"Smart markets": harnessing the potential of new technologies for endemic and emerging infectious disease surveillance in traditional food markets.

Emerging and endemic zoonotic diseases continue to threaten human and animal health, our social fabric, and the global economy. Zoonoses frequently emerge from congregate interfaces where multiple animal species and humans coexist, including farms and markets. Traditional food markets are widespread across the globe and create an interface where domestic and wild animals interact among themselves and with humans, increasing the risk of pathogen spillover. Despite decades of evidence linking markets to disease outbreaks across the world, there remains a striking lack of pathogen surveillance programs that can relay timely, cost-effective, and actionable information to decision-makers to protect human and animal health. However, the strategic incorporation of environmental surveillance systems in markets coupled with novel pathogen detection strategies can create an early warning system capable of alerting us to the risk of outbreaks before they happen. Here, we explore the concept of "smart" markets that utilize continuous surveillance systems to monitor the emergence of zoonotic pathogens with spillover potential.IMPORTANCEFast detection and rapid intervention are crucial to mitigate risks of pathogen emergence, spillover and spread-every second counts. However, comprehensive, active, longitudinal surveillance systems at high-risk interfaces that provide real-time data for action remain lacking. This paper proposes "smart market" systems harnessing cutting-edge tools and a range of sampling techniques, including wastewater and air collection, multiplex assays, and metagenomic sequencing. Coupled with robust response pathways, these systems could better enable Early Warning and bolster prevention efforts.

"Hospitals respond to demand. Public health needs to respond to risk": health system lessons from a case study of northern Queensland's COVID-19 surveillance and response.

BACKGROUND: The vast region of northern Queensland (NQ) in Australia experiences poorer health outcomes and a disproportionate burden of communicable diseases compared with urban populations in Australia. This study examined the governance of COVID-19 surveillance and response in NQ to identify strengths and opportunities for improvement. METHODS: The manuscript presents an analysis of one case-unit within a broader case study project examining systems for surveillance and response for COVID-19 in NQ. Data were collected between October 2020-December 2021 comprising 47 interviews with clinical and public health staff, document review, and observation in organisational settings. Thematic analysis produced five key themes. RESULTS: Study findings highlight key strengths of the COVID-19 response, including rapid implementation of response measures, and the relative autonomy of NQ's Public Health Units to lead logistical decision-making. However, findings also highlight limitations and fragility of the public health system more generally, including unclear accountabilities, constraints on local community engagement, and workforce and other resourcing shortfalls. These were framed by state-wide regulatory and organisational incentives that prioritise clinical health care rather than disease prevention, health protection, and health promotion. Although NQ mobilised an effective COVID-19 response, findings suggest that NQ public health systems are marked by fragility, calling into question the region's preparedness for future pandemic events and other public health crises. CONCLUSIONS: Study findings highlight an urgent need to improve governance, resourcing, and political priority of public health in NQ to address unmet needs and ongoing threats.

Metagenomic and Molecular Detection of Novel Fecal Viruses in Free-Ranging Agile Wallabies.

The agile wallaby (Notamacropus agilis) is one of the most abundant marsupial species in northern Queensland and a competent host for the zoonotic Ross River virus. Despite their increased proximity and interactions with humans, little is known about the viruses carried by these animals, and whether any are of conservation or zoonotic importance. Metagenomics and molecular techniques were used in a complementary manner to identify and characterize novel viruses in the fecal samples of free-ranging agile wallabies. We detected a variety of novel marsupial-related viral species including agile wallaby atadenovirus 1, agile wallaby chaphamaparvovirus 1-2, agile wallaby polyomavirus 1-2, agile wallaby associated picobirnavirus 1-9, and a known macropod gammaherpesvirus 3. Phylogenetic analyses indicate that most of these novel viruses would have co-evolved with their hosts (agile wallabies). Additionally, non-marsupial viruses that infect bacteria (phages), plants, insects, and other eukaryotes were identified. This study highlighted the utility of non-invasive sampling as well as the integration of broad-based molecular assays (consensus PCR and next generation sequencing) for monitoring the emergence of potential pathogenic viruses in wildlife species. Furthermore, the novel marsupial viruses identified in this study will enrich the diversity of knowledge about marsupial viruses, and may be useful for developing diagnostics and vaccines.

