Standardized Sampling Protocol Research Articles

Development and evaluation of a standardised sampling protocol to determine the effect of cleaning in the pig sty

BackgroundAll-in, all-out with strict hygienic routines is necessary in modern pig production. Furthermore, a standardised, validated method is needed to quantitatively control the effect of these hygiene protocols. This study aimed to establish a reproducible and reliable sampling method to assess cleaning of the pig pen.MethodsSterilised pig faeces were mixed with indicator bacteria (i.e. Enterococcus hirae, Escherichia coli, Pseudomonas aeruginosa and Staphylococcus aureus) and spread out in a controlled environment. The retrieval rate of three different sampling methods were evaluated; swabbing by (i) a cloth and (ii) a sponge, analysed by standardised bacterial culture and counting of colony-forming units, and (iii) a cotton swab analysed by adenosine triphosphate (ATP) bioluminescence. Two time-points were evaluated during the study; after drying overnight and after manual scraping of the surfaces. To determine sample-to-sample variability, sampling by the cloth and the cotton swab was carried out after manual scraping and further, after high-pressure washing with cold water.ResultsSampling by the cloth and the sponge showed few differences in in the number of CFU obtained before and after the manual scraping (retrieval rate), whereas the swabs, measuring ATP bioluminescence, showed a very high retrieval rate. Sample-to-sample variability was low for all three methods.ConclusionsIn conclusion, to sample pens for the presence of bacteria, the cloth was assessed as the preferable material, being cheap, easy, specific, and approachable, and with a low sample-to-sample variability. The ATP measurement could have potential for use when evaluating the cleaning of stables, however, threshold values for evaluating the cleaning of a pig sty needs to be developed.

Evaluation of Fish Biodiversity in Estuaries Through Environmental DNA Metabarcoding: A Comprehensive Review

Traditional methods face difficulties in capturing elusive species and navigating the complexities of estuarine environments. The adoption of eDNA metabarcoding has revolutionized biodiversity assessment, offering non-invasive and comprehensive sampling. Challenges specific to estuaries, such as sediment loads and salinity fluctuations, were initially addressed through improved DNA extraction, optimized primers, and specialized bioinformatics tools. The integration of multiple genetic markers and quantitative eDNA approaches has enhanced resolution, enabling the detection of rare and cryptic species. Standardized sampling protocols and validation through traditional methods contribute to the reliability of eDNA metabarcoding. This technology holds promise for informing conservation and management strategies in estuarine ecosystems by providing timely and cost-effective biodiversity data.

Impact of carbon dioxide exposures on sleep latency among healthy volunteers: A randomized order, paired crossover study, evidence from the multiple sleep latency test

BackgroundDaytime sleepiness affects work efficiency, occupational safety, and public health. Although previous studies have reported an association between environmental carbon dioxide (eCO2) and daytime sleepiness, it has been challenging to draw a firm conclusion due to the lack of standardized sampling and profiling protocols. ObjectiveWe examined the effect of pure CO2 exposure at 5000 (ppm, parts per million) on daytime sleepiness. MethodsEleven healthy participants (males of 24 ± 3 years, mean ± SD) completed a four-nap multiple sleep latency test (MSLT) protocol in the environmentally controlled chamber under two conditions: the CO2 condition (4851 ± 229 ppm) and the Control condition (1102 ± 204 ppm). The subjective sleepiness level and cognitive performances were also evaluated using the Stanford Sleepiness Scale (SSS) questionnaire, Psychomotor Vigilance Test (PVT), and Stroop test after each nap session. ResultsA significant reduction in sleep latency was observed in the CO2 exposure condition (Control vs. CO2 = 13.1 ± 3.3 min vs. 9.7 ± 3.2 min). The subjective sleepiness scores were also significantly higher in the CO2 exposure condition than in the Control condition (Control vs. CO2 = 2.7 ± 0.5 vs. 4.7 ± 0.8). Cognitive responses after naps showed no significant difference across conditions. ConclusionThis study revealed that exposure to environmental CO2 at a concentration as high as the upper safety limit at work sites significantly shortened the sleep latency and enhanced subjective sleepiness during naps in the MSLT without affecting cognitive responses after each exposure. Our results demonstrated that exposure to high environmental CO2 induces daytime sleepiness that potentially compromises work efficiency and safety.

