Rapid, in-field deployable, avian influenza virus haemagglutinin characterisation tool using MinION technology.

Outbreaks of avian influenza virus (AIV) from wild waterfowl into the poultry industry is of upmost significance and is an ongoing and constant threat to the industry. Accurate surveillance of AIV in wild waterfowl is critical in understanding viral diversity in the natural reservoir. Current surveillance methods for AIV involve collection of samples and transportation to a laboratory for molecular diagnostics. Processing of samples using this approach takes more than three days and may limit testing locations to those with practical access to laboratories. In potential outbreak situations, response times are critical, and delays have implications in terms of the spread of the virus that leads to increased economic cost. This study used nanopore sequencing technology for in-field sequencing and subtype characterisation of AIV strains collected from wild bird faeces and poultry. A custom in-field virus screening and sequencing protocol, including a targeted offline bioinformatic pipeline, was developed to accurately subtype AIV. Due to the lack of optimal diagnostic MinION packages for Australian AIV strains the bioinformatic pipeline was specifically targeted to confidently subtype local strains. The method presented eliminates the transportation of samples, dependence on internet access and delivers critical diagnostic information in a timely manner.

Australia as a global sink for the genetic diversity of avian influenza A virus.

Most of our understanding of the ecology and evolution of avian influenza A virus (AIV) in wild birds is derived from studies conducted in the northern hemisphere on waterfowl, with a substantial bias towards dabbling ducks. However, relevant environmental conditions and patterns of avian migration and reproduction are substantially different in the southern hemisphere. Through the sequencing and analysis of 333 unique AIV genomes collected from wild birds collected over 15 years we show that Australia is a global sink for AIV diversity and not integrally linked with the Eurasian gene pool. Rather, AIV are infrequently introduced to Australia, followed by decades of isolated circulation and eventual extinction. The number of co-circulating viral lineages varies per subtype. AIV haemagglutinin (HA) subtypes that are rarely identified at duck-centric study sites (H8-12) had more detected introductions and contemporary co-circulating lineages in Australia. Combined with a lack of duck migration beyond the Australian-Papuan region, these findings suggest introductions by long-distance migratory shorebirds. In addition, on the available data we found no evidence of directional or consistent patterns in virus movement across the Australian continent. This feature corresponds to patterns of bird movement, whereby waterfowl have nomadic and erratic rainfall-dependant distributions rather than consistent intra-continental migratory routes. Finally, we detected high levels of virus gene segment reassortment, with a high diversity of AIV genome constellations across years and locations. These data, in addition to those from other studies in Africa and South America, clearly show that patterns of AIV dynamics in the Southern Hemisphere are distinct from those in the temperate north.

In Vitro and In Vivo Characterization of a Pigeon Paramyxovirus Type 1 Isolated from Domestic Pigeons in Victoria, Australia 2011.

Significant mortalities of racing pigeons occurred in Australia in late 2011 associated with a pigeon paramyxovirus serotype 1 (PPMV-1) infection. The causative agent, designated APMV-1/pigeon/Australia/3/2011 (P/Aus/3/11), was isolated from diagnostic specimens in specific pathogen free (SPF) embryonated eggs and was identified by a Newcastle Disease virus (NDV)-specific RT-PCR and haemagglutination inhibition (HI) test using reference polyclonal antiserum specific for NDV. The P/Aus/3/11 strain was further classified as PPMV-1 using the HI test and monoclonal antibody 617/161 by HI and phylogenetic analysis of the fusion gene sequence. The isolate P/Aus/3/11 had a slow haemagglutin-elution rate and was inactivated within 45 min at 56 °C. Cross HI tests generated an R value of 0.25, indicating a significant antigenic difference between P/Aus/3/11 and NDV V4 isolates. The mean death time (MDT) of SPF eggs infected with the P/Aus/3/11 isolate was 89.2 hr, characteristic of a mesogenic pathotype, consistent with other PPMV-1 strains. The plaque size of the P/Aus/3/11 isolate on chicken embryo fibroblast (CEF) cells was smaller than those of mesogenic and velogenic NDV reference strains, indicating a lower virulence phenotype in vitro and challenge of six-week-old SPF chickens did not induce clinical signs. However, sequence analysis of the fusion protein cleavage site demonstrated an 112RRQKRF117 motif, which is typical of a velogenic NDV pathotype. Phylogenetic analysis indicated that the P/Aus/3/11 isolate belongs to a distinct subgenotype within class II genotype VI of avian paramyxovirus type 1. This is the first time this genotype has been detected in Australia causing disease in domestic pigeons and is the first time since 2002 that an NDV with potential for virulence has been detected in Australia.

