics of these phylogenetic clusters varied considerably by cluster size. Pairs were more likely to be composed of people living with HIV from a single country of birth (p = 0.032). Larger clusters (n≥3) were more likely to contain people born in Australian/New Zealand (p = 0.002), migrants from more than one country of birth (p = 0.013) and viral subtype-B, the most common subtype in Australia (p = 0.006). Pairs were significantly more likely to contain females (p = 0.037) and less likely to include HIV diagnoses with male-to-male sexual contact reported as a possible exposure (p<0.001) compared to larger clusters (n≥3). CONCLUSION: Migrants appear to be at elevated risk of HIV acquisition, in part due to intimate relationships between migrants from the same country of origin, and in part due to risks associated with the broader Australian HIV epidemic. However, there was no evidence of large transmission clusters driven by heterosexual transmission between migrants. A multipronged approach to prevention of HIV among migrants is warranted.

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.

A national study of the molecular epidemiology of HIV-1 in Australia 2005-2012.

INTRODUCTION: Rates of new HIV-1 diagnoses are increasing in Australia, with evidence of an increasing proportion of non-B HIV-1 subtypes reflecting a growing impact of migration and travel. The present study aims to define HIV-1 subtype diversity patterns and investigate possible HIV-1 transmission networks within Australia. METHODS: The Australian Molecular Epidemiology Network (AMEN) HIV collaborating sites in Western Australia, South Australia, Victoria, Queensland and western Sydney (New South Wales), provided baseline HIV-1 partial pol sequence, age and gender information for 4,873 patients who had genotypes performed during 2005-2012. HIV-1 phylogenetic analyses utilised MEGA V6, with a stringent classification of transmission pairs or clusters (bootstrap ≥98%, genetic distance ≤1.5% from at least one other sequence in the cluster). RESULTS: HIV-1 subtype B represented 74.5% of the 4,873 sequences (WA 59%, SA 68.4%, w-Syd 73.8%, Vic 75.6%, Qld 82.1%), with similar proportion of transmission pairs and clusters found in the B and non-B cohorts (23% vs 24.5% of sequences, p = 0.3). Significantly more subtype B clusters were comprised of ≥3 sequences compared with non-B clusters (45.0% vs 24.0%, p = 0.021) and significantly more subtype B pairs and clusters were male-only (88% compared to 53% CRF01_AE and 17% subtype C clusters). Factors associated with being in a cluster of any size included; being sequenced in a more recent time period (p<0.001), being younger (p<0.001), being male (p = 0.023) and having a B subtype (p = 0.02). Being in a larger cluster (>3) was associated with being sequenced in a more recent time period (p = 0.05) and being male (p = 0.008). CONCLUSION: This nationwide HIV-1 study of 4,873 patient sequences highlights the increased diversity of HIV-1 subtypes within the Australian epidemic, as well as differences in transmission networks associated with these HIV-1 subtypes. These findings provide epidemiological insights not readily available using standard surveillance methods and can inform the development of effective public health strategies in the current paradigm of HIV prevention in Australia.

Studies of measles viruses circulating in Australia between 1999 and 2001 reveals a new genotype.

Nineteen distinct measles virus (MV) strains associated with nine different genotypes were identified in five Australian states (Victoria, New South Wales, Queensland, Northern Territory and Western Australia) between 1999 and 2001. One of the strains identified is likely to represent a new genotype within the clade D viruses (proposed to be d9). No evidence for an indigenous MV strain was found. When epidemiologic information associated with the index case was available for the outbreaks, it usually supported introduction of the virus from overseas, with the main source being South East Asia. Changes in the circulation of MV in Australia since the early 1970s were also observed. Prior to the introduction of measles vaccine, the majority of the population acquired immunity through infection with wild-type virus in early childhood. Nowadays in Australia, young adults are at most risk of infection. The age range of cases in the study period was from 1 month to 48 years, with the majority (59%) of cases from individuals aged 18-30 years.

Novel mutations in the thymidine kinase and DNA polymerase genes of acyclovir and foscarnet resistant herpes simplex viruses infecting an immunocompromised patient.

