ProbeTools: designing hybridization probes for targeted genomic sequencing of diverse and hypervariable viral taxa

Background Sequencing viruses in many specimens is hindered by excessive background material from hosts, microbiota, and environmental organisms. Consequently, enrichment of target genomic material is necessary for practical high-throughput viral genome sequencing. Hybridization probes are widely used for enrichment in many fields, but their application to viral sequencing faces a major obstacle: it is difficult to design panels of probe oligo sequences that broadly target many viral taxa due to their rapid evolution, extensive diversity, and genetic hypervariability. To address this challenge, we created ProbeTools, a package of bioinformatic tools for generating effective viral capture panels, and for assessing coverage of target sequences by probe panel designs in silico. In this study, we validated ProbeTools by designing a panel of 3600 probes for subtyping the hypervariable haemagglutinin (HA) and neuraminidase (NA) genome segments of avian-origin influenza A viruses (AIVs). Using in silico assessment of AIV reference sequences and in vitro capture on egg-cultured viral isolates, we demonstrated effective performance by our custom AIV panel and ProbeTools’ suitability for challenging viral probe design applications. Results Based on ProbeTool’s in silico analysis, our panel provided broadly inclusive coverage of 14,772 HA and 11,967 NA reference sequences. For each reference sequence, we calculated the percentage of nucleotide positions covered by our panel in silico; 90% of HA and NA references sequences had at least 90.8 and 95.1% of their nucleotide positions covered respectively. We also observed effective in vitro capture on a representative collection of 23 egg-cultured AIVs that included isolates from wild birds, poultry, and humans and representatives from all HA and NA subtypes. Forty-two of forty-six HA and NA segments had over 98.3% of their nucleotide positions significantly enriched by our custom panel. These in vitro results were further used to validate ProbeTools’ in silico coverage assessment algorithm; 89.2% of in silico predictions were concordant with in vitro results. Conclusions ProbeTools generated an effective panel for subtyping AIVs that can be deployed for genomic surveillance, outbreak prevention, and pandemic preparedness. Effective probe design against hypervariable AIV targets also validated ProbeTools’ design and coverage assessment algorithms, demonstrating their suitability for other challenging viral capture applications.Medicine, Faculty ofNon UBCPathology and Laboratory Medicine, Department ofPopulation and Public Health (SPPH), School ofReviewedFacultyResearche

Molecular epidemiology of Giardia spp. in different hosts and watersheds

Giardia lamblia has been problematic in British Columbia (BC) since the 1980s, having been the etiological agent in 13 of the 29 documented waterborne outbreaks in the province. Despite improvements to drinking water facilities, giardiasis continues to occur at higher rates in BC compared to the rest of Canada. This study aimed to address knowledge gaps with regard to the occurrence and molecular epidemiology of Giardia isolates, to help address the higher occurrence of giardiasis in British Columbia. This study was conducted in three steps. First, tools were developed and validated to genotype G.lamblia isolates into groups/genotypes called Assemblages. These tools were first applied to a library of archived G.lamblia isolates collected from patients, animals and water sources in British Columbia. These same tools were then applied to water samples collected in the Salmon River watershed in the Township of Langley, BC, the second stage of the study. The occurrence and characteristics of isolates collected in the Salmon River were then compared to isolates collected in the Grand River watershed, an intensely developed mixed-urban watershed in Ontario, for the third stage of the study. In the first study, it was determined that 18s rRNA nested PCR with sequencing was the most appropriate molecular epidemiological tools to study G.lamblia in water supplies. Using a combination of molecular methods and USEPA Method 1623, it was determined that the majority of isolates in the Salmon River watershed were potentially infectious to humans (belonging to Assemblages A and B), with Assemblage A occurring most frequently. In contrast, Assemblage B was the most frequently detected genotype in the Grand River watershed, a more intensely developed watershed. Analysis of rainfall and sequence data suggests that G.lamblia isolates could have originated from sewage effluent or septic tanks, which was confirmed with the occurrence of Cryptosporidium hominis. Non-zoonotic G.lamblia isolates occurred more frequently in the Grand River watershed than in the Salmon River watershed but this water source still represents a threat to public health. Although challenging to incorporate molecular analyses into environmental monitoring, suggestions are made for the most effective linking of molecular analyses into Method 1623.Medicine, Faculty ofPathology and Laboratory Medicine, Department ofGraduat

Looking Upstream: Findings from Focus Groups on Public Perceptions of Source Water Quality in British Columbia, Canada.