Longitudinal comparison of bacterial pathogen seropositivity among wet market vendors in the Lao People's Democratic Republic.

Wild animal trade for human consumption is a global issue, involving complex interactions between economics, culture, food security and conservation. Whilst being a biodiversity issue, it is also a major public health concern, with recent epidemics and pandemics of zoonotic pathogens linked to interactions with wildlife. At three time points, between March 2017 and June 2018, a longitudinal sero-survey of 150 market vendors from three wet markets in Laos (selling vegetables, domestic animal meat and/or wildlife meat) was conducted to determine if vendors had been differentially exposed to three endemic bacterial pathogens - Orientia tsutsugamushi, Rickettsia typhi, and Leptospira spp. A total of 367 serum samples were tested by IgG enzyme-linked immunosorbent assay (ELISA) and immunofluorescence assay (IFA, for scrub typhus group (STG) and typhus group (TG) only). Among vendors, 32.7% were IgG-positive for at least one pathogen, 13.3% sero-converted during the study. Multi-season occupancy modelling for STG indicated a significantly higher prevalence of STG IgG in vegetable vendors (27.3%) and wildlife vendors (28.4%) than in domestic animal meat vendors (6.9 %, p=0.05), and higher in Phonsavanh market (OR=9.6, p=0.03) compared to Lak Sao and Salavan markets. Estimated mean incidence was 57 cases per 10,000 per 7.5-month period. For TG, vendor age had a significant effect on prevalence (OR=1.04, p=0.006), estimated mean incidence was 64 cases per 10,000 per season (7.5-month period). Despite individuals selling domestic meat having a higher prevalence of Leptospira infections than those that did not (11.6% versus 4.5%), the difference was not significant. Whilst this study has a number of limitations, including vendors changing what food types they sold and no investigation of exposure outside of markets, the finding that the risk of exposure of vendors to zoonotic pathogens may be associated with types of food sold for human consumption warrants further investigation.

Draft Genome Sequence of a Novel Adenovirus Recovered from the Metagenome of Agile Wallabies.

Here, we report the draft genome sequence of a novel agile wallaby adenovirus that was detected in the fecal metagenome of agile wallabies. The genome is 31,512 bp long, with a G+C content of 34.4%. Currently, the pathogenic and zoonotic potential of this novel virus is unknown.

Development of subfamily-based consensus PCR assays for the detection of human and animal herpesviruses.

Consensus PCR assays that can be used to sensitively detect several herpesvirus (HV) species across the different subfamilies were developed in this study. Primers containing degenerate bases were designed to amplify regions of the DNA polymerase (DPOL) gene of alpha- and gamma-HVs, and the glycoprotein B (gB) gene of beta-HVs in a singleplex, non-nested touchdown PCR format. The singleplex touchdown consensus PCR (STC-PCR) was used to amplify the DNA of eight human and 24 animal HVs. The assay was able to detect the lowest DNA dilution of 10-5 for alpha-HVs and 10-3 for beta- and gamma-HVs. In comparison, lowest detection limits of 10-5, 10-3, and 10-2 were obtained for alpha-, beta-, and gamma-HVs respectively when a nested PCR was used. The findings in this study suggest that the STC-PCR assays can be employed for the molecular surveys and clinical detection of novel and known HVs.

Japanese Encephalitis Enzootic and Epidemic Risks across Australia.

Japanese encephalitis virus (JEV) is an arboviral, encephalitogenic, zoonotic flavivirus characterized by its complex epidemiology whose transmission cycle involves reservoir and amplifying hosts, competent vector species and optimal environmental conditions. Although typically endemic in Asia and parts of the Pacific Islands, unprecedented outbreaks in both humans and domestic pigs in southeastern Australia emphasize the virus' expanding geographical range. To estimate areas at highest risk of JEV transmission in Australia, ecological niche models of vectors and waterbirds, a sample of piggery coordinates and feral pig population density models were combined using mathematical and geospatial mapping techniques. These results highlight that both coastal and inland regions across the continent are estimated to have varying risks of enzootic and/or epidemic JEV transmission. We recommend increased surveillance of waterbirds, feral pigs and mosquito populations in areas where domestic pigs and human populations are present.