Optimal inventorying and monitoring of taxonomic, phylogenetic and functional diversity.

Comparable data is essential to understand biodiversity patterns. While assemblage or community inventorying requires comprehensive sampling, monitoring focuses on as few components as possible to detect changes. Quantifying species, their evolutionary history, and the way they interact requires studying changes in taxonomic (TD), phylogenetic (PD) and functional diversity (FD). Here we propose a method for the optimization of sampling protocols for inventorying and monitoring assemblages or communities across these three diversity dimensions taking sampling costs into account. We used Iberian spiders and Amazonian bats as two case-studies. The optimal combination of methods for inventorying and monitoring required optimizing the accumulation curve of α-diversity and minimizing the difference between sampled and estimated β-diversity (bias), respectively. For Iberian spiders, the optimal combination for TD, PD and FD allowed sampling at least 50% of estimated diversity with 24 person-hours of fieldwork. The optimal combination of six person-hours allowed reaching a bias below 8% for all dimensions. For Amazonian bats, surveying all the 12 sites with mist-nets and 0 or 1 acoustic recorders was the optimal combination for almost all diversity types, resulting in >89% of the diversity and <10% bias with roughly a third of the cost. Only for phylogenetic α-diversity, the best solution was less clear and involved surveying both with mist nets and acoustic recorders. The widespread use of optimized and standardized sampling protocols and regular repetition in time will radically improve global inventory and monitoring of biodiversity. We strongly advocate for the global adoption of sampling protocols for both inventory and monitoring of taxonomic, phylogenetic and functional diversity.

Addition of a Novel Qualitative Technique to Standard Quantitative Practices for Evaluation of Hazardous Drug Exposure in a Canadian Hospital Setting.

Current recommendations from regulatory authorities suggest quantitative surface sampling for detection of hazardous drugs at least once every 6 months. A more frequent and efficient process for hazardous drug testing might reduce the safety risks associated with exposure to these agents. The primary objective was to assess the findings of surface testing based on traditional quantitative sampling methods relative to the findings of qualitative surface sample testing with the BD HD Check system. The secondary objectives included assessment of the ease of integrating qualitative sampling into pharmacy protocols and identification of opportunities to enhance patient and staff education and safety. Samples from 23 unique surfaces were tested concurrently once a month for 5 months using a quantitative surface sampling method and the qualitative BD HD Check system on adjacent 12 inch × 12 inch (30.5 cm × 30.5 cm) surface areas. The presence or absence of cyclophosphamide, methotrexate, and/or doxorubicin contamination was assessed by each of the 2 testing methods. The BD HD Check system was also assessed for ease of use and efficiency. Ten areas of contamination were identified over the 5-month period. Nine were detected by the BD HD Check system and one by the quantitative system. The BD HD Check system was easy to use, with results available in less than 10 minutes per area tested. The BD HD Check system allows for more timely identification of surface contamination with hazardous drugs than the standard sampling protocol. The discrepancy in results between the 2 methods of hazardous drug surface sampling requires further investigation.

Salmonellae in the air environment: A review

Salmonella bacteria, zoonotic pathogen, are frequently transmitted through food and water, producing toxins responsible for typhoid and paratyphoid fever. Bio-aerosols are growing concern as pathogenic bacteria could be transmitted to the ambient air, causing potential health hazards. The airborne transmission of pathogenic microorganisms should be considered as a route of infections. Salmonella have been found in rare counts in the air but indicate their ability to survive in the atmosphere. Physical, biological and environmental stressors affect survival of airborne microorganisms. The infectivity of airborne salmonella bacteria is determined by their pathogenicity, infective dose and individual health conditions. The accurate assessment of the presence of Salmonella in aerosols is a problem due to synergistically influence of many uncontrollable environmental conditions and lack of standardized sampling and analysis protocol. Knowledge on airborne Salmonella and factors influence their viability is critical to understand their potential health risk associated and the related-control measures. This review focuses on the scarcely data published concerning the presence of salmonella bacteria in the air environment, survivability, sampling and analysis methods, and control strategy.

