The clinical utility and costs of whole-genome sequencing to detect cancer susceptibility variants-a multi-site prospective cohort study.

BACKGROUND: Many families and individuals do not meet criteria for a known hereditary cancer syndrome but display unusual clusters of cancers. These families may carry pathogenic variants in cancer predisposition genes and be at higher risk for developing cancer. METHODS: This multi-centre prospective study recruited 195 cancer-affected participants suspected to have a hereditary cancer syndrome for whom previous clinical targeted genetic testing was either not informative or not available. To identify pathogenic disease-causing variants explaining participant presentation, germline whole-genome sequencing (WGS) and a comprehensive cancer virtual gene panel analysis were undertaken. RESULTS: Pathogenic variants consistent with the presenting cancer(s) were identified in 5.1% (10/195) of participants and pathogenic variants considered secondary findings with potential risk management implications were identified in another 9.7% (19/195) of participants. Health economic analysis estimated the marginal cost per case with an actionable variant was significantly lower for upfront WGS with virtual panel ($8744AUD) compared to standard testing followed by WGS ($24,894AUD). Financial analysis suggests that national adoption of diagnostic WGS testing would require a ninefold increase in government annual expenditure compared to conventional testing. CONCLUSIONS: These findings make a case for replacing conventional testing with WGS to deliver clinically important benefits for cancer patients and families. The uptake of such an approach will depend on the perspectives of different payers on affordability.

Community input in a genomic health implementation program: Perspectives of a community advisory group.

Consumer and community engagement (CCE) in the implementation of genomics into health services and associated research is needed to ensure that changes benefit the affected patients. Queensland Genomics was a program to implement genomics into a public health service. We describe its Community Advisory Group's (CAG) structure and function and provide recommendations based on the CAG members' perspectives. The CAG provided advice to the Queensland Genomics program and its projects in an advisory capacity. The CAG was also resourced to develop and lead community-focused activities. Key enablers for CAG included; diversity of CAG members' skills and experience, adequate resourcing, and the CAG's ability to self-determine their direction. The CAG experienced limitations due to a lack of mechanisms to implement CCE in the Program's projects. Here, we provide insights and commentary on this CAG, which will be useful for other initiatives seeking to undertake CCE in genomic research and health care.

Ask the people: developing guidelines for genomic research with Aboriginal and Torres Strait Islander peoples.

In health and medical research, guidelines are a set of statements and recommendations, whereby experts or stakeholders assess published literature to generate practical advice for a specific audience. This emphasis on guidelines development with expert consultation and published literature is not practical or inclusive when working in disciplines with minimal data and addressing issues that concern under-represented communities. Here we describe the process used for developing guidelines for the conduct of genomic research projects in partnership with Aboriginal and Torres Strait Islander peoples. A new technology with individual and community level ethical and social implications, and First Nations peoples with cultural and community expectations for research. We developed the guidelines through a consultation process that used participatory action research to engage with various stakeholders during multiple rounds of tailored activities. The end product, 'Genomic Partnerships: Guidelines for Genomics Research with Aboriginal and Torres Strait Islander peoples of Queensland' reflects the needs of the end-users and perspectives of the Aboriginal and Torres Strait Islander peoples, communities and organisations that participated. Through this process, we have identified recommendations for developing guidelines with other under-represented communities.

Queensland Genomics: an adaptive approach for integrating genomics into a public healthcare system.

The establishment of genomics in health care systems has been occurring for the past decade. It is recognised that implementing genomics within a health service is challenging without a system-wide approach. Globally, as clinical genomics implementation programs have matured there is a growing body of information around program design and outcomes. Program structures vary depending on local ecosystems including the health system, politics and funding availability, however, lessons from other programs are important to the design of programs in different jurisdictions. Here we describe an adaptive approach to the implementation of genomics into a publicly funded health care system servicing a population of 5.1 million people. The adaptive approach enabled flexibility to facilitate substantial changes during the program in response to learnings and external factors. We report the benefits and challenges experienced by the program, particularly in relation to the engagement of people and services, and the design of both individual projects and the program as a whole.

'CTRL': an online, Dynamic Consent and participant engagement platform working towards solving the complexities of consent in genomic research.