In association with the development of new microbial tests for source water quality (SWQ), focus groups with members of the public were conducted to gain insight into their perceptions of SWQ, behaviours and contaminants they think pose the greatest threat to its quality, and what/how they want to know about SWQ. Discussions revealed a low concern about SWQ in general, and in particular about microbial contamination. Participants identified behaviours that threaten SWQ, barriers to changing behaviour and suggestions for inducing change. A strong desire was expressed for water quality information to be interpreted and communicated in terms of how SWQ may impact human health and how their actions should be altered in response to test results. The information can be used to inform communication strategies and possibly impact policies associated with water quality testing and implementation of new tests. More broadly, awareness of the public's understanding and beliefs about source water can be used in working with the public to adopt water-friendly behaviours, influence the content and methods of communicating with the public about water issues and water quality, and could contribute to the direction of future research and investment into water technologies to align with the public's priorities

Microbial water quality communication : public and practitioner insights from British Columbia, Canada

This work examines the communication interactions of water suppliers and health authorities with the general public regarding microbial source water quality for recreational and drinking water. We compare current approaches to risk communication observable in British Columbia (BC), Canada, with best practices derived from the communications literature, finding significant gaps between theory and practice. By considering public views and government practices together, we identify key disconnects, leading to the conclusion that at present, neither the public’s needs nor public health officials’ goals are being met. We find: (1) there is a general lack of awareness and poor understanding by the public of microbial threats to water and the associated health implications; (2) the public often does not know where to find water quality information; (3) public information needs are not identified or met; (4) information sharing by authorities is predominantly one-way and reactive (crisis-oriented); and (5) the effectiveness of communications is not evaluated. There is a need for both improved public understanding of water quality-related risks, and new approaches to ensure information related to water quality reaches audiences. Overall, greater attention should be given to planning and goal setting related to microbial water risk communication.Arts, Faculty ofMedicine, Faculty ofNon UBCGeography, Department ofPathology and Laboratory Medicine, Department ofPopulation and Public Health (SPPH), School ofResources, Environment and Sustainability (IRES), Institute forReviewedFacultyGraduat

Watershed communities.

<p>Watershed communities.</p

Number of focus group participants, by community and sex.

<p>Number of focus group participants, by community and sex.</p

A comparative analysis of current microbial water quality risk assessment and management practices in British Columbia and Ontario, Canada

Bacteria, protozoa and viruses are ubiquitous in aquatic environments and may pose threats to water quality for both human and ecosystem health. Microbial risk assessment and management in the water sector is a focus of governmental regulation and scientific inquiry; however, stark gaps remain in their application and interpretation. This paper evaluates how water managers practice microbial risk assessment and management in two Canadian provinces (BC and Ontario). We assess three types of entities engaged in water management along the source-to-tap spectrum (watershed agencies, water utilities, and public health authorities). We analyze and compare the approaches used by these agencies to assess and manage microbial risk (including scope, frequency, and tools). We evaluate key similarities and differences, and situate them with respect to international best practices derived from literatures related to microbial risk assessment and management. We find considerable variability in microbial risk assessment frameworks and management tools in that approaches 1) vary between provinces; 2) vary within provinces and between similar types of agencies; 3) have limited focus on microbial risk assessment for ecosystem health and 4) diverge considerably from the literature on best practices.We find that risk assessments that are formalized, routine and applied system-wide (i.e. from source-to-tap) are limited. We identify key limitations of current testing methodologies and looking forward consider the outcomes of this research within the context of new developments in microbial water quality monitoring such as tests derived from genomics and metagenomics based research.Medicine, Faculty ofScience, Faculty ofOther UBCNon UBCPathology and Laboratory Medicine, Department ofResources, Environment and Sustainability (IRES), Institute forReviewedFacult

