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Background: Every year, 60% of deaths from diarrhoeal disease occur in low and middle-income countries due to inadequate water, sanitation, and hygiene. In these countries, diarrhoeal diseases are the second leading cause of death in children under five, excluding neonatal deaths. The approximately 100,000 people residing in the Bentiu Internally Displaced Population (IDP) camp in South Sudan have previously experienced water, sanitation, and hygiene outbreaks, including an ongoing Hepatitis E outbreak in 2021. This study aimed to assess the gaps in Water, Sanitation, and Hygiene (WASH), prioritise areas for intervention, and advocate for the improvement of WASH services based on the findings. Methods: A cross-sectional lot quality assurance sampling (LQAS) survey was conducted in ninety-five households to collect data on water, sanitation, and hygiene (WASH) coverage performance across five sectors. Nineteen households were allocated to each sector, referred to as supervision areas in LQAS surveys. Probability proportional to size sampling was used to determine the number of households to sample in each sector block selected using a geographic positioning system. One adult respondent, familiar with the household, was chosen to answer WASH-related questions, and one child under the age of five was selected through a lottery method to assess the prevalence of WASH-related disease morbidities in the previous two weeks. The data were collected using the KoBoCollect mobile application. Data analysis was conducted using R statistical software and a generic LQAS Excel analyser. Crude values, weighted averages, and 95% confidence intervals were calculated for each indicator. Target coverage benchmarks set by program managers and WASH guidelines were used to classify the performance of each indicator. Results: The LQAS survey revealed that five out of 13 clean water supply indicators, eight out of 10 hygiene and sanitation indicators, and two out of four health indicators did not meet the target coverage. Regarding the clean water supply indicators, 68.9% (95% CI 60.8%-77.1%) of households reported having water available six days a week, while 37% (95% CI 27%-46%) had water containers in adequate condition. For the hygiene and sanitation indicators, 17.9% (95% CI 10.9%-24.8%) of households had handwashing points in their living area, 66.8% (95% CI 49%-84.6%) had their own jug for cleansing after defaecation, and 26.4% (95% CI 17.4%-35.3%) of households had one piece of soap. More than 40% of households wash dead bodies at funerals and wash their hands in a shared bowl. Households with sanitary facilities at an acceptable level were 22.8% (95% CI 15.6%-30.1%), while 13.2% (95% CI 6.6%-19.9%) of households had functioning handwashing points at the latrines. Over the previous two weeks, 57.9% (95% CI 49.6–69.7%) of households reported no diarrhoea, and 71.3% (95% CI 62.1%-80.6%) reported no eye infections among children under five. Conclusion: The camp's hygiene and sanitation situation necessitated immediate intervention to halt the hepatitis E outbreak and prevent further WASH-related outbreaks and health issues. The LQAS findings were employed to advocate for interventions addressing the WASH gaps, resulting in WASH and health actors stepping in. [ABSTRACT FROM AUTHOR]

Diversity studies of aquatic picoplankton (bacterioplankton) communities using size-class filtration, DNA extraction, PCR and sequencing of phylogenetic markers, require a robust methodological pipeline, since biases have been demonstrated essentially at all levels, including DNA extraction, primer choice and PCR. Even different filtration volumes of the same plankton sample and, thus, different biomass loading of the filters, can distort the sequencing results. In this study, we designed an Arduino microcontroller-based flowmeter that records the decrease of initial (maximal) flowrate as proxy for increasing biomass loading and clogging of filters during plankton filtration. The device was tested using freshwater plankton of Lake Constance, and total DNA was extracted and an 16S rDNA amplicon was sequenced. We confirmed that different filtration volumes used for the same water sample affect the sequencing results. Differences were visible in alpha and beta diversities and across all taxonomic ranks. Taxa most affected were typical freshwater Actinobacteria and Bacteroidetes, increasing up to 38% and decreasing up to 29% in relative abundance, respectively. In another experiment, a lake water sample was filtered undiluted and three-fold diluted, and each filtration was stopped once the flowrate had reduced to 50% of initial flowrate, hence, at the same degree of filter clogging. The three-fold diluted sample required three-fold filtration volumes, while equivalent amounts of total DNA were extracted and differences across all taxonomic ranks were not statistically significant compared to the undiluted controls. In conclusion, this work confirms a volume/biomass-dependent bacterioplankton filtration bias for sequencing-based community analyses and provides an improved procedure for controlling biomass loading during filtrations and recovery of equivalent amounts of DNA from samples independent of the plankton density. The application of the device can also avoid the distorting of sequencing results as caused by the plankton filtration bias. [ABSTRACT FROM AUTHOR]

Erosion poses a significant threat to oceanic beaches worldwide. To combat this threat, management agencies often utilize renourishment, which supplements eroded beaches with offsite sand. This process can alter the physical characteristics of the beach and can influence the presence and abundance of microbial communities. In this study, we examined how an oceanic beach renourishment project may have impacted the presence and abundance of Escherichia coli (E. coli), a common bacteria species, and sand grain size, a sediment characteristic that can influence bacterial persistence. Using an observational field approach, we quantified the presence and abundance of E. coli in sand (from sub-tidal, intertidal, and dune zones on the beach) and water samples at study sites in both renourished and non-renourished sections of Folly Beach, South Carolina, USA in 2014 and 2015. In addition, we also measured how renourishment may have impacted sand grain size by quantifying the relative frequency of grain sizes (from sub-tidal, intertidal, and dune zones on the beach) at both renourished and non-renourished sites. Using this approach, we found that E. coli was present in sand samples in all zones of the beach and at each of our study sites in both years of sampling but never in water samples. Additionally, we found that in comparison to non-renourished sections, renourished sites had significantly higher abundances of E. coli and coarser sand grains in the intertidal zone, which is where renourished sand is typically placed. However, these differences were only present in 2014 and were not detected when we resampled the study sites in 2015. Collectively, our findings show that E. coli can be commonly found in this sandy beach microbial community. In addition, our results suggest that renourishment has the potential to alter both the physical structure of the beach and the microbial community but that these impacts may be short-lived. [ABSTRACT FROM AUTHOR]

Dams and weirs impede the continuity of rivers and transit of migratory fish. To overcome this obstacle, fishways are installed worldwide; however, management after installation is important. The Miyanaka Intake Dam has three fish ladders with different flow velocities and discharges and has been under adaptive management since 2012. Fish catch surveys, conducted as an adaptive management strategy, place a heavy burden on fish. Furthermore, a large number of investigators must be mobilized during the 30-day investigation period. Thus, a monitoring method using environmental DNA that exerts no burden on fish and requires only a few surveyors (to obtain water samples) and an in-house analyst was devised; however, its implementation in a fishway away from the point of analysis and with limited flow space and its effective water sampling frequency have not been reported. Therefore, in 2019, we started a trial aiming to evaluate the methods and application conditions of environmental DNA surveys for the continuous and long-term monitoring of various fish fauna upstream and downstream of the Miyanaka Intake Dam. To evaluate the fish fauna, the results of an environmental DNA survey (metabarcoding method) for 2019 to 2022 were compared to those of a catch survey in the fishway from 2012 to 2022. The results confirmed the use of environmental DNA surveys in evaluating the contribution of fishways to biodiversity under certain conditions and introduced a novel method for sample collection. [ABSTRACT FROM AUTHOR]

The role of glycosylated hemoglobin (HbA1c) in youth is largely unclear. The aims of this study are to investigate the distribution and potential determinants of HbA1c among a population-based sample of adolescents. The German Health Interview and Examination Survey for Children and Adolescents (KiGGS) Wave 2 includes a nationwide representative sample of 0-17-year-old participants. For this evaluation, data from a randomly selected subgroup aged 14–17 years and without diagnosed diabetes was included (n = 857). Percentile-based HbA1c values (measured at laboratory in whole blood samples by high performance liquid chromatography) were calculated to examine HbA1c distribution. Multivariable linear regression analyses were performed to investigate factors (age, sex, parental socioeconomic status, body mass index (BMI), birth weight, smoking, alcohol consumption, healthy food diversity, sport activity, oral contraceptive use) associated with HbA1c. The mean HbA1c level was 5.2% (minimum: 3.9%, P10: 4.8%, P50: 5.1%, P90: 5.5%, maximum: 6.7%). Overall, 2.8% of adolescents had an HbA1c value in the prediabetic range (5.7–6.4%) and 0.1% had an undiagnosed diabetes (≥6.5%). Multivariable regression analysis showed an inverse association of age with HbA1c (17 vs. 14 years: ß: -1.18; 95% CI -2.05, -0.31). Higher HbA1c values were observed for higher BMI-standard deviation scores (SDS) (ß: 0.24; 95% CI -0.04, 0.52) and smoking (ß: 0.73; 95% CI -0.12, 1.57), but these tendencies were non-significant. In sex-stratified analysis, smoking and birth weight were significantly associated with HbA1c in boys. Among adolescents without diagnosed diabetes in Germany, HbA1c values ranged from 3.9% to 6.7%. To ensure health in adulthood, the influence of determinants on HbA1c levels in younger age should be further investigated. [ABSTRACT FROM AUTHOR]

Typhoid-conjugate vaccines (TCVs) provide an opportunity to reduce the burden of typhoid fever, caused by Salmonella Typhi, in endemic areas. As policymakers design vaccination strategies, accurate and high-resolution data on disease burden is crucial. However, traditional blood culture-based surveillance is resource-extensive, prohibiting its large-scale and sustainable implementation. Salmonella Typhi is a water-borne pathogen, and here, we tested the potential of Typhi-specific bacteriophage surveillance in surface water bodies as a low-cost tool to identify where Salmonella Typhi circulates in the environment. In 2021, water samples were collected and tested for the presence of Salmonella Typhi bacteriophages at two sites in Bangladesh: urban capital city, Dhaka, and a rural district, Mirzapur. Salmonella Typhi-specific bacteriophages were detected in 66 of 211 (31%) environmental samples in Dhaka, in comparison to 3 of 92 (3%) environmental samples from Mirzapur. In the same year, 4,620 blood cultures at the two largest pediatric hospitals of Dhaka yielded 215 (5%) culture-confirmed typhoid cases, and 3,788 blood cultures in the largest hospital of Mirzapur yielded 2 (0.05%) cases. 75% (52/69) of positive phage samples were collected from sewage. All isolated phages were tested against a panel of isolates from different Salmonella Typhi genotypes circulating in Bangladesh and were found to exhibit a diverse killing spectrum, indicating that diverse bacteriophages were isolated. These results suggest an association between the presence of Typhi-specific phages in the environment and the burden of typhoid fever, and the potential of utilizing environmental phage surveillance as a low-cost tool to assist policy decisions on typhoid control. Author summary: The WHO prequalified two typhoid conjugate vaccines for use to reduce the burden of typhoid fever. As policymakers design vaccination strategies, accurate, high-resolution estimates of typhoid burden are crucial for efficient use of the vaccines. Typhoid burden can vary widely; for example, in Bangladesh, burden is high in the urban capital city Dhaka, but is 100-fold lower in the rural site, Mirzapur. Optimal local data rely on traditional blood-culture-based surveillance, which is expensive and often unavailable. With the knowledge that Salmonella Typhi, cause of typhoid fever, is a water-borne pathogen, we tested an environmental surveillance tool that detects bacteriophages (viruses) against Salmonella Typhi in environmental water bodies using simple assays. Testing of 303 water samples from Dhaka and Mirzapur showed a 10-fold lower abundance of bacteriophages in Mirzapur, depicting a correlation with typhoid burden in the community. This low-cost surveillance can be employed in different regions to generate rapid data on typhoid burden for evidence-based introduction of vaccines and tracking their impact upon rollout. [ABSTRACT FROM AUTHOR]