Mercury contamination is an invisible threat to declining migratory shorebirds along the East Asian-Australasian Flyway

Exposure to pollutants is a potentially crucial but overlooked driver of population declines in shorebirds along the East Asian-Australasian Flyway. We combined knowledge of moult strategy and life history with a standardised sampling protocol to assess mercury (Hg) contamination in 984 individuals across 33 migratory shorebird species on an intercontinental scale. Over one-third of the samples exceeded toxicity benchmarks. Feather Hg was best explained by moulting region, while habitat preference (coastal obligate vs. non-coastal obligate), the proportion of invertebrates in the diet and foraging stratum (foraging mostly on the surface vs. at depth) also contributed, but were less pronounced. Feather Hg was substantially higher in South China (Mai Po and Leizhou), Australia and the Yellow Sea than in temperate and Arctic breeding ranges. Non-coastal obligate species (Tringa genus) frequently encountered in freshwater habitats were at the highest risk. It is important to continue and expand biomonitoring research to assess how other pollutants might impact shorebirds.

The Potential of Fecal Volatile Organic Compound Analysis for the Early Diagnosis of Late-Onset Sepsis in Preterm Infants: A Narrative Review.

Early diagnosis and treatment of late-onset sepsis (LOS) is crucial for survival, but challenging. Intestinal microbiota and metabolome alterations precede the clinical onset of LOS, and the preterm gut is considered an important source of bacterial pathogens. Fecal volatile organic compounds (VOCs), formed by physiologic and pathophysiologic metabolic processes in the preterm gut, reflect a complex interplay between the human host, the environment, and microbiota. Disease-associated fecal VOCs can be detected with an array of devices with various potential for the development of a point-of-care test (POCT) for preclinical LOS detection. While characteristic VOCs for common LOS pathogens have been described, their VOC profiles often overlap with other pathogens due to similarities in metabolic pathways, hampering the construction of species-specific profiles. Clinical studies have, however, successfully discriminated LOS patients from healthy individuals using fecal VOC analysis with the highest predictive value for Gram-negative pathogens. This review discusses the current advancements in the development of a non-invasive fecal VOC-based POCT for early diagnosis of LOS, which may potentially provide opportunities for early intervention and targeted treatment and could improve clinical neonatal outcomes. Identification of confounding variables impacting VOC synthesis, selection of an optimal detection device, and development of standardized sampling protocols will allow for the development of a novel POCT in the near future.

Potential Environmental Reservoirs of Candida auris: A Systematic Review.

Candida auris, a multidrug-resistant yeast, poses significant challenges in healthcare settings worldwide. Understanding its environmental reservoirs is crucial for effective control strategies. This systematic review aimed to review the literature regarding the natural and environmental reservoirs of C. auris. Following the PRISMA guidelines, published studies until October 2023 were searched in three databases: PubMed, Web of Science, and Scopus. Information regarding the origin, sampling procedure, methods for laboratory identification, and antifungal susceptibility was collected and analyzed. Thirty-three studies published between 2016 and 2023 in 15 countries were included and analyzed. C. auris was detected in various environments, including wastewater treatment plants, hospital patient care surfaces, and natural environments such as salt marshes, sand, seawater, estuaries, apples, and dogs. Detection methods varied, with molecular techniques often used alongside culture. Susceptibility profiles revealed resistance patterns. Phylogenetic studies highlight the potential of environmental strains to influence clinical infections. Despite methodological heterogeneity, this review provides valuable information for future research and highlights the need for standardized sampling and detection protocols to mitigate C. auris transmission.