Field Verification of an African Swine Fever Virus Loop-Mediated Isothermal Amplification (LAMP) Assay During an Outbreak in Timor-Leste.

Recent outbreaks of African swine fever virus (ASFV) have seen the movement of this virus into multiple new regions with devastating impact. Many of these outbreaks are occurring in remote, or resource-limited areas, that do not have access to molecular laboratories. Loop-mediated isothermal amplification (LAMP) is a rapid point of care test that can overcome a range of inhibitors. We outline further development of a real-time ASFV LAMP, including field verification during an outbreak in Timor-Leste. To increase field applicability, the extraction step was removed and an internal amplification control (IAC) was implemented. Assay performance was assessed in six different sample matrices and verified for a range of clinical samples. A LAMP detection limit of 400 copies/rxn was determined based on synthetic positive control spikes. A colourmetric LAMP assay was also assessed on serum samples. Comparison of the LAMP assay to a quantitative polymerase chain reaction (qPCR) was performed on clinical ASFV samples, using both serum and oral/rectal swabs, with a substantial level of agreement observed. The further verification of the ASFV LAMP assay, removal of extraction step, implementation of an IAC and the assessment of a range of sample matrix, further support the use of this assay for rapid in-field detection of ASFV.

Avian influenza infection dynamics under variable climatic conditions, viral prevalence is rainfall driven in waterfowl from temperate, south-east Australia.

Understanding Avian Influenza Virus (AIV) infection dynamics in wildlife is crucial because of possible virus spill over to livestock and humans. Studies from the northern hemisphere have suggested several ecological and environmental drivers of AIV prevalence in wild birds. To determine if the same drivers apply in the southern hemisphere, where more irregular environmental conditions prevail, we investigated AIV prevalence in ducks in relation to biotic and abiotic factors in south-eastern Australia. We sampled duck faeces for AIV and tested for an effect of bird numbers, rainfall anomaly, temperature anomaly and long-term ENSO (El-Niño Southern Oscillation) patterns on AIV prevalence. We demonstrate a positive long term effect of ENSO-related rainfall on AIV prevalence. We also found a more immediate response to rainfall where AIV prevalence was positively related to rainfall in the preceding 3-7 months. Additionally, for one duck species we found a positive relationship between their numbers and AIV prevalence, while prevalence was negatively or not affected by duck numbers in the remaining four species studied. In Australia largely non-seasonal rainfall patterns determine breeding opportunities and thereby influence bird numbers. Based on our findings we suggest that rainfall influences age structures within populations, producing an influx of immunologically naïve juveniles within the population, which may subsequently affect AIV infection dynamics. Our study suggests that drivers of AIV dynamics in the northern hemisphere do not have the same influence at our south-east Australian field site in the southern hemisphere due to more erratic climatological conditions.

Does online learning click with rural nurses? A qualitative study.

OBJECTIVE: To explore the factors that influence rural nurses engagement with online learning within a rural health district in New Sound Wales (NSW), Australia. DESIGN: This qualitative study based on appreciative inquiry methodology used semi-structured interviews with managers and nurses. Purposive sampling methods were used to recruit facility managers, whereas convenience sampling was used to recruit nurses in 2012-2013. SETTING: Three public health facilities in rural NSW. PARTICIPANTS: Fourteen nurses were involved in the study, including Health Service Managers (n = 3), Nurse Unit Manager (n = 1), Clinical Nurse Specialists (n = 3), Registered Nurses (n = 2), Enrolled Nurses (n = 2) and Assistant in Nursing (n = 3). MAIN OUTCOME MEASURE: The research found that online learning works well when there is accountability for education being undertaken by linking to organisational goals and protected time. Nurses in this study valued the ability to access and revisit online learning at any time. However, systems that are hard to access or navigate and module design that did not provide a mechanism for users to seek feedback negatively affected their use and engagement. CONCLUSION: This study demonstrates that rural nurses' engagement with online learning would be enhanced by a whole of system redesign in order to deliver a learning environment that will increase satisfaction, engagement and learning outcomes.