Background: Environmental surveillance, using detection of Salmonella Typhi DNA, has emerged as a potentially useful tool to identify typhoid-endemic settings; however, it is relatively costly and requires molecular diagnostic capacity. We sought to determine whether S. Typhi bacteriophages are abundant in water sources in a typhoid-endemic setting, using low-cost assays. Methodology: We collected drinking and surface water samples from urban, peri-urban and rural areas in 4 regions of Nepal. We performed a double agar overlay with S. Typhi to assess the presence of bacteriophages. We isolated and tested phages against multiple strains to assess their host range. We performed whole genome sequencing of isolated phages, and generated phylogenies using conserved genes. Findings: S. Typhi-specific bacteriophages were detected in 54.9% (198/361) of river and 6.3% (1/16) drinking water samples from the Kathmandu Valley and Kavrepalanchok. Water samples collected within or downstream of population-dense areas were more likely to be positive (72.6%, 193/266) than those collected upstream from population centers (5.3%, 5/95) (p=0.005). In urban Biratnagar and rural Dolakha, where typhoid incidence is low, only 6.7% (1/15, Biratnagar) and 0% (0/16, Dolakha) river water samples contained phages. All S. Typhi phages were unable to infect other Salmonella and non-Salmonella strains, nor a Vi-knockout S. Typhi strain. Representative strains from S. Typhi lineages were variably susceptible to the isolated phages. Phylogenetic analysis showed that S. Typhi phages belonged to the class Caudoviricetes and clustered in three distinct groups. Conclusions: S. Typhi bacteriophages were highly abundant in surface waters of typhoid-endemic communities but rarely detected in low typhoid burden communities. Bacteriophages recovered were specific for S. Typhi and required Vi polysaccharide for infection. Screening small volumes of water with simple, low-cost (~$2) plaque assays enables detection of S. Typhi phages and should be further evaluated as a scalable tool for typhoid environmental surveillance. Author summary: Typhoid fever is a major health issue in low- and middle-income countries, causing illness and death. The World Health Organization recommends the use of typhoid-conjugate vaccines to combat its spread. However, limited data on typhoid prevalence in these countries hinders effective vaccination programs. Environmental surveillance, which involves detecting Salmonella Typhi DNA in the environment, has emerged as a method to identify high-risk areas for typhoid transmission. Nevertheless, this approach is costly and requires infrastructure often unavailable in many communities. To address this, we conducted a study in Nepal to explore a low-cost and sustainable typhoid surveillance method. We examined typhoid bacteriophages in surface and drinking water across communities with varying population densities and typhoid burdens. Phages were easily detectable in communities with high typhoid burdens but less prevalent in areas with lower burdens. The isolated phages exhibited specificity for S. Typhi and were effective against different bacterial strains in Nepal. This study demonstrates that phage-based assays can be a simple and affordable tool for typhoid environmental surveillance. This method can help identify areas that require vaccination prioritization and interventions for clean water and sanitation. By utilizing phage-based assays, health authorities can better understand typhoid transmission and implement targeted control measures. [ABSTRACT FROM AUTHOR]

Mesoscale oceanographic features, including eddies, have the potential to alter productivity and other biogeochemical rates in the ocean. Here, we examine the microbiome of a cyclonic, Gulf Stream frontal eddy, with a distinct origin and environmental parameters compared to surrounding waters, in order to better understand the processes dominating microbial community assembly in the dynamic coastal ocean. Our microbiome-based approach identified the eddy as distinct from the surround Gulf Stream waters. The eddy-associated microbial community occupied a larger area than identified by temperature and salinity alone, increasing the predicted extent of eddy-associated biogeochemical processes. While the eddy formed on the continental shelf, after two weeks both environmental parameters and microbiome composition of the eddy were most similar to the Gulf Stream, suggesting the effect of environmental filtering on community assembly or physical mixing with adjacent Gulf Stream waters. In spite of the potential for eddy-driven upwelling to introduce nutrients and stimulate primary production, eddy surface waters exhibit lower chlorophyll a along with a distinct and less even microbial community, compared to the Gulf Stream. At the population level, the eddy microbiome exhibited differences among the cyanobacteria (e.g. lower Trichodesmium and higher Prochlorococcus) and in the heterotrophic alpha Proteobacteria (e.g. lower relative abundances of specific SAR11 phylotypes) versus the Gulf Stream. However, better delineation of the relative roles of processes driving eddy community assembly will likely require following the eddy and surrounding waters since inception. Additionally, sampling throughout the water column could better clarify the contribution of these mesoscale features to primary production and carbon export in the oceans. [ABSTRACT FROM AUTHOR]

Environmental DNA (eDNA) metabarcoding has been increasingly used to monitor the community assemblages of a wide variety of organisms. Here, we test the efficacy of eDNA metabarcoding to assess the composition of Iberian freshwater fishes, one of the most endangered groups of vertebrates in Spain. For this purpose, we sampled 12 sampling sites throughout one of Spain's largest basins, the Duero, which is home to approximately 70% of the genera and 30% of the primary freshwater fish in Spain. We sampled these sampling sites in the summer by using electrofishing, a traditional sampling method, and eDNA metabarcoding of river water samples using the mitochondrial 12S rRNA gene (12S) as a marker. We also resampled four of these sampling sites in autumn by eDNA. We compared the results obtained through eDNA metabarcoding with those of electrofishing surveys (ones conducted for the present study and past ones) and assessed the suitability of 12S as an eDNA metabarcoding marker for this group of freshwater fishes. We found that the 12S fragment, analysed for 25 Iberian species, showed sufficient taxonomic resolution to be useful for eDNA approaches, and even showed population-level differences in the studied populations across the tissue samples for Achondrostoma arcasii. In most cases, a greater number of species was detected through eDNA metabarcoding than through electrofishing. Based on our results, eDNA metabarcoding is a powerful tool to study the freshwater fish composition in the Iberian Peninsula and to unmask cryptic diversity. However, we highlight the need to generate a local genetic database for 12S gene for such studies and to interpret the results with caution when studying only mitochondrial DNA. Finally, our survey shows that the high detection sensitivity of eDNA metabarcoding and the non-invasiveness of this method allows it to act as a detection system for species of low abundance, such as early invasive species or species in population decline, two key aspects of conservation management of Spanish freshwater fishes. [ABSTRACT FROM AUTHOR]

Background: Serum or whole blood collection, processing, transport and storage still present significant challenges in low resource settings where mass surveillance is required to sustain disease elimination. Therefore, in this study, we explored the diagnostic efficacy of dried blood spots (DBS) as a minimally invasive and potentially cost-effective alternative sampling technique to whole blood sampling procedures for subsequent detection of Leishmania donovani antibodies or DNA. Methodology and principal findings: Archived serum, DNA samples from whole blood of visceral leishmaniasis (VL) cases and healthy controls, and DBS from corresponding cases and controls, were used. Both molecular and serological assays were optimized to detect L. donovani antibodies or DNA in DBS elute and results were compared against those obtained with whole blood. Serological assays (both rK28 ELISA and rK39 ELISA) of DBS samples showed sensitivity and specificity of 100% and had excellent agreement with results from whole blood samples (kappa value ranged from 0.98–1). Bland-Altman analysis of OD values from rK28-ELISA with DBS elute and patients' serum showed an excellent agreement (ICC = 0.9) whereas a good agreement (ICC = 0.8) was observed in the case of rK39-ELISA. However, qPCR and RPA of DBS samples had a diminished sensitivity of 76% and 68%, respectively, and poor agreement was observed with the whole blood samples. Conclusion: Our results demonstrate that DBS offer excellent diagnostic efficiency for serological assays and represent a viable alternative to whole blood sampling procedures. Author summary: Since the inception of the National Kala-azar Elimination Program (NKEP) in 2005, visceral leishmaniasis (VL) cases have declined significantly in Bangladesh. Bangladesh has achieved the elimination targets set by the WHO and continuous strides have been enforced by the government and non-government organizations in the present maintenance phase. To sustain the outcomes of the NKEP, move towards a zero-transmission goal and subvert the endgame challenges, an effective surveillance strategy is a primary focus. As part of this strategy, we examined whether using dried blood spots (DBS), rather than whole blood samples as currently recommended, could serve as a minimally invasive sampling technique prior to subsequent diagnostic tests. We determined the accuracy of molecular and serological assays using DBS samples compared with whole blood samples. While using DBS, excellent diagnostic accuracy was observed for serological assays (rK28 ELISA and rK39 ELISA), whereas sensitivity and specificity using molecular assays for DNA detection (qPCR and RPA) declined in comparison to whole blood. Given the low cost and field feasibility of DBS, we believe this simple sample collection technique coupled with serology-based detection of L. donovani meets the requirements for mass surveillance towards detecting residual VL transmission. [ABSTRACT FROM AUTHOR]

Introduction: Salmonella Typhi and Salmonella Paratyphi, fecal-oral transmitted bacterium, have temporally and geographically heterogeneous pathways of transmission. Previous work in Kathmandu, Nepal implicated stone waterspouts as a dominant transmission pathway after 77% of samples tested positive for Salmonella Typhi and 70% for Salmonella Paratyphi. Due to a falling water table, these spouts no longer provide drinking water, but typhoid fever persists, and the question of the disease's dominant pathway of transmission remains unanswered. Methods: We used environmental surveillance to detect Salmonella Typhi and Salmonella Paratyphi A DNA from potential sources of transmission. We collected 370, 1L drinking water samples from a population-based random sample of households in the Kathmandu and Kavre Districts of Nepal between February and October 2019. Between November 2019 and July 2021, we collected 380, 50mL river water samples from 19 sentinel sites on a monthly interval along the rivers leading through the Kathmandu and Kavre Districts. We processed drinking water samples using a single qPCR and processed river water samples using differential centrifugation and qPCR at 0 and after 16 hours of liquid culture enrichment. A 3-cycle threshold (Ct) decrease of Salmonella Typhi or Salmonella Paratyphi, pre- and post-enrichment, was used as evidence of growth. We also performed structured observations of human-environment interactions to understand pathways of potential exposure. Results: Among 370 drinking water samples, Salmonella Typhi was detected in 7 samples (1.8%) and Salmonella Paratyphi A was detected in 4 (1.0%) samples. Among 380 river water samples, Salmonella Typhi was detected in 171 (45%) and Salmonella Paratyphi A was detected in 152 (42%) samples. Samples located upstream of the Kathmandu city center were positive for Salmonella Typhi 12% of the time while samples from locations in and downstream were positive 58% and 67% of the time respectively. Individuals were observed bathing, washing clothes, and washing vegetables in the rivers. Implications: These results suggest that drinking water was not the dominant pathway of transmission of Salmonella Typhi and Salmonella Paratyphi A in the Kathmandu Valley in 2019. The high degree of river water contamination and its use for washing vegetables raises the possibility that river systems represent an important source of typhoid exposure in Kathmandu. Author summary: Understanding the dominant route of transmission of a pathogen is important for designing and implementing effective control strategies. Salmonella Typhi and Paratyphi A, which cause enteric fever, infect approximately 10 million people and cause over 100,000 deaths annually. In the Kathmandu Valley, prior work suggested ancient stone spouts used for drinking water were often contaminated and driving transmission of the diseases. However, many of these spouts no longer function, and people are still getting sick, suggesting other possible dominant pathways for enteric fever transmission. We tested drinking water from households in this area as well as local river water and found that only 7 drinking water samples were positive for Salmonella Typhi and 4 were positive for Salmonella Paratyphi A. We also tested river water and found many samples (>40%) tested positive for these bacteria. River water samples were not often positive upstream of Kathmandu city center (12% positive for Salmonella Typhi) but were often positive within the city center (58% positive for Salmonella Typhi) and in rural areas up to 10 km downstream of the city (67% positive for Salmonella Typhi). During sample collection, individuals were observed interacting with rivers by walking in them, washing clothes and washing vegetables for sale in markets. This study shows that drinking water may not be a primary driver of enteric fever transmission in the Kathmandu Valley, but that sewage contaminated river water may be a way disease transmits into the wider population. [ABSTRACT FROM AUTHOR]