Foodborne Botulism, Canada, 2006–2021

During 2006–2021, Canada had 55 laboratory-confirmed outbreaks of foodborne botulism, involving 67 cases. The mean annual incidence was 0.01 case/100,000 population. Foodborne botulism in Indigenous communities accounted for 46% of all cases, which is down from 85% of all cases during 1990–2005. Among all cases, 52% were caused by botulinum neurotoxin type E, but types A (24%), B (16%), F (3%), and AB (1%) also occurred; 3% were caused by undetermined serotypes. Four outbreaks resulted from commercial products, including a 2006 international outbreak caused by carrot juice. Hospital data indicated that 78% of patients were transferred to special care units and 70% required mechanical ventilation; 7 deaths were reported. Botulinum neurotoxin type A was associated with much longer hospital stays and more time spent in special care than types B or E. Foodborne botulism often is misdiagnosed. Increased clinician awareness can improve diagnosis, which can aid epidemiologic investigations and patient treatment

Hands off the Mink! Using Environmental Sampling for SARS-CoV-2 Surveillance in American Mink

Throughout the COVID-19 pandemic, numerous non-human species were shown to be susceptible to natural infection by SARS-CoV-2, including farmed American mink. Once infected, American mink can transfer the virus from mink to human and mink to mink, resulting in a high rate of viral mutation. Therefore, outbreak surveillance on American mink farms is imperative for both mink and human health. Historically, disease surveillance on mink farms has consisted of a combination of mortality and live animal sampling; however, these methodologies have significant limitations. This study compared PCR testing of both deceased and live animal samples to environmental samples on an active outbreak premise, to determine the utility of environmental sampling. Environmental sampling mirrored trends in both deceased and live animal sampling in terms of percent positivity and appeared more sensitive in some low-prevalence instances. PCR CT values of environmental samples were significantly different from live animal samples’ CT values and were consistently high (mean CT = 36.2), likely indicating a low amount of viral RNA in the samples. There is compelling evidence in favour of environmental sampling for the purpose of disease surveillance, specifically as an early warning tool for SARS-CoV-2; however, further work is needed to ultimately determine whether environmental samples are viable sources for molecular epidemiology investigations

The utility of SARS-CoV-2 genomic data for informative clustering under different epidemiological scenarios and sampling

Objectives: Clustering pathogen sequence data is a common practice in epidemiology to gain insights into the genetic diversity and evolutionary relationships among pathogens. We can find groups of cases with a shared transmission history and common origin, as well as identifying transmission hotspots. Motivated by the experience of clustering SARS-CoV-2 cases using whole genome sequence data during the COVID-19 pandemic to aid with public health investigation, we investigated how differences in epidemiology and sampling can influence the composition of clusters that are identified. Methods: We performed genomic clustering on simulated SARS-CoV-2 outbreaks produced with different transmission rates and levels of genomic diversity, along with varying the proportion of cases sampled. Results: In single outbreaks with a low transmission rate, decreasing the sampling fraction resulted in multiple, separate clusters being identified where intermediate cases in transmission chains are missed. Outbreaks simulated with a high transmission rate were more robust to changes in the sampling fraction and largely resulted in a single cluster that included all sampled outbreak cases. When considering multiple outbreaks in a sampled jurisdiction seeded by different introductions, low genomic diversity between introduced cases caused outbreaks to be merged into large clusters. If the transmission and sampling fraction, and diversity between introductions was low, a combination of the spurious break-up of outbreaks and the linking of closely related cases in different outbreaks resulted in clusters that may appear informative, but these did not reflect the true underlying population structure. Conversely, genomic clusters matched the true population structure when there was relatively high diversity between introductions and a high transmission rate. Conclusion: Differences in epidemiology and sampling can impact our ability to identify genomic clusters that describe the underlying population structure. These findings can help to guide recommendations for the use of pathogen clustering in public health investigations.Medicine, Faculty ofNon UBCPathology and Laboratory Medicine, Department ofReviewedFacultyPostdoctoralGraduat