Validation of vacuum extraction protocol for larval sampling of spotted-wing drosophila in blueberries

For spotted-wing drosophila, Drosophila suzukii Matsumura, the standard larval sampling protocol from the fruits involves immersing the fruits in hot water, salt, or sugar solution followed by enumerating the larvae that exit from the fruit. In this study, using the fruit samples collected from commercial blueberry farms, we validated the efficacy of a novel D. suzukii larval sampling technique, the vacuum extraction protocol. The larval extraction efficacy of the vacuum extraction method was compared with the salt extraction method using paired fruit samples collected from three different fields for four consecutive weeks. Overall, larval counts detected through the vacuum extraction were significantly higher than those detected through salt extraction in two out of three blueberry fields. Additionally, among the five blueberry fruit sample volumes tested (118, 177, 237, 355, and 473 mL), we identified 355 mL (12 fl. oz.) as the optimum fruit sample volume for vacuum extraction that maximized sampling precision for unit sampling cost. Since D. suzukii larvae exited from fruits after vacuum extraction remain alive and active, a delay in separating the larvae from the sample with coffee filter extraction may result in larval movement back into the fruit flesh. However, compared with immediate processing, delaying coffee filter extraction up to 60 minutes after vacuum incubating the fruit samples at -98 kPa for 60 minutes did not significantly reduce the larval count. Overall, the results of validation trials indicate that vacuum extraction is a promising sampling method for growers to detect D. suzukii larval infestation in blueberries.

Relative and absolute difference in soil organic carbon stocks in grassland soils in Ireland: Impact of rock fragments, bulk density and calculation methods

On-farm management measures can sequester carbon by removing carbon dioxide from the atmosphere and storing it securely for long periods, in our soils. Estimates of soil organic carbon (SOC) stocks differ by calculation methods in terms of how they account for the coarse >2 mm fraction (i.e. rock fragments) of the soil. Irish grassland soils have variable volumetric stone contents and the extent of overestimation is unknown. Without best practice guidance, calculations of SOC in these soils are vulnerable to overestimation. The objectives of the present study for grassland soils in Ireland were to: 1) calculate mean SOC stocks using different methods by soil horizon and volumetric stone content class; 2) highlight relative differences in SOC stocks between these calculation methods and 3) compare normalised layer (Ly, fixed depth) versus horizon (Hz) type derived sampling data in terms of SOC stocks, to establish a best practice standardised sampling protocol. Five methods (M, 1–3 overestimate SOC stocks, whereas 4–5 are equivalent and are closest to reality) to calculate SOC stocks were used in combination with three soil databases i.e. Irish Soil Information System (I-SIS), the Heavy Soils Programme (HSP) and the Soil Quality Assessment Research Project (SQUARE). Objectives 1 and 2 were assessed using all three databases across methods, whereas objective 3 used the SQUARE database and M4. These data were sorted into volumetric stone content (%) classes (< 5%, 5–10%, 10–20%, 20–30% and > 30%) and sampling horizon (O, A, E, A/B, B, B/C, and C) with SOC stocks calculated using M1–5 equations. Results confirmed the high volumetric stone content assumption of grassland soils in Ireland (exception O horizons). Volumetric stone contents ranged from 0% to 36% for all horizons. When M1–5 are considered the average relative difference in SOC stocks between M1 and M5 was 18.9% (max: 388%), M2 and M5 was 7.7% (max: 60.7%) and M3 and M5 was 8.9 (max: 310%). In terms of Ly versus Hz sampling, no significant differences were found for the A horizons sampled. However, in Ireland, best practice should still utilise elements from both Ly (using an auger to get fixed depth soil samples; analysed for organic C) and Hz (using a soil profile pit to extract representative BD cores and to gather bulk samples for volumetric stone content analysis) approaches in the field. Laboratory methods used must fit with the needs and assumptions of equations M4/5 to calculate SOC stocks (thereby minimising overestimation and indeed underestimation).

Responses of the macroinvertebrate community to urban wastewater pollution in the upper Ouémé Basin in Benin

In Benin, most of urban wastewaters are discharged into rivers without any prior treatment. The objective of this study was to assess the effects of urban wastewater on the macroinvertebrate communities of the upper Ouémé River in Benin. To address this question, 30 stations located on five rivers were monitored in the dry and the wet seasons. For each station and each season, 12 samples of macroinvertebrates following standardized French multi-habitat sampling protocol were collected and physico-chemical parameters were recorded. Three types of stations were chosen on each river: two control stations located upstream of the wastewater discharge points, two stations impacted in the urban area and receiving urban wastewaters, and two stations downstream of the wastewater discharge points to measure the resilience of a set of river characteristics. Urban wastewater impacted the water quality by mainly increasing electrical conductivity and the nutrient concentrations. Wastewaters also deeply impacted the diversity and the composition of the invertebrate community. The Indval index highlighted three indicator taxa for the control stations (Caenidae, Baetidae and Ephemerellidae), one for the impacted stations (Chironomidae), and two for the downstream stations (Libellulidae and Lestidae). We also observed ecosystem resilience a few hundred meters downstream of the discharge points. These results challenge managers on the degradation of river water quality in the upper Ouémé River, but also reveal good self-purification capacities of the watercourses likely to promote the resilience of these ecosystems.