In cut flowers, xylem occlusion or blockage by bacteria negatively affects water balance and postharvest quality. Many studies have used culture-based methods to examine bacterial populations in vase water and their effects on flower longevity. It is still unclear if and how bacterial communities at the 16S rRNA gene (16S) level change during the vase period and how such change might correlate with postharvest longevity. This study compared the sequences of 16S amplicons from 4 different types of flowers and their vase water over the course of 7 days (Rosa spp., Gerbera jamesonii, and two Lilium varieties). The relative abundance of plant chloroplast and mitochondria 16S decreased significantly over the course 7 days in all 4 flowers as bacterial diversity increased. Richness and evenness of the bacterial communities increased over time, as did the number of rare taxa and phylogenetic diversity. Bacterial communities varied with time, as well as by flower source, types, and sample location (water, stem surface, whole stem). Some taxa, such as Enterobacteriacea and Bradyhizobiaceae decreased significantly over time while others such as Pseudomonas spp. increased. For example, Pseudomonas veronii, implicated in soft rot of calla lily, increased in both whole stem samples and water samples from Gerbera jamesonii. Erwinia spp., which includes plant pathogenic species, also increased in water samples. This work highlights the dynamic and complex nature of bacterial succession in the flower vase ecosystem. More work is needed to understand if and how bacterial community structure can be managed to improve cut flower vase life. [ABSTRACT FROM AUTHOR]

Coastal estuarine systems may hold a large number of microplastic particles, which predators often mistake for prey. This study estimated the encounter rates between microplastics (alleged prey) and zooplankton having different feeding modes, trophic positions, swimming velocities, and perception distances, under calm and turbulent conditions, and during two seasons. Surface water samples were taken at 10/12 sites of the Sontecomapan lagoon, southern Gulf of Mexico, to quantify microplastic concentration. Zooplankton organisms considered were copepods, chaetognaths, and luciferids, common organisms in the lagoon. In June, at surface waters and during calm conditions, mean encounter rates were 1.5, 2450, and 980 particles per individual per hour, that is, for copepods, chaetognaths, and luciferids, respectively. When the wind blows (0.8 m s-1) encounter rates were 1.2, 1.4, and 2.6 times higher than in calm conditions. In October, mean encounter rates under calm conditions were 0.2, 355, and 142 particles per individual per hour, for copepods, chaetognaths, and luciferids; these values increase 1.3, 1.6, and 3.3 times when the wind blows (1.12 m s-1). The major number of encounters in June was due to a higher concentration of microplastics, despite the lower turbulent velocity. Regarding their trophic position, we propose that secondary consumers (chaetognaths and luciferids) are more affected because they could eat microplastics via contaminated prey or accidentally ingest them owing to confusion in the motion signals, especially under turbulent conditions. Another consequence of encounters could be the entanglement of microplastics in the body of the animals, especially in those with complex morphology, such as crustaceans. Encounters between zooplankton and microplastics do not always result in ingestion or entanglement, but the encounters are the first step in the case of occurrence. [ABSTRACT FROM AUTHOR]

Cipangopaludina chinensis Gray 1833 is an East Asian freshwater snail and invasive species in many parts of the world (Global Invasive Species Database, 2022). Within the UK, it was first found at the Pevensey Levels, Sussex, and has since been reported at a second site at Southampton Common, Hampshire. Both sites are designated as Sites of Special Scientific Interest (SSSI) for their wildlife importance. Although the impacts of this species within the UK have not yet been investigated several exotic parasites of the snail have been reported and research suggests that its presence can negatively impact native snail species. This is especially important at the Pevensey Levels due to the presence of the rare freshwater mollusc Anisus vorticulus (Little Whirlpool Rams's-horn snail). Here, we have developed a qPCR-based eDNA assay for the detection of C. chinensis and compared water samples tested for eDNA with results from manual survey of the ditches at the Pevensey Levels. Our eDNA analysis exhibited an overall observed percentage agreement of 80% with a kappa coefficient of agreement between manual and eDNA surveys of 0.59 (95% CI 0.31 to 0.88). Some samples which were qPCR negative for C. chinensis were positive by manual survey, and vice versa revealing the potential for improved overall detection rates when using a combination of manual and eDNA methodologies. eDNA analysis can therefore augment manual survey techniques for C. chinensis as a relatively quick and inexpensive tool for collecting presence and distribution data that could be used to inform further manual surveys and control measures within the ditches. [ABSTRACT FROM AUTHOR]

Background: In Brazilian Amazon, deforestation and other anthropogenic activities as a consequence of human occupation have created new and artificial larval habitats for anopheline mosquitoes, providing conditions for oviposition, development and expansion of malaria vector populations. Objectives: This study aimed to structurally characterize and describe the entomological and limnological parameters of Anopheles larval habitats from a malaria region in northern Brazilian Amazon. Methods: Fifty-two larval habitats were investigated in the District of Ilha de Santana, in the Brazilian state of Amapá, comprising fish farming tanks, ponds, and streams. For entomological parameters, the immature larvae were collected monthly from July 2019 to June 2020. For limnological parameters, the water samples were collected from 20 larval habitats during the sampling period. The data were analyzed using Generalized Linear Models, Multivariate analyses, and Kruskal-Wallis tests. Results: Fifty habitats were positive for Anopheles larvae and a total of nine species were collected. The fish farming tanks had the highest abundance of larvae compared with ponds and streams. Anopheles darlingi, Anopheles nuneztovari s.l. and Anopheles triannulatus were collected in 94% of the larval habitats and showed the highest positivity index. The degree of shade and the type of water of the breeding sites were important factors for the presence of the main malaria vector, A. darlingi. This species was the most affected by pH, total dissolved solids, electrical conductivity, and nitrate. Conclusions: Our findings indicate that fish farming tanks are major contributors to vector anopheline abundance and malaria transmission. Vector control strategies focused on these habitats are urgently needed. [ABSTRACT FROM AUTHOR]

The molecular detection of DNA fragments that are shed into the environment (eDNA) has become an increasingly applied tool used to inventory biological communities and to perform targeted species surveys. This method is particularly useful in habitats where it is difficult or not practical to visually detect or trap the target organisms. Central Texas Eurycea salamanders inhabit both surface and subterranean aquatic environments. Subterranean surveys are challenging or infeasible, and the detection of salamander eDNA in water samples is an appealing survey technique for these situations. Here, we develop and validate an eDNA assay using quantitative PCR for E. chisholmensis, E. naufragia, and E. tonkawae. These three species are federally threatened and constitute the Septentriomolge clade that occurs in the northern segment of the Edwards Aquifer. First, we validated the specificity of the assay in silico and with DNA extracted from tissue samples of both target Septentriomolge and non-target amphibians that overlap in distribution. Then, we evaluated the sensitivity of the assay in two controls, one with salamander-positive water and one at field sites known to be occupied by Septentriomolge. For the salamander-positive control, the estimated probability of eDNA occurrence (ψ) was 0.981 (SE = 0.019), and the estimated probability of detecting eDNA in a qPCR replicate (p) was 0.981 (SE = 0.011). For the field control, the estimated probability of eDNA occurring at a site (ψ) was 0.938 (95% CRI: 0.714–0.998). The estimated probability of collecting eDNA in a water sample (θ) was positively correlated with salamander relative density and ranged from 0.371 (95% CRI: 0.201–0.561) to 0.999 (95% CRI: 0.850– > 0.999) among sampled sites. Therefore, sites with low salamander density require more water samples for eDNA evaluation, and we determined that our site with the lowest estimated θ would require seven water samples for the cumulative collection probability to exceed 0.95. The estimated probability of detecting eDNA in a qPCR replicate (p) was 0.882 (95% CRI: 0.807–0.936), and our assay required two qPCR replicates for the cumulative detection probability to exceed 0.95. In complementary visual encounter surveys, the estimated probability of salamanders occurring at a known-occupied site was 0.905 (SE = 0.096), and the estimated probability of detecting salamanders in a visual encounter survey was 0.925 (SE = 0.052). We additionally discuss future research needed to refine this method and understand its limitations before practical application and incorporation into formal survey protocols for these taxa. [ABSTRACT FROM AUTHOR]

The vast diversity of microalgae imposes the challenge of identifying them through the most common and economical identification method, morphological identification, or through using the more recent molecular-level identification tools. Here we report an approach combining enrichment and metagenomic molecular techniques to enhance microalgae identification and identify microalgae diversity from environmental water samples. From this perspective, we aimed to identify the most suitable culturing media and molecular approach (using different primer sets and reference databases) for detecting microalgae diversity. Using this approach, we have analyzed three water samples collected from the River Nile on several enrichment media. A total of 37 microalgae were identified morphologically to the genus level. While sequencing the three-primer sets (16S rRNA V1-V3 and V4-V5 and 18S rRNA V4 region) and aligning them to three reference databases (GG, SILVA, and PR2), a total of 87 microalgae were identified to the genus level. The highest eukaryotic microalgae diversity was identified using the 18S rRNA V4 region and alignment to the SILVA database (43 genera). The two 16S rRNA regions sequenced added to the eukaryotic microalgae identification, 26 eukaryotic microalgae. Cyanobacteria were identified through the two sequenced 16S rRNA regions. Alignment to the SILVA database served to identify 14 cyanobacteria to the genera level, followed by Greengenes, 11 cyanobacteria genera. Our multiple-media, primer, and reference database approach revealed a high microalgae diversity that would have been overlooked if a single approach had been used over the other. [ABSTRACT FROM AUTHOR]