The complexities of the informed consent process for participating in research in genomic medicine are well-documented. Inspired by the potential for Dynamic Consent to increase participant choice and autonomy in decision-making, as well as the opportunities for ongoing participant engagement it affords, we wanted to trial Dynamic Consent and to do so developed our own web-based application (web app) called CTRL (control). This paper documents the design and development of CTRL, for use in the Australian Genomics study: a health services research project building evidence to inform the integration of genomic medicine into mainstream healthcare. Australian Genomics brought together a multi-disciplinary team to develop CTRL. The design and development process considered user experience; security and privacy; the application of international standards in data sharing; IT, operational and ethical issues. The CTRL tool is now being offered to participants in the study, who can use CTRL to keep personal and contact details up to date; make consent choices (including indicate preferences for return of results and future research use of biological samples, genomic and health data); follow their progress through the study; complete surveys, contact the researchers and access study news and information. While there are remaining challenges to implementing Dynamic Consent in genomic research, this study demonstrates the feasibility of building such a tool, and its ongoing use will provide evidence about the value of Dynamic Consent in large-scale genomic research programs.

Evaluation of a Genetics Education Program for Health Interpreters: A Pilot Study.

Health Interpreters enable effective communication between health practitioners and patients with limited knowledge of the predominant language. This study developed and evaluated a training session introducing Health Interpreters to genetics. The online training was delivered multiple times as a single 2-h session comprising lectures and activities. Participants completed questionnaires (pre-, post-, and 6-months follow-up) to assess the impact of training on knowledge, attitude, self-efficacy, and self-reported practice behaviour. Questionnaires were analysed using descriptive statistics, Fisher's Exact, or independent t-test. In total, 118 interpreters participated in the training sessions. Respondent knowledge improved, with gains maintained at 6-months (p < 0.01). There were no changes in self-efficacy, and attitudes. Training did not change self-reported practice behaviour, but there was notable pre-existing variability in participants' methods of managing unknown genetic words. Most respondents agreed that training was useful (93%) and relevant (79%) to their work. More respondents reported learning more from the case study activity (86%) than the group activity (58%). Health Interpreters found the training acceptable and demonstrated sustained improvement in knowledge of genetic concepts. Increased delivery of this training and associated research is needed to assess findings in a larger cohort and to measure the impact on patients.

Queensland Consumers' Awareness and Understanding of Clinical Genetics Services.

As genetic testing becomes increasingly utilized in health care, consumer awareness and understanding is critical. Both are reported to be low in Australia, though there are limited studies to date. A consumer survey assessed perceived knowledge, awareness and attitudes toward genetic medicine, prior to consumers' genomics forums in Queensland in 2018 and 2019. Data was analyzed using t-test and Mann-Whitney U tests analysis to detect any associations between sociodemographic factors and familiarity or attitudes. This highly educated and experienced health consumer cohort reported they were significantly more familiar with the healthcare system generally than genetic medicine specifically (p < 0.0001). Consumers perceived that genetic testing would be significantly more important in the future than it is currently (p < 0.00001). Consumers agreed that genetic testing should be promoted (91.4%), made available (100%), better funded (94.2%), and offered to all pregnant women (81.6%). The preferred learning modality about genetics was internet sites (62.7%) followed by talks/presentations (30.8%). Benefits of genetic testing, reported in qualitative responses, included the potential for additional information to promote personal control and improve healthcare. Perceived concerns included ethical implications (including privacy and discrimination), and current limitations of science, knowledge and/or practice. This study demonstrates that even knowledgeable consumers have little familiarity with genetic medicine but are optimistic about its potential benefits. Ethical concerns, particularly concerns regarding genetic discrimination should inform legislation and policy. Consumers are supportive of online resources in increasing genomic literacy.

Sharing genomic data from clinical testing with researchers: public survey of expectations of clinical genomic data management in Queensland, Australia.

BACKGROUND: There has been considerable investment and strategic planning to introduce genomic testing into Australia's public health system. As more patients' genomic data is being held by the public health system, there will be increased requests from researchers to access this data. It is important that public policy reflects public expectations for how genomic data that is generated from clinical tests is used. To inform public policy and discussions around genomic data sharing, we sought public opinions on using genomic data contained in medical records for research purposes in the Australian state of Queensland. METHODS: A total of 1494 participants completed an online questionnaire between February and May 2019. Participants were adults living in Australia. The questionnaire explored participant preferences for sharing genomic data or biological samples with researchers, and concerns about genomic data sharing. RESULTS: Most participants wanted to be given the choice to have their genomic data from medical records used in research. Their expectations on whether and how often they needed to be approached for permission on using their genomic data, depended on whether the data was identifiable or anonymous. Their willingness to sharing data for research purposes depended on the type of information being shared, what type of research would be undertaken and who would be doing the research. Participants were most concerned with genomics data sharing that could lead to discrimination (insurance and employment), data being used for marketing, data security, or commercial use. CONCLUSIONS: Most participants were willing to share their genomic data from medical records with researchers, as long as permission for use was sought. However, the existing policies related to this process in Queensland do not reflect participant expectations for how this is achieved, particularly with anonymous genomics data. This inconsistency may be addressed by process changes, such as inclusion of research in addition to clinical consent or general research data consent programs.