Meso- and microplastic composition, distribution patterns and drivers: A snapshot of plastic pollution on Brazilian beaches

Pollution by plastics is a worldwide problem on par with climatic change and biological invasions. In coastal sediments, plastic particles tend to accumulate and persist over the long term. We assessed the plastic pollution using a standardized surface sediment sampling protocol on 22 sandy beaches along >4600 km of the Brazilian coast. The abundance, size, color, type, and polymeric composition of all meso- and microplastic items found in the surveys were processed to disclose spatial patterns of distribution and pollution associated drivers. A General Linear Model (GLM) was run to investigate how the predictor variables influenced overall beach plastic amounts and by plastic type and size class. Overall, 3114 plastic items were found, with microplastics comprising just over half of all items (54 %). Most items were either white (60 %) or blue (13 %), while polystyrene foam (45 %) and fragments (39 %) comprised the most abundant plastic types. The principal polymers were Polyethylene (40 %) and Polypropylene (32 %). The analyses indicated that the distribution of plastic litter along beaches is determined by three predictive variables: the distance to the nearest estuary (−), tourism (+), and the number of inhabitants in the nearest urban center (+). Tourist (highly-visited) beaches and those near estuarine runoffs or urban centers presented the highest plastic pollution rates. The unveiling of plastic pollution patterns through a large-scale systematic survey is essential for future management guidance and science-based decisions for mitigating and solving the plastic pollution crisis.

Evaluating the effect of moss functional traits and sampling on elemental concentrations in Pleurozium schreberi and Ptilium crista-castrensis in Eastern Canada (Québec) black spruce forest

Characterizing atmospheric depositions allows evaluating the impact of air pollution on ecosystems, human health, and the economy. It also informs decision-makers about the implementation of regulations improving environmental quality. Biomonitoring uses organisms, such as mosses, as proxies to assess the presence of atmospheric contaminants (e.g., metals). This approach is cost-efficient and does not require complicated infrastructure or scientific skills, making it suitable for large-scale monitoring initiatives and citizen-based campaigns. Therefore, precise sampling protocols are needed to limit bias. Biomonitoring data remains scarce in North America, compared to e.g., Europe, and there is a need to develop large-scale and long-term biomonitoring initiatives to record current and future atmospheric depositions. As there is no standardized international sampling protocol, this study assessed the impact of parameters known to affect the elemental concentration of mosses, using samples collected along a 1000-km transect in Eastern Canada (Quebec) from 2016 to 2022. We specifically examined the effects of species, stem color, canopy opening, time of sampling, and stem length on 18 elements. Non-parametric statistical tests indicate that these factors have significant effects on some metals, but differences are generally low (<30 %), except for stem length. These results suggest that sampling protocols can be flexible in terms of species, canopy opening, time of sampling, and stem color. However, normalizing the length of the stems analyzed is required to account for differences in growth rates between sites. Moreover, since no large-scale biomonitoring campaign using mosses has been conducted in Eastern Canada, this paper also provides the first elemental baseline for moss in the region.

An overview of DNA methylation markers for early detection of gastric cancer: current status, challenges, and prospects.