The oomycete Aphanomyces invadans causes epizootic ulcerative syndrome (EUS), a World Organization for Animal Health (WOAH)-listed disease that has seriously impacted a wide range of fish worldwide. Currently, only three conventional polymerase chain reaction (PCR) assays are recommended for the detection of A. invadans. The robust quantitative PCR (qPCR) assay has recently become more important due to its highly accurate nature and the applicability of qPCR-based environmental DNA (eDNA) detection in the monitoring of pathogens in aquatic environments. Therefore, in this study, we developed a novel TaqMan probe-based qPCR method to sensitively and quantitatively detect A. invadans. The assay limit of detection was determined using 10-fold serial dilutions of linearized A. invadans plasmid. Assay sensitivity was assessed in the presence of interfering substances and compared to three WOAH-listed primers using the mycelia and zoospores of A. invadans with and without fish muscle tissue. The assay specificity was also theoretically and experimentally assessed against other oomycetes, fish muscle tissue, and water samples. The assay's repeatability and reproducibility were determined. In this study, the limit of detection of the developed assay was 7.24 copies of A. invadans genomic DNA per reaction (95% confidence interval (CI): 2.75 to 19.05 copies/reaction). The assay showed the same sensitivity in the presence of other substances. Compared to the WOAH-recommended PCR assays, this assay had 10-times higher sensitivity for all tested samples. There were no cross-reactions with other closely related oomycetes, fish muscle, or water samples, indicating that the assay was highly specific for A. invadans. The repeatability and reproducibility tests showed little variation, ranging from 0.1–0.9% and 0.04–1.1%, respectively, indicating the high consistency, repeatability, and reliability of the developed assay. This highly rapid, sensitive, specific, and consistent EUS qPCR assay would be of importance in transboundary disease management and the monitoring of pathogens in aquatic environments. [ABSTRACT FROM AUTHOR]

Radon (222Rn), an inert gas, is considered a silent killer due to its carcinogenic characteristics. Dhaka city is situated on the banks of the Buriganga River, which is regarded as the lifeline of Dhaka city because it serves as a significant source of the city's water supply for domestic and industrial purposes. Thirty water samples (10 tap water from Dhaka city and 20 surface samples from the Buriganga River) were collected and analyzed using a RAD H2O accessory for 222Rn concentration. The average 222Rn concentration in tap and river water was 1.54 ± 0.38 Bq/L and 0.68 ± 0.29 Bq/L, respectively. All the values were found below the maximum contamination limit (MCL) of 11.1 Bq/L set by the USEPA, the WHO-recommended safe limit of 100 Bq/L, and the UNSCEAR suggested range of 4–40 Bq/L. The mean values of the total annual effective doses due to inhalation and ingestion were calculated to be 9.77 μSv/y and 4.29 μSv/y for tap water and river water, respectively. Although all these values were well below the permissible limit of 100 μSv/y proposed by WHO, they cannot be neglected because of the hazardous nature of 222Rn, especially considering their entry to the human body via inhalation and ingestion pathways. The obtained data may serve as a reference for future 222Rn-related works. [ABSTRACT FROM AUTHOR]

Background: Leptospirosis, a life-threatening disease in humans and animals, is one of the most widespread global zoonosis. Contaminated soil and water are the major transmission sources in humans and animals. Clusters of disease outbreaks are common during rainy seasons. Methodology/Principal findings: In this study, to detect the presence of Leptospira, we applied PCR, direct metagenomic sequencing, and enrichment culture followed by PCR and metagenomic sequencing on water and soil samples. Direct sequencing and enrichment cultures followed by PCR or sequencing effectively detected pathogenic and nonpathogenic Leptospira compared to direct PCR and 16S amplification-based metagenomic sequencing in soil or water samples. Among multiple culture media evaluated, Ellinghausen-McCullough-Johnson-Harris (EMJH) media containing antimicrobial agents was superior in recovering and detecting Leptospira from the environmental samples. Our results show that enrichment culture followed by PCR can be used to confirm the presence of pathogenic Leptospira in environmental samples. Additionally, metagenomic sequencing on enrichment cultures effectively detects the abundance and diversity of Leptospira spp. from environmental samples. Conclusions/Significance: The selection of methodology is critical when testing environmental samples for the presence of Leptospira. Selective enrichment culture improves Leptospira detection efficacy by PCR or metagenomic sequencing and can be used successfully to understand the presence and diversity of pathogenic Leptospira during environmental surveillance. Author summary: Leptospirosis, a life-threatening disease in humans and animals, is one of the most widespread global zoonosis. Contaminated soil and water are major sources of transmission in humans and animals. For this reason, clusters of disease outbreaks are common during the rainy season. In this study, Leptospira enrichment cultures followed by PCR and sequencing detected pathogenic and nonpathogenic Leptospira in soil and water samples. However, pathogenic and intermediate groups of Leptospira were more prevalent in soil samples tested. Metagenomic sequencing on enrichment culture was effective in detecting the abundance and diversity of various Leptospira spp. in environmental samples. Soil samples in proximity to water may be an ideal niche for Leptospira growth and survival and may be an appropriate sample of choice for testing. [ABSTRACT FROM AUTHOR]

Background: Schistosomiasis is a parasitic disease that is endemic in 78 countries and affects almost 240 million people worldwide. It has been acknowledged that an integrated approach that goes beyond drug treatment is needed to achieve control and eventual elimination of the disease. Improving hygiene has been encouraged by World Health Assembly, and one aspect of good hygiene is using soap during water-contact activities, such as bathing and doing laundry. This hygiene practice might directly reduce the skin exposure to cercariae at transmission sites. A systematic review was carried out to investigate the efficacy of soap against schistosome cercariae and to identify the knowledge gaps surrounding this topic. Methodology: Six online databases were searched between 5th and 8th July of 2021. Records returned from these databases were screened to remove duplicates, and the remaining records were classified by reading titles, abstracts, and full texts to identify the included studies. The results were categorised into two groups based on two different protective mechanisms of soap (namely, damage to cercariae and protection of skin). Conclusions: Limited research has been conducted on the efficacy of soap against schistosome cercariae and only 11 studies met the criteria to be included in this review. The review demonstrates that soap has the potential of protecting people against schistosome cercariae and there are two protective aspects: (1) soap affects cercariae adversely; (2) soap on the skin prevents cercariae from penetrating the skin, developing into adult worms and producing eggs. Both aspects of protection were influenced by many factors, but the differences in the reported experimental conditions, such as the cercarial endpoint measurement used and the cercaria numbers used per water sample, lead to low comparability between the previous studies. This review indicates that more evidence is needed to inform hygiene advice for people living in schistosomiasis endemic areas. Author summary: Schistosomiasis affects millions of people living in low- and middle-income countries lacking safe access to water, sanitation and hygiene (WASH). Infection occurs through skin-contact with water containing schistosome cercariae which are released from the snail intermediate hosts. The disease is mainly controlled through drug treatment with praziquantel in humans, but reinfection is possible if contact with cercaria-contaminated water continues. Despite all the efforts that have been made, this disease remains a major public health problem in disease-endemic regions. The use of soap during water contact, as part of good hygiene practice, may be an effective complementary control method as it might reduce the penetration of schistosome cercariae into the skin, thereby reducing the likelihood of reinfection. We conducted a systematic review to summarise previous research into the efficacy of soap against schistosome cercariae, and to identify current knowledge gaps to inform future studies. The information gathered from this review can provide policymakers with preliminary scientific evidence (e.g. the soap types, soap amounts, exposure times that are needed) to further emphasise the importance of effective soap usage in reducing the spread of schistosomiasis. [ABSTRACT FROM AUTHOR]

The diversity among bacteriophages depends on different factors like ecology, temperature conditions and genetic pool. Current study focused on isolation, identification and diversity of phages from 34 sewage water samples collected from two different wastewater treatment plants (WWTPs), King Saud University wastewater treatment plants (KSU-WWTP) and Manfoha wastewater treatment plants (MN-WWTP) in Riyadh, Saudi Arabia. Samples were analyzed by PCR and Next Generation Sequencing (NGS). Siphoviridae, Podoviridae and Myoviridae families were detected by family-specific PCR and highest prevalence of Myoviridae 29.40% was found at MN-WWTP followed by 11.76% at KSU-WWTP. Siphoviridae was detected 11.76% at MN-WWTP and 5.88% at KSU-WWTP. Lowest prevalence for Podoviridae family (5.88%) was recorded at MN-WWTP. Significant influence of temporal variations on prevalence of Myoviridae and Siphoviridae was detected in both WWTP and MN-WWTP, respectively. Highest phage prevalence was obtained in August (75%), followed by September (50%). Highest phage prevalence was recorded at a temperature range of 29–33°C. Significant influence of temperature on the prevalence of Myoviridae phages was detected at MN-WWTP. Four bacteriophages with various abundance levels were identified by NGS. Cronobacter virus Esp2949-1 was found first time with highest abundance (4.41%) in wastewater of Riyadh. Bordetella virus BPP1 (4.14%), Dickeya virus Limestone (1.55%) and Ralstonia virus RSA1 (1.04%) were also detected from samples of MN-WWTP. Highest occurrence of Bordetella virus BPP1 (67%) and (33.33%) was recorded at KSU-WWTP and MN-WWTP, respectively. Highest Bordetella virus BPP1 occurrence was recorded in September (50%) followed by August (40%). The findings of study showed new insights of phage diversity from wastewater sources and further large-scale data studies are suggested for comprehensive understanding. [ABSTRACT FROM AUTHOR]

Modern nautilids (Nautilus and Allonautilus) have often been studied by paleontologists to better understand the anatomy and ecology of fossil relatives. Because direct observations of these animals are difficult, the analysis of light stable isotopes (C, O) preserved in their shells has been employed to reveal their habitat and life history. We aim to (1) reconstruct the habitat depth of Nautilus macromphalus and (2) decipher the fraction of metabolic carbon in its shell by analyzing oxygen and carbon isotopes (δ18O, δ13C) in the septa of two specimens in combination with analyses of water samples from the area. Additionally, we investigate whether morphological changes during ontogeny are reflected in the isotopic values of the shells. Results reveal that the patterns of change of δ18O and δ13C in the septa of N. macromphalus pre- and post-hatching are consistent with previous studies. Values of δ18Owater range from 0.7 to 1.4‰ (VSMOW), with a maximum value coincident with a salinity maximum at ~150 m. We use the temperature and δ18Owater profiles to calculate equilibrium values of δ18Oaragonite with depth. Comparing these values with the measured δ18O of the septa shows that the habitat depth of N. macromphalus is ~140 m pre-hatching and ~370 m post-hatching. Using δ13C of shell carbonate and published data on metabolic carbon, the fraction of metabolic carbon is reconstructed as ~21% and 14% pre- and post-hatching, respectively. The reconstructed depth pre-hatching is slightly shallower than in N. pompilius from the Philippines and Fiji, but the post-hatching depth is similar. However, it is important to emphasize that these estimates represent average over time and space because nautilus is a mobile animal. Lastly, the changes in morphological parameters and the changes in δ13C and δ18O during ontogeny do not coincide except at hatching and at the onset of maturity. [ABSTRACT FROM AUTHOR]

Water molecules at the protein-small molecule interface often form hydrogen bonds with both the small molecule ligand and the protein, affecting the structural integrity and energetics of a binding event. The inclusion of these 'bridging waters' has been shown to improve the accuracy of predicted docked structures; however, due to increased computational costs, this step is typically omitted in ligand docking simulations. In this study, we introduce a resource-efficient, Rosetta-based protocol named "PlaceWaters" to predict the location of explicit interface bridging waters during a ligand docking simulation. In contrast to other explicit water methods, this protocol is independent of knowledge of number and location of crystallographic waters in homologous structures. We test this method on a diverse protein-small molecule benchmark set in comparison to other Rosetta-based protocols. Our results suggest that this coarse-grained, structure-based approach quickly and accurately predicts the location of bridging waters, improving our ability to computationally screen drug candidates. [ABSTRACT FROM AUTHOR]