The relative contribution of causal factors in the transition from infection to clinical chlamydial disease.

Chlamydia is a major bacterial pathogen in humans and animals globally. Yet 80% of infections never progress to clinical disease. Decades of research have generated an interconnected network linking pathogen, host, and environmental factors to disease expression, but the relative importance of these and whether they account for disease progression remains unknown. To address this, we used structural equation modeling to evaluate putative factors likely to contribute to urogenital and ocular chlamydial disease in the koala (Phascolarctos cinereus). These factors include Chlamydia detection, load, and ompA genotype; urogenital and ocular microbiomes; host sex, age, weight, body condition; breading season, time of year; location; retrovirus co-infection; and major histocompatibility complex class II (MHCII) alleles. We show different microbiological processes underpin disease progression at urogenital and ocular sites. From each category of factors, urogenital disease was most strongly predicted by chlamydial PCR detection and load, koala body condition and environmental location. In contrast, ocular disease was most strongly predicted by phylum-level Chlamydiae microbiome proportions, sampling during breeding season and co-infection with koala retrovirus subtype B. Host MHCII alleles also contributed predictive power to both disease models. Our results also show considerable uncertainty remains, suggesting major causal mechanisms are yet to be discovered.

Dysbiosis of the Vaginal Microbiota and Higher Vaginal Kynurenine/Tryptophan Ratio Reveals an Association with Chlamydia trachomatis Genital Infections.

The natural course of Chlamydia trachomatis urogenital tract infections varies between individuals. While protective immunity can occur, some women can become reinfected, contributing to the development of severe pathology. While the reasons for these differences are unknown, an individual's response to induced interferon-γ (IFN-γ) is suggested to be critical. IFN-γ induction of the enzyme indoleamine 2,3-dioxygenase, which depletes tryptophan, may be the key. One hypothesis suggests that indole-producing bacteria in the vaginal microbiota can provide a substrate for the Chlamydia to synthesize tryptophan, rescuing the Chlamydia from host IFN-γ attack. We studied a cohort of 25 women who were either, Chlamydia negative, Chlamydia positive with a single infection, or Chlamydia positive with repeated infection, to test our hypothesis. We characterized their vaginal microbiota, cytokine response, as well as their tryptophan, kynurenine and indole concentrations directly in vaginal secretions. We found that C. trachomatis urogenital tract infections either initial or repeat infections, were associated with elevated vaginal kynurenine/tryptophan ratios, primarily as a result of elevated kynurenine levels. In addition, vaginal microbiota of community state type (CST) IV showed significantly lower vaginal tryptophan levels compared to CST I and III, which might be related to a higher abundance of indole producers found within this group. Furthermore, we found a higher abundance of indole producers in women who cleared their Chlamydia infection post antibiotic treatment. This study demonstrates for the first time in vivo, the association between high vaginal kynurenine/tryptophan ratios and C. trachomatis infections. In addition, tryptophan depletion was associated with vaginal microbiota of CST IV.

Group G Streptococcus Induces an Autoimmune Carditis Mediated by Interleukin 17A and Interferon γ in the Lewis Rat Model of Rheumatic Heart Disease.

Acute rheumatic fever and rheumatic heart disease (ARF/RHD) have long been described as autoimmune sequelae of Streptococcus pyogenes or group A streptococcal (GAS) infection. Both antibody and T-cell responses against immunodominant GAS virulence factors, including M protein, cross-react with host tissue proteins, triggering an inflammatory response leading to permanent heart damage. However, in some ARF/RHD-endemic regions, throat carriage of GAS is low. Because Streptococcus dysgalactiae subspecies equisimilis organisms, also known as ß-hemolytic group C streptococci and group G streptococci (GGS), also express M protein, we postulated that streptococci other than GAS may have the potential to initiate or exacerbate ARF/RHD. Using a model initially developed to investigate the uniquely human disease of ARF/RHD, we have discovered that GGS causes interleukin 17A/interferon γ-induced myocarditis and valvulitis, hallmarks of ARF/RHD. Remarkably the histological, immunological, and functional changes in the hearts of rats exposed to GGS are identical to those exposed to GAS. Furthermore, antibody cross-reactivity to cardiac myosin was comparable in both GGS- and GAS-exposed animals, providing additional evidence that GGS can induce and/or exacerbate ARF/RHD.