Background: Gastric cancer (GC) is one of the most common malignancies, with a low 5-year survival rate. However, if diagnosed at an early stage, it can be cured by endoscopic treatment and has a good prognosis. While gastrointestinal X-ray and upper endoscopy are used as national GC screening methods in some GC high-risk countries, such as Japan and Korea, their radiation exposure, invasiveness, and high cost suggest that they are not the optimal tools for early detection of GC in many countries. Therefore, a cost-effective, and highly accurate method for GC early detection is urgently needed in clinical settings. DNA methylation plays a key role in cancer progression and metastasis and has been demonstrated as a promising marker for cancer early detection. Aims and methods: This review provides a comprehensive overview of the current status of DNA methylation markers associated with GC, the assays developed for GC early detection, challenges in methylation marker discovery and application, and the future prospects of utilizing methylation markers for early detection of GC. Through our analysis, we found that the currently reported DNA methylation markers related to GC are mainly in the early discovery stage. Most of them have only been evaluated in tissue samples. The majority of non-invasive assays developed based on blood lack standardized sampling protocols, pre-analytical procedures, and multicenter validation, and they exhibit insufficient sensitivity for early-stage GC detection. Meanwhile, the reported GC DNA methylation markers are generally considered pan-cancer markers. Conclusion: Therefore, future endeavors should focus on identifying additional methylation markers specific to GC and establishing non-invasive diagnostic assays that rely on these markers. These assays should undergo multicenter, large-scale prospective validation in diverse populations.

Usage and Microbial Safety of Shared and Unshared Excreta Disposal Facilities in Developing Countries: The Case of a Ghanaian Rural District

Sharing facilities with other households offers the most realistic opportunity for access to sanitation for many households in low-income settings. However, questions remain about the safety of shared toilets, including those shared at the household level. This study sought to compare the usage and microbial safety of household-level shared and unshared toilets in a Ghanaian rural district to investigate any association between their microbial safety and sharing status. A semi-structured questionnaire was used to collect data on the user characteristics of the sampled toilets, while common contact surfaces (door handles and toilet seats) were assessed for faecal contamination following standard swab sampling and analytical protocols. The results of the study indicate that sharing toilets affords about 90% more household-level access to sanitation as compared to single-household toilets. Toilet sharing mostly occurred between two households, with a maximum user population of 14 per toilet. Generally, there was a high prevalence of faecal contamination on the door handles and seats of both shared and unshared toilets, but this had no association with the sharing status of the toilets. The median concentration of Escherichia coli (E. coli) on the door handles and seats of shared toilets was 34.3 × 105 and 103.2 × 105 CFU/mL, respectively, as compared to 54.7 × 105 and 125.0 × 105 CFU/mL, respectively, on unshared toilets. In conclusion, the sharing of toilets at the household level nearly doubles access to sanitation at home without necessarily exposing the users to a higher risk of faecal–oral disease transmission.

Characterizing the heterogeneity of eastern hemlock xylem water isotopic compositions: Implications for the design of plant water uptake studies

AbstractXylem water isotopic compositions (2H, 18O; δXYLEM) can be used to estimate plant water uptake depths; however, environmental heterogeneity in these measurements may prevent reaching robust conclusions. Bayesian mixing models used to estimate plant water uptake depths often assume that measurements of δXYLEM and candidate water uptake sources are normally and identically distributed. We tested if δXYLEM measured across 30 Eastern hemlock (Tsuga canadensis (L.) Carrière) trees met these assumptions. Bootstrap simulations suggested that the distributions of hemlock δXYLEM data were non‐normal in March, April, June, and July and that between 15 and 26 hemlock δXYLEM samples were required to reject the assumption of normality. In June, July, and August, δXYLEM was significantly predicted by a multivariate linear regression with tree sapwood depth or elevation, rejecting the assumption of independently distributed observations. A comparison of dry season hemlock water uptake depth estimates between a Bayesian mixing model and a process‐based ecohydrological model calibration showed differences, with the Bayesian model estimating a substantially greater proportion of shallow water uptake. These results highlight the need for standardized field sampling protocols for δXYLEM and analytical methods that will lead to more robust estimates of plant water uptake depths. These findings also suggest that water uptake functions conditioned on landscape and tree structural variables could substantially advance the representation of plants in ecohydrological models.