This document is a correction notice for an article titled "Electron microscope loading and in situ nanoindentation of water ice at cryogenic temperatures" published in the journal PLoS ONE. The correction addresses an error in the first sentence of the Elastic modulus and hardness measurements subsection of the Results and Discussion section. The corrected sentence provides accurate information about the nanoindentation process. Additionally, the correction notice mentions that Figure 3 in the article is incorrect and provides a corrected version of the graph. The authors of the article are Renelle Dubosq, Eric Woods, Baptiste Gault, and James P. Best. [Extracted from the article]

Fine bubbles (FBs) have attracted significant attention in several research fields. Although some reports have argued that FB dispersion is useful as an oxygen (gas) carrier, only a few reports have examined its properties as an oxygen carrier using experimental data. As one of the reasons for this, there are no standard methods for measuring the oxygen content in FB dispersions. Conventional oxygen measurement methods have certain drawbacks in accuracy or speed; thus, it is difficult to use oxygen content as the primary outcome. In this study, we introduce a Clark-type polarographic oxygen electrode device (OXYG1-PLUS) for oxygen measurement, allowing the dilution of FB dispersion without the influence of ambient air and the adhesion of FBs on the electrode surface due to its special shape. First, the accuracy of our dilution method was evaluated using pure water as a sample, and it was confirmed that our method could measure with an accuracy of ±0.5 mg/L from the results with conventional dissolved oxygen meters. Second, the oxygen content in FB dispersion was evaluated with our method and a chemical titration method (Winkler's method), and it was found that our method could measure the oxygen content in FB dispersions quantitively. This method satisfies the easiness (4 steps) and quickness (within 8 min) for a wide range of oxygen contents (0 to 332 mg/L, theoretical range) with low coefficient variation (< 4.7%) and requires a small sample volume (50–500 μL); thus, it is a useful method for measuring the oxygen in FB dispersions. [ABSTRACT FROM AUTHOR]

Human leptospirosis involves the classic epidemiological triad (agent, host and environment); hence the investigations should include the knowledge on Leptospira within the animals and the environment. The objectives of this study are to explore the abundance of Leptospira in different climate zones of Sri Lanka and to describe the presence of Leptospira in the same water source at serial time points. First, water and soil samples were collected from different parts of Sri Lanka (Component-1); second, water sampling continued only in the dry zone (Component-2). Finally, serial water sampling from ten open wells was performed at five different time points (Component-3). Quantitative PCR of water and metagenomic sequencing of soil were performed to detect Leptospira. Three replicates for each sample were used for PCR testing, and positive result of two or more replicates was defined as 'strongly positive,' and one positive replicate was defined as positive. In the water and soil sample analysis in the whole country (Component-1), two out of 12 water sites were positive, and both were situated in the wet zone. Very small quantities of the genus Leptospira were detected by 16 amplicon analysis of soil in all 11 sites. In the dry zone water sample analysis (Component-2), only samples from 6 out of 26 sites were positive, of which one site was strongly positive. In the serial sample analysis (Component-3), Six, five, four, five, and six wells were positive in serial measurements. All wells were positive for at least one time point, while only one well was positive for all five time points. Proximity to the tank and greater distances from the main road were associated with strong positive results for Leptospira (P<0.05). The presence of Leptospira was not consistent, indicating the variable abundance of Leptospira in the natural environment. This intermittent nature of positivity could be explained by the repetitive contamination by animal urine. [ABSTRACT FROM AUTHOR]

Fluro(quinolones) is an important class of antibiotic used widely in both human and veterinary medicine. Resistance to fluro(quinolones) can be acquired by either chromosomal point mutations or plasmid-mediated quinolone resistance (PMQR). There is a lack of studies on the prevalence of PMQR in organisms from environmental sources in Bangladesh. In this study, we investigated the occurrence of PMQR genes in E. coli from various water sources and analysed associations between multi-drug resistance (MDR) and resistance to extended spectrum β-lactam antibiotics. We analysed 300 E. coli isolates from wastewaters of urban live-bird markets (n = 74) and rural households (n = 80), rural ponds (n = 71) and river water samples (n = 75) during 2017–2018. We isolated E. coli by filtering 100 ml of water samples through a 0.2μm cellulose membrane and incubating on mTEC agar media followed by identification of isolated colonies using biochemical tests. We selected one isolate per sample for detection of PMQR genes by multiplex PCR and tested for antibiotic susceptibility by disc diffusion. Clonal relatedness of PMQR-positive isolates was evaluated by enterobacterial repetitive intergenic consensus-PCR (ERIC-PCR). About 66% (n = 199) of E. coli isolates harbored PMQR-genes, predominantly qnrS (82%, n = 164) followed by aac(6')-lb-cr (9%, n = 17), oqxAB (7%, n = 13), qnrB (6%, n = 11) and qepA (4%, n = 8). Around 68% (n = 135) of PMQR-positive isolates were MDR and 92% (n = 183) were extended spectrum β-lactamase (ESBL)-producing of which the proportion of positive samples was 87% (n = 159) for blaCTX-M-1' 34% (n = 62) for blaTEM, 9% (n = 16) for blaOXA-1,blaOXA-47 and blaCMY-2, and 2% (n = 4) for blaSHV. Further, 16% (n = 32) of PMQR-positive isolates were resistant to carbapenems of which 20 isolates carried blaNDM-1. Class 1 integron (int1) was found in 36% (n = 72) of PMQR-positive E. coli isolates. PMQR genes were significantly associated with ESBL phenotypes (p≤0.001). The presence of several PMQR genes were positively associated with ESBL and carbapenemase encoding genes such as qnrS with blaCTXM-1 (p<0.001), qnrB with blaTEM (p<0.001) and blaOXA-1 (p = 0.005), oqxAB and aac(6')-lb-cr with blaSHV and blaOXA-1 (p<0.001), qnrB with blaNDM-1 (p<0.001), aac(6')-lb-cr with blaOXA-47 (p<0.001) and blaNDM-1 (p = 0.002). Further, int1 was found to correlate with qnrB (p<0.001) and qepA (p = 0.011). ERIC-PCR profiles allowed identification of 84 of 199 isolates with 85% matching profiles which were further grouped into 33 clusters. Only 5 clusters had isolates (n = 11) with identical ERIC-PCR profiles suggesting that PMQR-positive E. coli isolates are genetically heterogeneous. Overall, PMQR-positive MDR E. coli were widely distributed in aquatic environments of Bangladesh indicating poor wastewater treatment and highlighting the risk of transmission to humans and animals. [ABSTRACT FROM AUTHOR]

Saprolegniasis is an important disease in freshwater aquaculture, and is associated with oomycete pathogens in the genus Saprolegnia. Early detection of significant levels of Saprolegnia spp. pathogens would allow informed decisions for treatment which could significantly reduce losses. This study is the first to report the development of loop-mediated isothermal amplification (LAMP) for the detection of Saprolegnia spp. and compares it with quantitative PCR (qPCR). The developed protocols targeted the internal transcribed spacer (ITS) region of ribosomal DNA and the cytochrome C oxidase subunit 1 (CoxI) gene and was shown to be specific only to Saprolegnia genus. This LAMP method can detect as low as 10 fg of S. salmonis DNA while the qPCR method has a detection limit of 2 pg of S. salmonis DNA, indicating the superior sensitivity of LAMP compared to qPCR. When applied to detect the pathogen in water samples, both methods could detect the pathogen when only one zoospore of Saprolegnia was present. We propose LAMP as a quick (about 20–60 minutes) and sensitive molecular diagnostic tool for the detection of Saprolegnia spp. suitable for on-site applications. [ABSTRACT FROM AUTHOR]

Low frequency electric fields were exposed to various water samples using platinum electrodes mounted near the water surface. Responses were monitored using a spectro-radiometer and a contact-angle goniometer. Treatment of DI (deionized), EZ (Exclusion Zone), and bulk water with certain electromagnetic frequencies resulted in a drop of radiance persisting for at least half an hour. Compared to DI water, however, samples of EZ and bulk water showed lesser radiance drop. Contact-angle goniometric results confirmed that when treated with alternating electric fields (E = 600 ± 150 V/m, f = 7.8 and 1000 Hz), droplets of EZ and bulk water acquired different charges. The applied electric field interacted with EZ water only when electrodes were installed above the chamber, but not beneath. Further, when DI water interacted with an electric field applied from above (E = 600 ± 150 V/m, f = 75 Hz), its radiance profile became similar to that of EZ water. Putting these last two findings together, one can say that application of an electric field on DI water from above (E = 600 ± 150 V/m, f = 7.8 to 75 Hz) may induce a molecular ordering in DI water similar to that of EZ water. [ABSTRACT FROM AUTHOR]

On 4th December 2020, a sudden outbreak, with neurological symptoms like seizures, loss of consciousness etc., was reported in a town from south India. By 3rd day about 400 people were involved. A multi disciplinary team from our institute visited the site to investigate the outbreak. Based on the case history and clinical examination of the patients, the team suspected a probable diagnosis of an acute pesticide, heavy metal and/or mycotoxin exposure for which, biological samples (blood, urine) were collected from those who reported the symptoms as well as from a few who did not report symptoms (controls). To identify the source, water and food samples were collected. The samples were subjected to ICP-MS for heavy metal analysis, LC-MS/MS for pesticide analysis, microbiological analysis and ELISA-Kit method for aflatoxins if any. Clinical and dietary details were collected from a total of 112 participants, of which, 103 cases (77 active cases at Hospital and 26 recovered cases from community) and 9 were controls. A total of 109 biological samples, 36 water samples and food samples were collected. The mean age of the study participants was 29.2 years. Among cases, Seizures were seen in 84%, loss of consciousness in 66%, mental confusion in 35%, pinpoint pupil in 11%. Triazophos (organophosphate) pesticide was present in 74% of Blood samples and its metabolites were present in 98% of the urine samples collected from the cases. All the ten heavy metals investigated including lead, mercury and nickel were found to be within permissible limits except for a few samples. No presence of mycotoxins was observed in Food samples. Water samples which included Head pump and reservoir were free from pesticides; however, all water samples from households of cases had triazophos pesticide with a mean concentration of 1.00 ug/L. Thus, it was concluded that, the probable cause of outbreak was Triazophos (Organophosphate) pesticide contamination in water at the Household level. Regular surveillance for the presence of residual pesticides in soil, water and food with heightened vigour is recommended to prevent future outbreaks. [ABSTRACT FROM AUTHOR]