Analytical sensitivity of COVID-19 rapid antigen tests: A case for a robust reference standard.

Aggressive diagnostic testing remains an indispensable strategy for health and aged care facilities to prevent the transmission of SARS-CoV-2 in vulnerable populations. The preferred diagnostic platform has shifted towards COVID-19 rapid antigen tests (RATs) to identify the most infectious individuals. As such, RATs are being manufactured faster than at any other time in our history yet lack the relevant quantitative analytics required to inform on absolute analytical sensitivity enabling manufacturers to maintain high batch-to-batch reproducibility, and end-users to accurately compare brands for decision making. Here, we describe a novel reference standard to measure and compare the analytical sensitivity of RATs using a recombinant GFP-tagged nucleocapsid protein (NP-GFP). Importantly, we show that the GFP tag does not interfere with NP detection and provides several advantages affording streamlined protein expression and purification in high yields as well as faster, cheaper and more sensitive quality control measures for post-production assessment of protein solubility and stability. Ten commercial COVID-19 RATs were evaluated and ranked using NP-GFP as a reference standard. Analytical sensitivity data of the selected devices as determined with NP-GFP did not correlate with those reported by the manufacturers using the median tissue culture infectious dose (TCID50) assay. Of note, TCID50 discordance has been previously reported. Taken together, our results highlight an urgent need for a reliable reference standard for evaluation and benchmarking of the analytical sensitivity of RAT devices. NP-GFP is a promising candidate as a reference standard that will ensure that RAT performance is accurately communicated to healthcare providers and the public.

Aerosol exposure of live bird market workers to viable influenza A/H5N1 and A/H9N2 viruses, Cambodia.

Live bird markets (LBMs) have been identified as key factors in the spread, persistence and evolution of avian influenza viruses (AIVs). In addition, these settings have been associated with human infections with AIVs of pandemic concern. Exposure to aerosolised AIVs by workers in a Cambodian LBM was assessed using aerosol impact samplers. LBM vendors were asked to wear an air sampler for 30 min per day for 1 week while continuing their usual activities in the LBM during a period of high AIV circulation (February) and a period of low circulation (May). During the period of high circulation, AIV RNA was detected from 100% of the air samplers using molecular methods and viable AIV (A/H5N1 and/or A/H9N2) was isolated from 50% of air samplers following inoculation into embryonated chicken eggs. In contrast, AIV was not detected by molecular methods or successfully isolated during the period of low circulation. This study demonstrates the increased risk of aerosol exposure of LBM workers to AIVs during periods of high circulation and highlights the need for interventions during these high-risk periods. Novel approaches, such as environmental sampling, should be further explored at key high-risk interfaces as a potentially cost-effective alternative for monitoring pandemic threats.

Diversity of Leptospira spp. in bats and rodents from Papua New Guinea.

Leptospirosis is the most common bacterial zoonosis globally. The pathogen, Leptospira spp., is primarily associated with rodent reservoirs. However, a wide range of other species has been implicated as reservoirs or dead-end hosts. We conducted a survey for Leptospira spp. in bats and rodents from Papua New Guinea. Kidney samples were collected from 97 pteropodid bats (five species), 37 insectivorous bats from four different families (six species) and 188 rodents (two species). Leptospires were detected in a high proportion of pteropodid bats, including Nyctimene cf. albiventer (35%), Macroglossus minimus (34%) and Rousettus amplexicaudatus (36%). Partial sequencing of the secY gene from rodent and bat leptospires showed host species clustering, with Leptospira interrogans and L. weilii detected in rodents and L. kirschneri and a potential novel species of Leptospira detected in bats. Further research is needed in Papua New Guinea and other locales in the Pacific region to gain a better understanding of the circulation dynamics of leptospires in reservoir species and the risks to public and veterinary health.

Genomic Sequencing of Dengue Virus Strains Associated with Papua New Guinean Outbreaks in 2016 Reveals Endemic Circulation of DENV-1 and DENV-2.