Agroecological zones influence maize infestation and damage severity by the fall armyworm (Spodoptera frugiperda [J. E. Smith, 1797]) in southwestern Nigeria

The fall armyworm (Spodoptera frugiperda) is an invasive and highly destructive insect pest that has caused extensive damage to maize in Africa since its first report on the continent in 2016. Information on fall armyworm infestation and damage within African agroecologies is essential for the development of appropriate pest management strategies, but these are scant in Nigeria. Consequently, in this study, fall armyworm infestation levels and severity of damage to maize in the three major maize-growing agro-ecological zones (humid forest, derived savanna, and southern guinea savanna) of southwestern Nigeria was investigated using standard field sampling protocols. Results showed that maize infestation and damage severity varied across agroecological zones, with the humid forest being the most impacted. Information provided will enhance decision-making for effective management of the fall armyworm in southwestern Nigeria.

Optimizing data quantity and quality for side-looking echosounder surveys in large rivers

The increased use of side-looking echosounder surveys in large rivers necessitates the creation and evaluation of standardized sampling protocols to effectively sample these habitats. Using data from five Ohio River pools, we evaluated the effects of habitat and transect direction on the quality and quantity of mobile, side-looking, split-beam echosounder data to determine how these factors may affect survey design in large rivers. Split beam echosounder data were collected using two multiplexing 200 kHz transducers offset in angle to maximize water column coverage. Using mixed-effects models, we evaluated the effects of habitat (main channel vs. tributary) and transect direction (upstream vs. downstream movement of the vessel) on fish density and the number of targets per fish track (series of acoustic targets from an individual fish). We also used a skewness test in conjunction with a Kolmogorov-Smirnov test to determine the distribution of fish orientations relative to the transducer and examine differences in the distribution of fish orientations among habitats, transect directions, and years. Mean fish densities were greater in tributary habitat relative to main channel habitat, whereas main channel habitat had a higher mean number of targets per fish track when compared to tributaries. Upstream-facing transects had greater mean fish density estimates and mean number of targets per fish track than downstream-facing transects. Although most fish were oriented perpendicular to the transducer, we identified habitat, transect direction, and annual differences in the distribution of fish orientations. These results suggest that researchers and managers that plan to use side-looking echosounders to assess fish populations in large rivers should ensure that standardized protocols include main channel and tributaries, and are expanded to encompass other relevant habitats (e.g., backwaters and side channels). Further, managers should use fish orientation data to select or develop TS-L equations that best matches the fish orientation distribution of each survey to ensure accurate density and size estimates.

Targeted locus amplification to develop robust patient-specific assays for liquid biopsies in pediatric solid tumors.

Liquid biopsies combine minimally invasive sample collection with sensitive detection of residual disease. Pediatric malignancies harbor tumor-driving copy number alterations or fusion genes, rather than recurrent point mutations. These regions contain tumor-specific DNA breakpoint sequences. We investigated the feasibility to use these breakpoints to design patient-specific markers to detect tumor-derived cell-free DNA (cfDNA) in plasma from patients with pediatric solid tumors. Regions of interest (ROI) were identified through standard clinical diagnostic pipelines, using SNP array for CNAs, and FISH or RT-qPCR for fusion genes. Using targeted locus amplification (TLA) on tumor organoids grown from tumor material or targeted locus capture (TLC) on FFPE material, ROI-specific primers and probes were designed, which were used to design droplet digital PCR (ddPCR) assays. cfDNA from patient plasma at diagnosis and during therapy was analyzed. TLA was performed on material from 2 rhabdomyosarcoma, 1 Ewing sarcoma and 3 neuroblastoma. FFPE-TLC was performed on 8 neuroblastoma tumors. For all patients, at least one patient-specific ddPCR was successfully designed and in all diagnostic plasma samples the patient-specific markers were detected. In the rhabdomyosarcoma and Ewing sarcoma patients, all samples after start of therapy were negative. In neuroblastoma patients, presence of patient-specific markers in cfDNA tracked tumor burden, decreasing during induction therapy, disappearing at complete remission and re-appearing at relapse. We demonstrate the feasibility to determine tumor-specific breakpoints using TLA/TLC in different pediatric solid tumors and use these for analysis of cfDNA from plasma. Considering the high prevalence of CNAs and fusion genes in pediatric solid tumors, this approach holds great promise and deserves further study in a larger cohort with standardized plasma sampling protocols.