There is a need for recent information on intermediate snail hosts of schistosomes in The Gambia as the latest studies were conducted over three decades ago. This study assessed the incidence, species diversity, distribution and infection status of schistosome intermediate snail hosts in the country. Malacological surveys were conducted in all 5 regions of The Gambia: Central River Region (CRR), Upper River Region (URR), Western Region (WR), Lower River Region (LRR) and North Bank Region (NBR). Sampling of snails was undertaken at 114 sites that included permanent water bodies such as streams (bolongs), rice fields, irrigation canals and swamps; and temporal (seasonal) laterite pools. Ecological and physicochemical factors of sites were recorded. Snails were identified morphologically and screened for schistosome infections using molecular techniques. Freshwater snails were found at more than 50% (60/114) of sites sampled. While three species of Bulinus were collected, no Biomphalaria snails were found in any of the sites sampled. Of the total 2877 Bulinus snails collected, 75.9% were identified as Bulinus senegalensis, 20.9% as Bulinus forskalii and 3.2% as Bulinus truncatus. Seasonal pools produced the largest number of snails, and CRR was the region with the largest number of snails. Bulinus senegalensis was found more in seasonal pools as opposed to permanent sites, where B. forskalii and B. truncatus were observed to thrive. Bulinus snails were more common in seasonal sites where aquatic vegetation was present. In permanent sites, the abundance of snails increased with increase in water temperature and decrease in water pH. Bulinus senegalensis was found infected with both S. haematobium and S. bovis, while B. forskalii and B. truncatus had only S. bovis infection. While the human parasite S. haematobium was restricted to just four sites, the livestock parasite S. bovis had a much more widespread geographical distribution across both CRR and URR. This new information on the distribution of intermediate snail hosts of schistosomes in The Gambia will be vital for the national schistosomiasis control initiative. Author summary: Several studies have been conducted on intermediate snail hosts of schistosomes in The Gambia between the 1950s and 1980s, but few reports of similar studies in recent times are available. After The Gambia initiated its national control programme for schistosomiasis in 2014, the need arose for more and current information on schistosomiasis and the intermediate snail hosts involved indirectly in its transmission. We undertook a malacological survey of freshwater sites in all five regions (CRR, URR, WR, LRR and NBR) of The Gambia. While no Biomplalaria snails were found in any of the 114 sites sampled, three species of Bulinus snails were found. Of the total 2877 Bulinus snails collected, 75.9% were identified as Bulinus senegalensis, 20.9% as Bulinus forskalii and 3.2% as Bulinus truncatus. CRR had the highest number ofsnails amongst the regions and seasonal pools yielded the most snails amongst the habitat types sampled. Habitat type, water temperature and pH of sites were observed to have an influence on snail abundance. All three Bulinus snails were found infected with schistosome parasites. The human parasite (Schistosoma haematobium) had a much more restricted distribution as opposed to the livestock parasite (Schistosoma bovis) which had a wider geographical range. The study provides the first malacological report of intermediate snail hosts of schistosomes in The Gambia in over 3 decades and will be useful to the national schistosomiasis control programme. [ABSTRACT FROM AUTHOR]

Background: Pakistan has been experiencing intervals of sporadic cases and localized outbreaks in the last two decades. No proper study has been carried out in order to find out the environmental burden of toxigenic V. cholerae as well as how temporal and environmental factors associated in driving cholera across the country. Methods: We tested waste water samples from designated national environment surveillance sites in Pakistan with RT-PCR assay. Multistage sampling technique were utilized for samples collection and for effective sample processing Bag-Mediated Filtration system, were employed. Results were analysed by district and month wise to understand the geographic distribution and identify the seasonal pattern of V. cholera detection in Pakistan. Results: Between May 2019, and February 2020, we obtained and screened 160 samples in 12 districts across Pakistan. Out of 16 sentinel environmental surveillance sites, 15 sites showed positive results against cholera toxigenic gene with mostly lower CT value (mean, 34±2) and have significant difference (p < 0.05). The highest number of positive samples were collected from Sindh in month of November, then in June it is circulating in different districts of Pakistan including four Provinces respectively. Conclusion: V. cholera detection do not follow a clear seasonal pattern. However, the poor sanitation problems or temperature and rainfall may potentially influence the frequency and duration of cholera across the country. Occurrence of toxigenic V. cholerae in the environment samples showed that cholera is endemic, which is an alarming for a potential future cholera outbreaks in the country. [ABSTRACT FROM AUTHOR]

While the number of published marine studies using environmental DNA (eDNA) has increased substantially in recent years, marine fish surveys are still scarce. To examine the potential for eDNA to support marine fisheries monitoring surveys, we optimized an eDNA isolation method, developed a multispecies assay and tested it on eDNA samples collected along the Pacific coast of the United States. Four commercial DNA extraction kits that exploit the capability of the nucleic acids binding a solid phase (two using a silica matrix and two magnetic beads) as well an organic separation method were tested. A species-specific multiplex qPCR assay was developed and tested to simultaneously target Pacific hake (Merluccius productus), Pacific lamprey (Entosphenus tridentatus) and eulachon (Thaleichthys pacificus). The specificity of the assay was tested in silico, in vitro and in natura. Environmental DNA isolation using phenol:chloroform:isoamyl purification with a phase lock was optimized and yielded the highest amount of total and target DNA and was used to extract 46 marine water samples for the detection of the three species of interest. The multiplex qPCR assay used in the quantification process was also optimized to provide convenience and accuracy. Pacific hake was present in 44% of the eDNA samples while the other two species were absent. Here, we present a complete workflow for the simultaneous detection and quantification of multiple marine fish species using eDNA. This workflow supports large-scale at-sea sampling efforts with preservation at ambient temperatures and has demonstrated DNA extraction efficiency and reliability. The multiplex qPCR assay is shown to be sensitive and specific for the purposes of simultaneously monitoring the relative abundance of multiple targeted fish species. [ABSTRACT FROM AUTHOR]

Imidacloprid is a neonicotinoid insecticide widely used in the production and cultivation of crops. In recent years, the extensive use of imidacloprid in agricultural production has resulted in large amounts of pesticide residues in agricultural products and the environment. Therefore, it is necessary to establish a rapid, accurate, sensitive and convenient method for detecting imidacloprid pesticide residues to ensure the safety of agricultural products and the environment. To clarify how to use the molecular imprinting method for the electrochemical rapid residue detection of imidacloprid. This paper selected reduced graphene oxide and gold nanoparticles as modifiers modified on screen-printed carbon electrodes (SPCE) chitosan as a functional monomer, and imidacloprid as template molecule to prepare molecularly imprinted polymer, and applied this sensor to the residue detection of imidacloprid. The results showed that the concentration of imidacloprid showed a good linear relationship with the peak response current, and the detection limit of imidacloprid was 0.5 μM, while the sensor had good repeatability and interference resistance. The recoveries of imidacloprid spiked on three samples, mango, cowpea and water, were in the range of 90–110% (relative standard deviation, RSD<5%), which proved the practicality and feasibility of the assay established in this paper. The results of this paper can be used as a basis for the research on the detection of imidacloprid pesticide residues in food or environment. [ABSTRACT FROM AUTHOR]

Free-living amoeba (FLA) is widely distributed in the natural environment. Since these amoebae are widely found in various waters, they pose an important public health problem. The aim of this study was to detect the presence of Acanthamoeba, B. mandrillaris, and N. fowleri in various water resources by qPCR in Izmir, Turkey. A total of (n = 27) 18.24% Acanthamoeba and (n = 4) 2.7% N. fowleri positives were detected in six different water sources using qPCR with ITS regions (ITS1) specific primers. The resulting concentrations varied in various water samples for Acanthamoeba in the range of 3.2x105-1.4x102 plasmid copies/l and for N. fowleri in the range of 8x103-11x102 plasmid copies/l. The highest concentration of Acanthamoeba and N. fowleri was found in seawater and damp samples respectively. All 27 Acanthamoeba isolates were identified in genotype level based on the 18S rRNA gene as T4 (51.85%), T5 (22.22%), T2 (14.81%) and T15 (11.11%). The four positive N. fowleri isolate was confirmed by sequencing the ITS1, ITS2 and 5.8S rRNA regions using specific primers. Four N. fowleri isolates were genotyped (three isolate as type 2 and one isolate as type 5) and detected for the first time from water sources in Turkey. Acanthamoeba and N. fowleri genotypes found in many natural environments are straightly related to human populations to have pathogenic potentials that may pose a risk to human health. Public health professionals should raise awareness on this issue, and public awareness education should be provided by the assistance of civil authorities. To the best of our knowledge, this is the first study on the quantitative detection and distribution of Acanthamoeba and N. fowleri genotypes in various water sources in Turkey. [ABSTRACT FROM AUTHOR]

Background: Loa loa and Mansonella perstans–the causative agents of loiasis and mansonellosis—are vector-borne filarial parasites co-endemic in sub-Saharan Africa. Diagnosis of both infections is usually established by microscopic analysis of blood samples. It was recently established that the odds for detecting Plasmodium spp. is higher in capillary (CAP) blood than in venous (VEN) blood. In analogy to this finding this analysis evaluates potential differences in microfilaraemia of L. loa and M. perstans in samples of CAP and VEN blood. Methods: Recruitment took place between 2015 and 2019 at the CERMEL in Lambaréné, Gabon and its surrounding villages. Persons of all ages presenting to diagnostic services of the research center around noon were invited to participate in the study. A thick smear of each 10 microliters of CAP and VEN blood was prepared and analysed by a minimum of two independent microscopists. Differences of log2-transformed CAP and VEN microfilaraemia were computed and expressed as percentages. Furthermore, odds ratios for paired data were computed to quantify the odds to detect microfilariae in CAP blood versus in VEN blood. Results: A total of 713 participants were recruited among whom 52% were below 30 years of age, 27% between 30–59 years of age and 21% above 60 years of age. Male-female ratio was 0.84. Among 152 participants with microscopically-confirmed L. loa infection median (IQR) microfilaraemia was 3,650 (275–11,100) per milliliter blood in CAP blood and 2,775 (200–8,875) in VEN blood (p<0.0001), while among 102 participants with M. perstans this was 100 (0–200) and 100 (0–200), respectively (p = 0.44). Differences in linear models amount up to an average of +34.5% (95% CI: +11.0 to +63.0) higher L. loa microfilaria quantity in CAP blood versus VEN blood and for M. perstans it was on average higher by +24.8% (95% CI: +0.0 to +60.5). Concordantly, the odds for detection of microfilaraemia in CAP samples versus VEN samples was 1.24 (95% CI: 0.65–2.34) and 1.65 (95% CI: 1.0–2.68) for infections with L. loa and M. perstans, respectively. Conclusion: This analysis indicates that average levels of microfilaraemia of L. loa are higher in CAP blood samples than in VEN blood samples. This might have implications for treatment algorithms of onchocerciasis and loiasis, in which exact quantification of L. loa microfilaraemia is of importance. Furthermore, the odds for detection of M. perstans microfilariae was higher in CAP than in VEN blood which may pre-dispose CAP blood for detection of M. perstans infection in large epidemiological studies when sampling of large blood quantities is not feasible. No solid evidence for a higher odds of L. loa microfilariae detection in CAP blood was revealed, which might be explained by generally high levels of L. loa microfilaraemia in CAP and VEN blood above the limit of detection of 100 microfilariae/ml. Yet, it cannot be excluded that the study was underpowered to detect a moderate difference. Author summary: Microfilaraemia of Loa loa and Mansonella perstans was investigated by light microscopy in paired thick smears of capillary and venous blood; each sample was prepared using a standardised quantity of 10 microliters of blood and analysed by a minimum of two independent microscopists. Microfilaraemia was on average +34.5% (95% CI: +11.0 to +63.0) higher in capillary than in venous blood samples for L. loa and +24.8% (95% CI: +0.0 to +60.5) for M. perstans. This might have implications for treatment algorithms of onchocerciasis and loiasis, in which exact quantification of L. loa microfilaraemia is of importance. Furthermore, the odds for detection of M. perstans microfilariae was 65% higher in capillary than in venous blood which may pre-dispose capillary blood for detection of M. perstans infection in large epidemiological studies when sampling of large blood quantities is not feasible. No solid evidence for a higher odds of L. loa microfilariae detection in capillary blood was revealed, which might be explained by generally high levels of L. loa microfilaraemia in capillary and venous blood. [ABSTRACT FROM AUTHOR]