Over the past decade, the Pacific region has experienced many arboviral outbreaks, including dengue, chikungunya, and Zika viruses. Papua New Guinea (PNG) has a high burden of arboviral diseases, but there is a paucity of knowledge about the epidemiology and circulation of these viruses in the country. In this study, we report investigations into suspected arboviral outbreaks of febrile disease in PNG from December 2015 to June 2017. DENV-1 and DENV-2 were the mostly commonly detected viruses, and low circulation of DENV-3 and ZIKV was also detected. DENV-4 and CHIKV were not detected during this period. Full genome sequencing of selected positive samples revealed that circulation was dominated by endemic indigenous strains belonging to DENV-1 (genotype IV) and DENV-2 (genotype C) that have been present in the country for up to a decade. A DENV-2 sublineage was also identified that has been associated with outbreaks of severe dengue in both PNG and the Solomon Islands.

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.

Zoonotic Pathogens in Wildlife Traded in Markets for Human Consumption, Laos.

We tested animals from wildlife trade sites in Laos for the presence of zoonotic pathogens. Leptospira spp. were the most frequently detected infectious agents, found in 20.1% of animals. Rickettsia typhi and R. felis were also detected. These findings suggest a substantial risk for exposure through handling and consumption of wild animal meat.

Whole genome sequence analysis of Salmonella Typhi in Papua New Guinea reveals an established population of genotype 2.1.7 sensitive to antimicrobials.

BACKGROUND: Typhoid fever, a systemic infection caused by Salmonella enterica serovar Typhi, remains a considerable public health threat in impoverished regions within many low- and middle-income settings. However, we still lack a detailed understanding of the emergence, population structure, molecular mechanisms of antimicrobial resistance (AMR), and transmission dynamics of S. Typhi across many settings, particularly throughout the Asia-Pacific islands. Here we present a comprehensive whole genome sequence (WGS) based overview of S. Typhi populations circulating in Papua New Guinea (PNG) over 30 years. PRINCIPLE FINDINGS: Bioinformatic analysis of 86 S. Typhi isolates collected between 1980-2010 demonstrated that the population structure of PNG is dominated by a single genotype (2.1.7) that appears to have emerged in the Indonesian archipelago in the mid-twentieth century with minimal evidence of inter-country transmission. Genotypic and phenotypic data demonstrated that the PNG S. Typhi population appears to be susceptible to former first line drugs for treating typhoid fever (chloramphenicol, ampicillin and co-trimoxazole), as well as fluoroquinolones, third generation cephalosporins, and macrolides. PNG genotype 2.1.7 was genetically conserved, with very few deletions, and no evidence of plasmid or prophage acquisition. Genetic variation among this population was attributed to either single point mutations, or homologous recombination adjacent to repetitive ribosomal RNA operons. SIGNIFICANCE: Antimicrobials remain an effective option for the treatment of typhoid fever in PNG, along with other intervention strategies including improvements to water, sanitation and hygiene (WaSH) related infrastructure and potentially the introduction of Vi-conjugate vaccines. However, continued genomic surveillance is warranted to monitor for the emergence of AMR within local populations, or the introduction of AMR associated genotypes of S. Typhi in this setting.

Seroprevalence of dengue, Zika, chikungunya and Ross River viruses across the Solomon Islands.

Across the Pacific, and including in the Solomon Islands, outbreaks of arboviruses such as dengue, chikungunya, and Zika are increasing in frequency, scale and impact. Outbreaks of mosquito-borne disease have the potential to overwhelm the health systems of small island nations. This study mapped the seroprevalence of dengue, Zika, chikungunya and Ross River viruses in 5 study sites in the Solomon Islands. Serum samples from 1,021 participants were analysed by ELISA. Overall, 56% of participants were flavivirus-seropositive for dengue (28%), Zika (1%) or both flaviviruses (27%); and 53% of participants were alphavirus-seropositive for chikungunya (3%), Ross River virus (31%) or both alphaviruses (18%). Seroprevalence for both flaviviruses and alphaviruses varied by village and age of the participant. The most prevalent arboviruses in the Solomon Islands were dengue and Ross River virus. The high seroprevalence of dengue suggests that herd immunity may be a driver of dengue outbreak dynamics in the Solomon Islands. Despite being undetected prior to this survey, serology results suggest that Ross River virus transmission is endemic. There is a real need to increase the diagnostic capacities for each of the arboviruses to support effective case management and to provide timely information to inform vector control efforts and other outbreak mitigation interventions.