The recent establishment of North American H10 lineage influenza viruses in Australian wild waterfowl and the evolution of Australian avian influenza viruses.

Influenza A H10N7 virus with a hemagglutinin gene of North American origin was detected in Australian chickens and poultry abattoir workers in New South Wales, Australia, in 2010 and in chickens in Queensland, Australia, on a mixed chicken and domestic duck farm in 2012. We investigated their genomic origins by sequencing full and partial genomes of H10 viruses isolated from wild aquatic birds and poultry in Australia and analyzed them with all available avian influenza virus sequences from Oceania and representative viruses from North America and Eurasia. Our analysis showed that the H10N7 viruses isolated from poultry were similar to those that have been circulating since 2009 in Australian aquatic birds and that their initial transmission into Australia occurred during 2007 and 2008. The H10 viruses that appear to have developed endemicity in Australian wild aquatic birds were derived from several viruses circulating in waterfowl along various flyways. Their hemagglutinin gene was derived from aquatic birds in the western states of the United States, whereas the neuraminidase was closely related to that from viruses previously detected in waterfowl in Japan. The remaining genes were derived from Eurasian avian influenza virus lineages. Our analysis of virological data spanning 40 years in Oceania indicates that the long-term evolutionary dynamics of avian influenza viruses in Australia may be determined by climatic changes. The introduction and long-term persistence of avian influenza virus lineages were observed during periods with increased rainfall, whereas bottlenecks and extinction were observed during phases of widespread decreases in rainfall. These results extend our understanding of factors affecting the dynamics of avian influenza and provide important considerations for surveillance and disease control strategies.

Molecular characterization and phylogenetic analysis of Murray Valley encephalitis virus and West Nile virus (Kunjin subtype) from an arbovirus disease outbreak in horses in Victoria, Australia, in 2011.

Virus was detected in the central nervous system (CNS) tissue of 11 horses from Victoria that died displaying neurological symptoms during an outbreak of disease in Australia in 2011. Five horses were identified as being infected with Murray Valley encephalitis virus (MVEV) and 6 as being infected with West Nile virus subtype Kunjin (WNV(KUN)). Analysis of partial sequence information from the NS5 and E genes indicated that the MVEVs within the samples were highly homogenous and all belonged to lineage I, which is enzootic to the tropical regions of northern Australia. Likewise, analysis of partial NS5 and E gene and full genome sequences indicated that the WNV(KUN) within the samples were also highly homogenous and clustered with WNV lineage 1, clade b, which is consistent with other WNV(KUN) isolates. Full genomes of 1 MVEV isolate and 2 WNV(KUN) isolates were sequenced and characterized. The genome sequences of Victorian WNV(KUN) are almost identical (3 amino acid differences) to that of the recently sequenced WNV isolate WNV(NSW2011). Metagenome sequencing directly from CNS tissue identified the presence of WNV(KUN) and MVEV within infected CNS tissue.

Surveillance and analysis of avian influenza viruses, Australia.

We investigated carriage of avian influenza viruses by wild birds in Australia, 2005-2008, to assess the risks to poultry industries and human health. We collected 21,858 (7,357 cloacal, 14,501 fecal) samples and detected 300 viruses, representing a detection rate of ≈1.4%. Rates were highest in autumn (March-May) and differed substantially between bird types, areas, and years. We typed 107 avian influenza viruses and identified 19 H5, 8 H7, and 16 H9 (40% of typed viruses). All were of low pathogenicity. These viruses formed clearly different phylogenetic clades to lineages from Eurasia or North America, suggesting the potential existence of Australian lineages. H7 viruses were similar to highly pathogenic H7 strains that caused outbreaks in poultry in Australia. Several periods of increased detection rates (numbers or subtypes of viruses) were identified. This study demonstrates the need for ongoing surveillance to detect emerging pathogenic strains and facilitate prevention of outbreaks.