BACKGROUND: Mutations in the thymidine kinase (TK) and DNA polymerase (pol) genes of herpes simplex virus (HSV) may confer resistance to antiviral drugs, particularly in the context of immunosuppression induced by infection with the human immunodeficiency virus (HIV). OBJECTIVES: To characterise the HSV type 2 (HSV-2) TK and DNA pol genes in an immunocompromised patient with clinical resistance to both acyclovir and foscarnet. STUDY DESIGN: The TK and DNA pol genes of isolates obtained over a 2-year period from an AIDS patient with severe genital herpes infection were characterised both phenotypically and genotypically. RESULTS: HSV strains that were acyclovir resistant/foscarnet sensitive, acyclovir sensitive/foscarnet sensitive and acyclovir resistant/foscarnet resistant were isolated during this time. The TK gene of all the acyclovir resistant isolates contained a large 969 bp deletion which extended into a downstream untranslated region. The foscarnet resistance was associated with an S725G mutation in a conserved region (region II) of the herpesvirus DNA pol gene. CONCLUSIONS: Clinical and virological suppression of the infection was not always associated with subsequent reactivation with wild-type virus. Mutations of the nature we describe have not previously been reported occurring simultaneously in HSV strains isolated from patients treated with acyclovir and foscarnet.

Molecular analysis of clinical isolates of acyclovir resistant herpes simplex virus.

We characterised the antiviral phenotype and genotype of 41 herpes simplex virus (HSV) strains from patients clinically resistant to acyclovir (ACV). Our results confirm recognised mutational sites as being major determinants of thymidine kinase (tk)-associated ACV resistance, in particular insertions and/or deletions at homopolymer stretches of Gs and Cs (59% of all isolates). Previously described amino acid substitutions in functional sites of the tk were also identified (7% of all isolates). In addition, we identified several stop codons in novel locations on the amino acid sequence (7% of all isolates) and amino acid substitutions (15% of all isolates) likely to be directly responsible for conferring resistance to ACV. When there were no mutations detected in the tk gene (12% of all isolates), mutations in the DNA polymerase gene likely to be important in the generation of resistant virus were identified.

Acquisition of HIV by African-born residents of Victoria, Australia: insights from molecular epidemiology.

African-born Australians are a recognised "priority population" in Australia's Sixth National HIV/AIDS Strategy. We compared exposure location and route for African-born people living with HIV (PLHIV) in Victoria, Australia, with HIV-1 pol subtype from drug resistance assays and geographical origin suggested by phylogenetic analysis of env gene. Twenty adult HIV positive African-born Victorian residents were recruited via treating doctors. HIV exposure details were obtained from interviews and case notes. Viral RNA was extracted from participant stored plasma or whole blood. The env V3 region was sequenced and compared to globally representative reference HIV-1 sequences in the Los Alamos National Library HIV Database. Twelve participants reported exposure via heterosexual sex and two via iatrogenic blood exposures; four were men having sex with men (MSM); two were exposed via unknown routes. Eight participants reported exposure in their countries of birth, seven in Australia, three in other countries and two in unknown locations. Genotype results (pol) were available for ten participants. HIV env amplification was successful in eighteen cases. HIV-1 subtype was identified in all participants: eight both pol and env; ten env alone and two pol alone. Twelve were subtype C, four subtype B, three subtype A and one subtype CRF02_AG. Reported exposure location was consistent with the phylogenetic clustering of env sequences. African Australians are members of multiple transnational social and sexual networks influencing their exposure to HIV. Phylogenetic analysis may complement traditional surveillance to discern patterns of HIV exposure, providing focus for HIV prevention programs in mobile populations.

Increasing diversity of Human Immunodeficiency Virus type 1 subtypes circulating in Australia.