Microbiota composition of the koala (Phascolarctos cinereus) ocular and urogenital sites, and their association with Chlamydia infection and disease.

Disease caused by Chlamydia pecorum is characterised by ocular and urogenital infections that can lead to blindness and infertility in koalas. However, koalas that are infected with C. pecorum do not always progress to disease. In other host systems, the influence of the microbiota has been implicated in either accelerating or preventing infections progressing to disease. This study investigates the contribution of koala urogenital and ocular microbiota to Chlamydia infection and disease in a free ranging koala population. Using univariate and multivariate analysis, it was found that reproductive status in females and sexual maturation in males, were defining features in the koala urogenital microbiota. Changes in the urogenital microbiota of koalas is correlated with infection by the common pathogen, C. pecorum. The correlation of microbiota composition and C. pecorum infection is suggestive of members of the microbiota being involved in the acceleration or prevention of infections progressing to disease. The analysis also suggests that multiple microbes are likely to be associated with this process of disease progression, rather than a single organism. While other Chlamydia-like organisms were also detected, they are unlikely to contribute to chlamydial disease as they are rare members of the urogenital and ocular microbiota communities.

Temporal Variation in Physiological Biomarkers in Black Flying-Foxes (Pteropus alecto), Australia.

Bats of the genus Pteropus (Pteropodidae) are recognised as the natural host of multiple emerging pathogenic viruses of animal and human health significance, including henipaviruses, lyssaviruses and ebolaviruses. Some studies have suggested that physiological and ecological factors may be associated with Hendra virus infection in flying-foxes in Australia; however, it is essential to understand the normal range and seasonal variability of physiological biomarkers before seeking physiological associations with infection status. We aimed to measure a suite of physiological biomarkers in P. alecto over time to identify any seasonal fluctuations and to examine possible associations with life-cycle and environmental stressors. We sampled 839 adult P. alecto in the Australian state of Queensland over a 12-month period. The adjusted population means of every assessed hematologic and biochemical parameter were within the reported reference range on every sampling occasion. However, within this range, we identified significant temporal variation in these parameters, in urinary parameters and body condition, which primarily reflected the normal annual life cycle. We found no evident effect of remarkable physiological demands or nutritional stress, and no indication of clinical disease driving any parameter values outside the normal species reference range. Our findings identify underlying temporal physiological changes at the population level that inform epidemiological studies and assessment of putative physiological risk factors driving Hendra virus infection in P. alecto. More broadly, the findings add to the knowledge of Pteropus populations in terms of their relative resistance and resilience to emerging infectious disease.

Routes of Hendra Virus Excretion in Naturally-Infected Flying-Foxes: Implications for Viral Transmission and Spillover Risk.

Pteropid bats or flying-foxes (Chiroptera: Pteropodidae) are the natural host of Hendra virus (HeV) which sporadically causes fatal disease in horses and humans in eastern Australia. While there is strong evidence that urine is an important infectious medium that likely drives bat to bat transmission and bat to horse transmission, there is uncertainty about the relative importance of alternative routes of excretion such as nasal and oral secretions, and faeces. Identifying the potential routes of HeV excretion in flying-foxes is important to effectively mitigate equine exposure risk at the bat-horse interface, and in determining transmission rates in host-pathogen models. The aim of this study was to identify the major routes of HeV excretion in naturally infected flying-foxes, and secondarily, to identify between-species variation in excretion prevalence. A total of 2840 flying-foxes from three of the four Australian mainland species (Pteropus alecto, P. poliocephalus and P. scapulatus) were captured and sampled at multiple roost locations in the eastern states of Queensland and New South Wales between 2012 and 2014. A range of biological samples (urine and serum, and urogenital, nasal, oral and rectal swabs) were collected from anaesthetized bats, and tested for HeV RNA using a qRT-PCR assay targeting the M gene. Forty-two P. alecto (n = 1410) had HeV RNA detected in at least one sample, and yielded a total of 78 positive samples, at an overall detection rate of 1.76% across all samples tested in this species (78/4436). The rate of detection, and the amount of viral RNA, was highest in urine samples (>serum, packed haemocytes >faecal >nasal >oral), identifying urine as the most plausible source of infection for flying-foxes and for horses. Detection in a urine sample was more efficient than detection in urogenital swabs, identifying the former as the preferred diagnostic sample. The detection of HeV RNA in serum is consistent with haematogenous spread, and with hypothesised latency and recrudesence in flying-foxes. There were no detections in P. poliocephalus (n = 1168 animals; n = 2958 samples) or P. scapulatus (n = 262 animals; n = 985 samples), suggesting (consistent with other recent studies) that these species are epidemiologically less important than P. alecto in HeV infection dynamics. The study is unprecedented in terms of the individual animal approach, the large sample size, and the use of a molecular assay to directly determine infection status. These features provide a high level of confidence in the veracity of our findings, and a sound basis from which to more precisely target equine risk mitigation strategies.