Care management for depression in primary care practice: findings from the RESPECT-Depression trial.

PURPOSE: This qualitative study examined the barriers to adopting depression care management among 42 primary care clinicians in 30 practices. METHODS: The RESPECT-Depression trial worked collaboratively with 5 large health care organizations (and 60 primary care practices) to implement and disseminate an evidence-based intervention. This study used semistructured interviews with 42 primary care clinicians from 30 practice sites, 18 care managers, and 7 mental health professionals to explore experience and perceptions with depression care management for patients. Subject selection in 4 waves of interviews was driven by themes emerging from ongoing data analysis. RESULTS: Primary care clinicians reported broad appreciation of the benefits of depression care management for their patients. Lack of reimbursement and the competing demands of primary care were often cited as barriers. These clinicians at many levels of initial enthusiasm for care management increased their enthusiasm after experiencing care management through the project. Psychiatric oversight of the care manager with suggestions for the clinicians was widely seen as important and appropriate by clinicians, care managers, and psychiatrists. Clinicians and care managers emphasized the importance of establishing effective communication among themselves, as well as maintaining a consistent and continuous relationship with the patients. The clinicians were selective in which patients they referred for care management, and there was wide variation in opinion about which patients were optimal candidates. Care managers were able to operate both from within a practice and more centrally when specific attention was given to negotiating communication strategies with a clinician. CONCLUSIONS: Care management for depression is an attractive option for most primary care clinicians. Lack of reimbursement remains the single greatest obstacle to more widespread adoption.

Recruitment and retention of Latinos in a primary care-based physical activity and diet trial: The Resources for Health study.

Mandates to include women and minority populations in research have heightened the need to identify successful recruitment strategies. This paper describes the recruitment and retention strategies used as part of a randomized controlled trial (RCT) of a physical activity and dietary intervention targeting low-income, predominantly Latino patients in a primary health care clinic. Data on the recruitment and retention rates and the representativeness of participants are presented. Strategies included hiring bilingual staff, translating and culturally adapting intervention materials and soliciting ongoing consultation from patients, clinicians and organizations providing services to the Latino community. The primary recruitment procedure involved letters from physicians followed by phone calls from project staff to patients identified from electronic medical records. Two hundred patients were recruited into the RCT (78% of those reached and eligible), with 69 and 81% reached for the 6-week and 6-month follow-ups, respectively. Women were more likely to both participate and remain in the trial, and there was a trend toward greater recruitment and retention of Spanish speakers. By engaging the community, clinicians and patients, this study was able to successfully recruit and retain a large proportion of this often difficult to reach Latino sub-population of patients with multiple chronic conditions.

Implementing a depression improvement intervention in five health care organizations: experience from the RESPECT-Depression trial.

A growing body of research provides strong evidence for the effectiveness of programs to improve the primary care of depression based on the chronic care model. At the same time these changes are difficult to sustain in their original research settings and more difficult to widely disseminate in primary care practice. The RESPECT-Depression trial tested an implementation and dissemination strategy by working through five community-based health care organizations (HCOs) to implement the Three Component Model (TCM) for improving depression care. This report describes the results of extensive interviews of project principals, health care program managers, depression care managers, and practicing primary care clinicians to understand the characteristics of organizations and the intervention components that were associated with implementation and dissemination of the TCM. In two of the organizations all 29 participating practices continued the TCM, while all 31 practices from the other three organizations did not. Successful continuation and dissemination appeared to be related to a broadly shared vision and commitment among all levels of the organization, clearly articulated by clinical leadership, for pursuing a systematic change strategy to improve chronic care that included, but extended beyond, depression, independent of clear evidence for cost-effectiveness of expanding depression management. Factors associated with inability to sustain the TCM included lack of a shared change strategy throughout the organization and inability to rationalize an economic model of depression care.