Characterization of HIV subtypes can provide a more comprehensive understanding of the epidemic within a distinct region, and when combined with notification data, may also be helpful in enhancing current HIV prevention strategies. In this study, we characterized 1056 HIV-positive individuals (948 males and 108 females) living in Victoria and whose infection was detected for the first time between 2005 and 2010 inclusive. HIV-1 strains were subtyped based on pol gene sequence. Phylogenetic analysis was performed on all non-B subtype sequences identified. Of the 1056 sequences analyzed, 825 were subtype B and 231 were non-B. Overall 6 HIV-1 subtypes, 6 circulating recombinant forms (CRFs), and 12 unique recombinant forms (URFs) were identified. Regardless of gender, the majority of individuals were infected with a subtype B virus (78%). Subtype B was dominant in males (n=806, 85%). In contrast, the majority of females were infected with non-B subtypes (n=89, 82%), in particular subtype C (n=48, 45%). Phylogenetic analysis of the non-B subtypes revealed that the majority of clustering, and thereby transmission, occurred with CRF01_AE strains. Despite the relatively high numbers identified in females there was very little clustering of subtype C viruses. Subtypes C and A1 both historically associated with heterosexual transmission, and CRF01_AE often associated with IVDU, were also associated with transmission within the MSM population, demonstrating the potential for non-B subtypes to expand into the MSM population. The observation of increasing numbers of females and heterosexual males infected with non-subtype B viruses, the majority imported through migration and travel to countries where there is a high prevalence of HIV, suggests a targeted public health message may be required to prevent further increases within these two groups.

HIV transmissions during seroconversion contribute significantly to new infections in men who have sex with men in Australia.

Transmission of HIV from recently infected individuals contributes to the number of new cases of infection, but the extent to which it occurs from those who are unaware of their infection is not known. Phylogenetic analysis was performed on 209 cases of acute HIV subtype B infection detected between January 2005 and September 2010, most of whom (88%) were men who have sex with men. Only new cases with an evolving Western blot profile confirmed by detection of HIV RNA were included. Subjects whose known dates of seroconversion were within 1 month of at least one other phylogenetically linked case identified during the 6 years of the study were then examined to estimate the prevalence of onward transmission. Almost 30% of cases could have acquired their infection from another person undergoing seroconversion within the same month. Temporal increases in the number of phylogenetically related strains within several clusters were demonstrated during the study, although the rate of increase varied. Transmission of HIV from individuals undergoing seroconversion is an important contributor to the number of new infections identified every year and very likely occurs before they have knowledge of their infection. Clusters of related HIV strains can grow at a disconcerting rate, demonstrating that more focused public health efforts are required to minimize further HIV transmissions within sexual networks.

Respiratory virus laboratory pandemic planning and surveillance in central Viet Nam, 2008-2010.

INTRODUCTION: Laboratory capacity is needed in central Viet Nam to provide early warning to public health authorities of respiratory outbreaks of importance to human health, for example the outbreak of influenza A(H1N1) pandemic in 2009. Polymerase chain reaction (PCR) procedures established as part of a capacity-building process were used to conduct prospective respiratory surveillance in a region where few previous studies have been undertaken. METHODS: Between October 2008 and September 2010, nose and throat swabs from adults and children (approximately 20 per week) presenting with an acute respiratory illness to the Ninh Hoa General Hospital were collected. Same-day PCR testing and result reporting for 13 respiratory viruses were carried out by locally trained scientists. RESULTS: Of 2144 surveillance samples tested, 1235 (57.6%) were positive for at least one virus. The most common were influenza A strains (17.9%), with pandemic influenza A(H1N1) 2009 and seasonal H3N2 strain accounting for 52% and 43% of these, respectively. Other virus detections included: rhinovirus (12.4%), enterovirus (8.9%), influenza B (8.3%), adenovirus (5.3%), parainfluenza (4.7%), respiratory syncytial virus (RSV) (3.9%), human coronavirus (3.0%) and human metapneumovirus (0.3%). The detection rate was greatest in the 0-5 year age group. Viral co-infections were identified in 148 (6.9%) cases. DISCUSSION: The outbreak in 2009 of the influenza A(H1N1) pandemic strain provided a practical test of the laboratory's pandemic plan. This study shows that the availability of appropriate equipment and molecular-based testing can contribute to important individual and public health outcomes in geographical locations susceptible to emerging infections.

A case of apparent resistance to all available antiretroviral drugs in a HIV-positive patient.

We describe a case of multidrug and class resistant HIV in a patient with psychological issues affecting his adherence. The complexities involved in subsequent treatment decisions when resistance interpretation algorithms are discordant are discussed.