Natural Hendra Virus Infection in Flying-Foxes - Tissue Tropism and Risk Factors.

Hendra virus (HeV) is a lethal zoonotic agent that emerged in 1994 in Australia. Pteropid bats (flying-foxes) are the natural reservoir. To date, HeV has spilled over from flying-foxes to horses on 51 known occasions, and from infected horses to close-contact humans on seven occasions. We undertook screening of archived bat tissues for HeV by reverse transcription quantitative polymerase chain reaction (RT-qPCR). Tissues were tested from 310 bats including 295 Pteropodiformes and 15 Vespertilioniformes. HeV was detected in 20 individual flying-foxes (6.4%) from various tissues including spleen, kidney, liver, lung, placenta and blood components. Detection was significantly higher in Pteropus Alecto and P. conspicillatus, identifying species as a risk factor for infection. Further, our findings indicate that HeV has a predilection for the spleen, suggesting this organ plays an important role in HeV infection. The lack of detections in the foetal tissues of HeV-positive females suggests that vertical transmission is not a regular mode of transmission in naturally infected flying-foxes, and that placental and foetal tissues are not a major source of infection for horses. A better understanding of HeV tissue tropism will strengthen management of the risk of spillover from flying-foxes to horses and ultimately humans.

Novel paramyxoviruses in Australian flying-fox populations support host-virus co-evolution.

Understanding the diversity of henipaviruses and related viruses is important in determining the viral ecology within flying-fox populations and assessing the potential threat posed by these agents. This study sought to identify the abundance and diversity of previously unknown paramyxoviruses (UPVs) in Australian flying-fox species (Pteropus alecto, Pteropus scapulatus, Pteropus poliocephalus and Pteropus conspicillatus) and in the Christmas Island species Pteropus melanotus natalis. Using a degenerative reverse transcription-PCR specific for the L gene of known species of the genus Henipavirus and two closely related paramyxovirus genera Respirovirus and Morbillivirus, we identified an abundance and diversity of previously UPVs, with a representative 31 UPVs clustering in eight distinct groups (100 UPVs/495 samples). No new henipaviruses were identified. The findings were consistent with a hypothesis of co-evolution of paramyxoviruses and their flying-fox hosts. Quantification of the degree of co-speciation between host and virus (beyond the scope of this study) would strengthen this hypothesis.

Discovering the recondite secondary metabolome spectrum of Salinispora species: a study of inter-species diversity.

Patterns of inter-species secondary metabolite production by bacteria can provide valuable information relating to species ecology and evolution. The complex nature of this chemical diversity has previously been probed via directed analyses of a small number of compounds, identified through targeted assays rather than more comprehensive biochemical profiling approaches such as metabolomics. Insights into ecological and evolutionary relationships within bacterial genera can be derived through comparative analysis of broader secondary metabolite patterns, and this can also eventually assist biodiscovery search strategies for new natural products. Here, we investigated the species-level chemical diversity of the two marine actinobacterial species Salinispora arenicola and Salinispora pacifica, isolated from sponges distributed across the Great Barrier Reef (GBR), via their secondary metabolite profiles using LC-MS-based metabolomics. The chemical profiles of these two species were obtained by UHPLC-QToF-MS based metabolic profiling. The resultant data were interrogated using multivariate data analysis methods to compare their (bio)chemical profiles. We found a high level of inter-species diversity in strains from these two bacterial species. We also found rifamycins and saliniketals were produced exclusively by S. arenicola species, as the main secondary metabolites differentiating the two species. Furthermore, the discovery of 57 candidate compounds greatly increases the small number of secondary metabolites previously known to be produced by these species. In addition, we report the production of rifamycin O and W, a key group of ansamycin compounds, in S. arenicola for the first time. Species of the marine actinobacteria harbour a much wider spectrum of secondary metabolites than suspected, and this knowledge may prove a rich field for biodiscovery as well as a database for understanding relationships between speciation, evolution and chemical ecology.