Arboviral Disease Outbreaks in the Pacific Islands Countries and Areas, 2014 to 2020: A Systematic Literature and Document Review.

Arthropod-borne diseases pose a significant public health threat, accounting for greater than 17% of infectious disease cases and 1 million deaths annually. Across Pacific Island countries and areas (PICs), outbreaks of dengue, chikungunya, and Zika are increasing in frequency and scale. Data about arbovirus outbreaks are incomplete, with reports sporadic, delayed, and often based solely on syndromic surveillance. We undertook a systematic review of published and grey literature and contacted relevant regional authorities to collect information about arboviral activity affecting PICs between October 2014 and June 2020. Our literature search identified 1176 unique peer-reviewed articles that were reduced to 25 relevant publications when screened. Our grey literature search identified 873 sources. Collectively, these data reported 104 unique outbreaks, including 72 dengue outbreaks affecting 19 (out of 22) PICs, 14 chikungunya outbreaks affecting 11 PICs, and 18 Zika outbreaks affecting 14 PICs. Our review is the most complete account of arboviral outbreaks to affect PICs since comparable work was published in 2014. It highlights the continued elevated level of arboviral activity across the Pacific and inconsistencies in how information about outbreaks is reported and recorded. It demonstrates the importance of a One-Health approach and the role that improved communication and reporting between different governments and sectors play in understanding the emergence, circulation, and transboundary risks posed by arboviral diseases.

The Ecology and Evolution of Japanese Encephalitis Virus.

Japanese encephalitis virus (JEV) is a mosquito-borne flavivirus mainly spread by Culex mosquitoes that currently has a geographic distribution across most of Southeast Asia and the Western Pacific. Infection with JEV can cause Japanese encephalitis (JE), a severe disease with a high mortality rate, which also results in ongoing sequalae in many survivors. The natural reservoir of JEV is ardeid wading birds, such as egrets and herons, but pigs commonly play an important role as an amplifying host during outbreaks in human populations. Other domestic animals and wildlife have been detected as hosts for JEV, but their role in the ecology and epidemiology of JEV is uncertain. Safe and effective JEV vaccines are available, but unfortunately, their use remains low in most endemic countries where they are most needed. Increased surveillance and diagnosis of JE is required as climate change and social disruption are likely to facilitate further geographical expansion of Culex vectors and JE risk areas.

Herpesviruses in Reptiles.

Since the 1970s, several species of herpesviruses have been identified and associated with significant diseases in reptiles. Earlier discoveries placed these viruses into different taxonomic groups on the basis of morphological and biological characteristics, while advancements in molecular methods have led to more recent descriptions of novel reptilian herpesviruses, as well as providing insight into the phylogenetic relationship of these viruses. Herpesvirus infections in reptiles are often characterised by non-pathognomonic signs including stomatitis, encephalitis, conjunctivitis, hepatitis and proliferative lesions. With the exception of fibropapillomatosis in marine turtles, the absence of specific clinical signs has fostered misdiagnosis and underreporting of the actual disease burden in reptilian populations and hampered potential investigations that could lead to the effective control of these diseases. In addition, complex life histories, sampling bias and poor monitoring systems have limited the assessment of the impact of herpesvirus infections in wild populations and captive collections. Here we review the current published knowledge of the taxonomy, pathogenesis, pathology and epidemiology of reptilian herpesviruses.

Gut microbiota composition in obese and non-obese adult relatives from the highlands of Papua New Guinea.

Obesity is a condition that results from an imbalance between energy intake and expenditure. Recently, obesity has been linked to differences in the composition of gut microbiota. To examine this association in Papua New Guinea (PNG) highlanders, fecal samples were collected from 18 adults; nine obese participants were paired with their non-obese relative. Amplification of the 16S rRNA gene targeting the V1-V2 region was performed on DNA extracts for each participant, with high-quality sequences selected and used for operational taxonomic unit clustering. The data showed Firmicutes and Bacteroidetes were the two dominant phyla, while at genus level Prevotella was the most dominant genus in all of the samples. Nonetheless, statistical evaluation of potential association between nutritional status and bacterial abundance at both phyla and genus levels showed no significant difference. Further studies, ideally in both rural and urban areas, are needed to evaluate the role of the gut microbiome in the occurrence of obesity in PNG and other resource-limited settings.