The use of sensitive methods is key for the detection of target taxa from trace amounts of environmental DNA (eDNA) in a sample. In this context, digital PCR (dPCR) enables direct quantification and is commonly perceived as more sensitive than endpoint PCR. However, endpoint PCR coupled with capillary electrophoresis (celPCR) potentially embodies a viable alternative as it quantitatively measures signal strength after PCR in Relative Fluorescence Units (RFU). Provided comparable levels of sensitivity are reached, celPCR permits the development of cost-efficient multiplex reactions, enabling the simultaneous detection of several target taxa. Here, we compared the sensitivity of singleplex and multiplex celPCR to dPCR for species-specific primer pairs amplifying mitochondrial DNA (COI) of fish species occurring in European freshwaters by analyzing dilution series of tissue extracts as well as field-collected water samples. Both singleplex and multiplex celPCR and dPCR displayed comparable sensitivity with reliable positive amplifications starting at two to 10 target DNA copies per μl extract. celPCR was suitable for quantifying target DNA and direct inference of copy numbers from RFU was possible after accounting for primer effects in linear mixed-effects models and calibration via dPCR. Furthermore, multiplex celPCR and dPCR were successfully used for the detection and quantification of fish-eDNA in field-collected water samples, confirming the results of the dilution series experiment and exemplifying the high sensitivity of the two approaches. The possibility of detection and quantification via multiplex celPCR is appealing for the cost-efficient screening of high sample numbers. The present results confirm the sensitivity of this approach thus enabling its application for future eDNA-based monitoring efforts. [ABSTRACT FROM AUTHOR]

Prevalence of toxigenic Vibrio cholerae O1 in aquatic reservoirs in Bangladesh apparently increases coinciding with the occurrence of seasonal cholera epidemics. In between epidemics, these bacteria persist in water mostly as dormant cells, known as viable but non-culturable cells (VBNC), or conditionally viable environmental cells (CVEC), that fail to grow in routine culture. CVEC resuscitate to active cells when enriched in culture medium supplemented with quorum sensing autoinducers CAI-1 or AI-2 which are signal molecules that regulate gene expression dependent on cell density. V. cholerae O1 mutant strains with inactivated cqsS gene encoding the CAI-1 receptor has been shown to overproduce AI-2 that enhance CVEC resuscitation in water samples. Since V. cholerae non-O1 non-O139 (non-cholera-vibrios) are abundant in aquatic ecosystems, we identified and characterized naturally occurring variant strains of V. cholerae non-O1 non-O139 which overproduce AI-2, and monitored their co-occurrence with V. cholerae O1 in water samples. The nucleotide sequence and predicted protein products of the cqsS gene carried by AI-2 overproducing variant strains showed divergence from that of typical V. cholerae O1 or non-O1 strains, and their culture supernatants enhanced resuscitation of CVEC in water samples. Furthermore, prevalence of V. cholerae O1 in the aquatic environment was found to coincide with an increase in AI-2 overproducing non-O1 non-O139 strains. These results suggest a possible role of non-cholera vibrios in the environmental biology of the cholera pathogen, in which non-O1 non-O139 variant strains overproducing AI-2 presumably contribute in resuscitation of the latent pathogen, leading to seasonal cholera epidemics. Importance. Toxigenic Vibrio cholerae which causes seasonal epidemics of cholera persists in aquatic reservoirs in endemic areas. The bacteria mostly exist in a dormant state during inter-epidemic periods, but periodically resuscitate to the active form. The resuscitation is enhanced by signal molecules called autoinducers (AIs). Toxigenic V. cholerae can be recovered from water samples that normally test negative for the organism in conventional culture, by supplementing the culture medium with exogenous AIs. V. cholerae belonging to the non-O1 non-O139 serogroups which do not cause cholera are also abundant in natural waters, and they are capable of producing AIs. In this study we characterized V. cholerae non-O1 non-O139 variant strains which overproduce an autoinducer called AI-2, and found that the abundance of the cholera pathogen in aquatic reservoirs correlates with an increase in the AI-2 overproducing strains. Our results suggest a probable role of these variant strains in the environmental biology and epidemiology of toxigenic V. cholerae, and may lead to novel means for surveillance, prevention and control of cholera. [ABSTRACT FROM AUTHOR]

Background: Bovine tuberculosis (bTB), caused by members of the Mycobacterium tuberculosis complex bacteria, mainly Mycobacterium bovis (M. bovis), is a major threat to public health and economic development. There has been no systematic epidemiological assessment concerning bTB in dairy cattle in China. Methodology/principal findings: Literature related to bTB in China was retrieved from China National Knowledge Infrastructure (CNKI), PubMed, ScienceDirect, VIP Chinese Journals Database, and Wan Fang Database to build the first meta-analysis for estimating the prevalence and infection moderators of bTB in dairy cattle in China. A total of 100 relevant studies published from 2010 to 2019 were included. We estimated the overall prevalence of bTB was 2.4% (95% CI: 2.1–2.8) during this decade. In the sampling year subgroup, the prevalence was lowest in 2017 or later at 0.8% (95% CI: 0.3–1.5). The lowest prevalence was 0.7% (95% CI: 0.5–1.0) in Northwestern China. The lowest prevalence was 2.1% (95% CI: 1.8–2.5) using SIT test. Heifer cows had the highest prevalence, which was 27.1% (95% CI: 9.7–49.2). The prevalence in scale farming was 3.7% (95% CI: 3.1–4.3), significantly higher than that in free-range farming (1.7%, 95% CI: 1.1–2.4). The prevalence of bTB was highest in summer at 4.0% (95% CI: 1.7–7.0). In addition, the influence of different geographical factors (altitude, longitude, latitude, precipitation, temperature, humidity) on the prevalence was analyzed. Conclusions/significance: The results showed that bTB was widespread in China but has been gradually reduced through concerted national intervention. It is suggested that different countries should formulate corresponding prevention and control measures according to the epidemic situation in its cattle industry. Enhanced monitoring of warm and humid areas may play an important role in reducing the incidence of bTB. In addition, when large-scale breeding is promoted, attention should be paid to standardizing breeding management and improving animal welfare to reduce the prevalence of bTB in cattle. Author summary: bTB is a neglected zoonotic disease caused by members of the Mycobacterium tuberculosis complex bacteria (M. bovis, mainly), which is also called "TB disease from bovine sources" when it occurs in humans. It is still widespread in China although the prevalence has gradually reduced through national intervention. Because beef and dairy products are important human dietary sources of protein, bTB has a great impact on public health and safety. We constructed the first meta-analysis to assess the epidemic of bTB in dairy cattle in China over the past 10 years and analyzed the potential moderators of bTB. A total of 100 studies were included. The results showed that bTB was common and unevenly distributed in China. The potential moderators affecting the bTB epidemic were region, sampling year, detection methods, age of dairy cattle, feeding mode, season, and certain geographical and climatic factors (longitude, precipitation, mean temperature, humidity, and altitude). These important findings may help in the formulation of policies to control the prevalence of bTB in cattle, thereby reducing the economic losses caused by bTB and reducing the threat of bTB to human health. [ABSTRACT FROM AUTHOR]

Leptospirosis is an emerging worldwide zoonotic disease, but the general biology of the causative agents is still poorly understood. Humans are an occasional host. The main risk factors are water-associated exposure during professional or recreational activities or during outbreaks in endemic areas. Detecting the presence of pathogenic bacteria in aquatic environments and their capacity to resist various inactivation processes are research fields that need to be further developed. In addition, the methods used for detecting and enumerating Leptospira still need to be improved. We aimed to describe a new quantitative polymerase chain reaction coupled to propidium monoazide treatment (PMAqPCR) that targets not only total Leptospira but also discriminates pathogenic from non-pathogenic Leptospira while also addressing PCR inhibitors, a frequently encountered problem when studying environmental water. In a second step, the killing efficiency of Leptospira to different treatments was tested and PMAqPCR compared to culture-based enumeration. This provided information about the effects of temperature, as well as ultraviolet and chlorine disinfection, that are both related to water treatment processes, in particular for the production of drinking water, on the persistence of both saprophytic and pathogenic Leptospira. Finally, PMAqPCR was used for the detection of Leptospira in freshwater samples for a proof-of-concept. In conclusion, our method could be used for routine freshwater monitoring and allows better evaluation of the presence of Leptospira, allowing evaluation of the bacterial dynamics in a designated area or assessment of the efficacy of water disinfection processes. [ABSTRACT FROM AUTHOR]

Nontuberculous mycobacteria (NTM) are waterborne pathogens commonly found in building water systems where they are a primary concern to vulnerable patient populations and can cause severe disease. The recovery of NTM from environmental samples can be a laborious undertaking and current pre-treatment methods and selective media lack sensitivity. We explored the use of the highly selective Rapidly Growing Mycobacteria (RGM) medium for culturing NTM from environmental water samples compared to existing methods. In total, 223 environmental water samples, including potable and non-potable water, were cultured for NTM using three culture media. In addition to direct culture on RGM medium, each sample was cultured on Middlebrook 7H10 medium and Mitchison 7H11 medium after pre-treatment with 0.2M KCl-HCl. Additionally, 33 distinct species of NTM were inoculated onto RGM medium and 7H10 medium in parallel to directly compare their growth. The use of RGM medium alone without pre-treatment provided a sensitivity (91%) comparable to that offered by culture on both 7H10 and 7H11 with acid pretreatment (combined sensitivity; 86%) with significantly less overgrowth and interference from other organisms on RGM medium. The average concentration of NTM observed on RGM medium alone was comparable to or greater than the NTM concentration on either medium alone or combined. Thirty-three species were examined in parallel and all tested strains of 27 of these species successfully grew on RGM medium, including 19 of 21 from the CDC's healthcare-associated infections species list. RGM medium was successful at recovering environmental NTM without a pre-treatment, greatly reducing labor and materials required to process samples. Simplification of culture processing for environmental NTM will allow for a better assessment of their presence in building water systems and the potential for reduced exposure of susceptible populations. [ABSTRACT FROM AUTHOR]

Wastewater based epidemiology (WBE) is increasingly used to provide decision makers with actionable data about community health. WBE efforts to date have primarily focused on sewer-transported wastewater in high-income countries, but at least 1.8 billion people in low- and middle-income countries (LMIC) use onsite sanitation systems such as pit latrines and septic tanks. Like wastewater, fecal sludges from such systems offer similar advantages in community pathogen monitoring and other epidemiological applications. To evaluate the distribution of enteric pathogens inside pit latrines–which could inform sampling methods for WBE in LMIC settings unserved by sewers–we collected fecal sludges from the surface, mid-point, and maximum-depth of 33 pit latrines in urban and peri-urban Malawi and analyzed the 99 samples for 20 common enteric pathogens via multiplex quantitative reverse transcription PCR. Using logistic regression adjusted for household population, latrine sharing, the presence of a concrete floor or slab, water source, and anal cleansing materials, we found no significant difference in the odds of detecting the 20 pathogens from the mid-point (adjusted odds ratio, aOR = 1.1; 95% confidence interval = 0.73, 1.6) and surface samples (aOR = 0.80, 95% CI = 0.54, 1.2) compared with those samples taken from the maximum depth. Our results suggest that, for the purposes of routine pathogen monitoring, pit latrine sampling depth does not strongly influence the odds of detecting enteric pathogens by molecular methods. A single sample from the pit latrines' surface, or a composite of surface samples, may be preferred as the most recent material contributed to the pit and may be easiest to collect. Author summary: Almost two billion people defecate into onsite sanitation facilities such as pit latrines. Evidence suggests these individuals are often at a disproportionately high risk of enteric infection compared to those served by piped sewerage. Public health surveillance in high-risk communities could monitor key pathogens in fecal sludges to inform which pathogens are circulating among those contributing waste. Representative surveys including multiple latrines could inform vaccination campaigns and interventions to limit the transmission of these pathogens, including eradication efforts, at a localized scale. A better understanding of how enteric pathogen detection may vary by pit latrine sampling depth would be useful to inform methods for fecal sludge collection. To refine methods for sampling in such surveys, we collected fecal sludges from the surface, mid-point, and maximum depth of pit latrines in Malawi. We found pit latrine depth did not significantly affect enteric pathogen detection by the methods we used. However, surface samples may be preferred due to their ease of collection and because the most recently deposited fecal material may be of greater interest in surveillance efforts that aim to inform pathogen carriage at the time of sampling. [ABSTRACT FROM AUTHOR]

Background: The impact of large scale Mass Drug Adminstration (MDA) of ivermectin on active onchocerciasis transmission by Simulium damnosum, which transmits the parasite O. volvulus is of great importance for onchocerciasis control programmes. We investigated in the Mbam river system area, the impact of MDA of ivermectin on entomological indices and also verify if there are river system factors that could have favoured the transmission of onchocerciasis in this area and contribute to the persistence of disease. We compared three independent techniques to detect Onchocerca larvae in blackflies and also analyzed the river system within 9 months post-MDA of ivermectin. Method: Simulium flies were captured before and after 1, 3, 6 and 9months of ivermectin-MDA. The biting rate was determined and 41% of the flies dissected while the rest were grouped into pools of 100 flies for DNA extraction. The extracted DNA was then subjected to O-150 LAMP and real-time PCR for the detection of infection by Onchocerca species using pool screening. The river system was analysed and the water discharge compared between rainy and dry seasons. Principal findings: We used human landing collection method (previously called human bait) to collect 22,274 adult female Simulium flies from Mbam River System. Of this number, 9,134 were dissected while 129 pools constituted for molecular screening. Overall biting and parous rates of 1113 flies/man/day and 24.7%, respectively, were observed. All diagnostic techniques detected similar rates of O. volvulus infection (P = 0.9252) and infectivity (P = 0.4825) at all monitoring time points. Onchocerca ochengi larvae were only detected in 2 of the 129 pools. Analysis of the river drainage revealed two hydroelectric dams constructed on the tributaries of the Mbam river were the key contributing factor to the high-water discharge during both rainy and dry seasons. Conclusion: Results from fly dissection (Microscopy), real-time PCR and LAMP revealed the same trends pre- and post-MDA. The infection rate with animal Onchocerca sp was exceptionally low. The dense river system generate important breeding sites that govern the abundance of Simulium during both dry and rainy seasons. Author summary: The presence of parasite strains that respond sub-optimally to an approved drug, favourable breeding sites for the vector and infected individual in an area, will surely provide conditions for continuous and persistent transmission of a disease despite a long-term control intervention. We investigated the impact of ivermectin on entomological indices within 9 months following a large-scale MDA in the Mbam river drainage. The river system factors that could have favoured abundance vector breeding and contribute to the persistence of disease transmission were also examined within the study period. We observed vector abundance and high entomological indices throughout the study period following ivermectin MDA. We also observed high water discharge along the main river of the drainage basin in both the rainy and dry seasons and this is due to the presence of two dams constructed upstream at Bamendjing and Mape to regularize the course of river Sanaga in view of generating hydroelectric power at Edea. Factors favouring continuous and persistent disease transmission are present in this drainage basin despite over 20 years of annual IVM-MDA. There is need for alternative control strategy in order to accelerate the fight against onchocerciasis in the area. [ABSTRACT FROM AUTHOR]

Monitoring aquatic species by identification of environmental DNA (eDNA) is becoming more common. To obtain quantitative eDNA datasets for individual species, organism-specific quantitative PCR (qPCR) assays are required. Here, we present detailed methodology of qPCR assay design and testing, including in silico, in vitro, and in vivo testing, and comment on the challenges associated with assay design and performance. We use the presented methodology to design assays for three important marine organisms common in the California Current Ecosystem (CCE): humpback whale (Megaptera novaeangliae), shortbelly rockfish (Sebastes jordani), and common murre (Uria aalge). All three assays have excellent sensitivity and high efficiencies ranging from 92% to 99%. However, specificities of the assays varied from species-specific in the case of common murre, genus-specific for the shortbelly rockfish assay, and broadly whale-specific for the humpback whale assay, which cross-amplified with other two other whale species, including one in a different family. All assays detected their associated targets in complex environmental water samples. [ABSTRACT FROM AUTHOR]

Introduction: Sources of infection of most cases of community-acquired Legionnaires' disease (CALD) are unknown. Objective: Identification of sources of infection of CALD. Setting: Berlin; December 2016–May 2019. Participants: Adult cases of CALD reported to district health authorities and consenting to the study; age and hospital matched controls. Main outcome measure: Percentage of cases of CALD with attributed source of infection. Methods: Analysis of secondary patient samples for monoclonal antibody (MAb) type (and sequence type); questionnaire-based interviews, analysis of standard household water samples for Legionella concentration followed by MAb (and sequence) typing of Legionella pneumophila serogroup 1 (Lp1) isolates; among cases taking of additional water samples to identify the infectious source as appropriate; recruitment of control persons for comparison of exposure history and Legionella in standard household water samples. For each case an appraisal matrix was filled in to attribute any of three source types (external (non-residence) source, residential non-drinking water (RnDW) source (not directly from drinking water outlet), residential drinking water (RDW) as source) using three evidence types (microbiological results, cluster evidence, analytical-comparative evidence (using added information from controls)). Results: Inclusion of 111 study cases and 202 controls. Median age of cases was 67 years (range 25–93 years), 74 (67%) were male. Among 65 patients with urine typable for MAb type we found a MAb 3/1-positive strain in all of them. Compared to controls being a case was not associated with a higher Legionella concentration in standard household water samples, however, the presence of a MAb 3/1-positive strain was significantly associated (odds ratio (OR) = 4.9, 95% confidence interval (CI) 1.7 to 11). Thus, a source was attributed by microbiological evidence if it contained a MAb 3/1-positive strain. A source was attributed by cluster evidence if at least two cases were exposed to the same source. Statistically significant general source types were attributed by calculating the population attributable risk (analytical-comparative evidence). We identified an external source in 16 (14%) cases, and RDW as source in 28 (25%). Wearing inadequately disinfected dentures was the only RnDW source significantly associated with cases (OR = 3.2, 95% CI 1.3 to 7.8) and led to an additional 8% of cases with source attribution, for a total of 48% of cases attributed. Conclusion: Using the appraisal matrix we attributed almost half of all cases of CALD to an infectious source, predominantly RDW. Risk for LD seems to be conferred primarily by the type of Legionella rather than the amount. Dentures as a new infectious source needs further, in particular, integrated microbiological, molecular and epidemiological confirmation. [ABSTRACT FROM AUTHOR]

Background: Food and water-borne illness caused by ingestion of (oo)cysts of Cryptosporidium and Giardia is one of the major health problems globally. Several methods are available to detect Giardia cyst and Cryptosporidium oocyst in food and water. Most of the available methods require a good laboratory facility and well-trained manpower and are therefore costly. There is a need of affordable and reliable method that can be easily implemented in resource limited settings. Methodology/Principle findings: We developed a smartphone based microscopic assay method to screen (oo)cysts of Cryptosporidium and Giardia contamination of vegetable and water samples. The method consisting of a ball lens of 1 mm diameter, white LED as illumination source and Lugols's iodine staining provided magnification and contrast capable of distinguishing (oo)cysts of Cryptosporidium and Giardia. The analytical performance of the method was tested by spike recovery experiments. The spike recovery experiments performed on cabbage, carrot, cucumber, radish, tomatoes, and water resulted in 26.8±10.3, 40.1±8.5, 44.4±7.3, 47.6±11.3, 49.2 ±10.9, and 30.2±7.9% recovery for Cryptosporidium, respectively and 10.2±4.0, 14.1±7.3, 24.2±12.1, 23.2±13.7, 17.1±13.9, and 37.6±2.4% recovery for Giardia, respectively. The spike recovery results are comparable with data obtained using commercial brightfield and fluorescence microscope methods. Finally, we tested the smartphone microscope system for detecting (oo)cysts on 7 types of vegetable (n = 196) and river water (n = 18) samples. Forty-two percent vegetable and thirty-nine percent water samples were found to be contaminated with Cryptosporidium oocyst. Similarly, thirty-one percent vegetable and thirty-three percent water samples were contaminated with Giardia cyst. Conclusions: The newly developed smartphone microscopic method showed comparable performance to commercial microscopic methods. The new method can be a low-cost and easy to implement alternative method for simultaneous detection of (oo)cysts in vegetable and water samples in resource limited settings. Author summary: Food and water-borne illness arising from the consumption of contaminated food and water are serious health hazards globally. Cryptosporidium and Giardia are the major food and water‒borne parasites. The infection occurs mainly by (oo)cyst phase of the parasites. People in developing countries are more vulnerable to these parasites where infection is more likely underdiagnosed and underreported due to limited resources for detection. There is need of a method that is affordable and easy to implement. In this study, we developed and optimized a novel smartphone microscope method that can detect and quantify the (oo)cyst of the parasites in food and water samples. The developed method is easy to implement and affordable and provides similar performance to the other commercially available microscopic methods. [ABSTRACT FROM AUTHOR]

Microwave heating of foods may alter their content. Yet, parents commonly heat infant formula in the microwave oven. The study aimed at understanding whether microwave heating of formula changes its fatty acid (FA) composition. Portions of infant formula were prepared and divided in three parts: control (sampled twice: at the start and after 30 minutes), microwave (sampled twice: after reaching 37°C and 50°C), and water bath (sampled twice: after reaching 37°C and 50°C). In thus obtained samples, a total profile of 25 FA was assessed using gas chromatography-mass spectrometry. Overall, fourteen portions were prepared, which were sampled 84 times yielding 2075 individual total FA level measurements. Few differences were identified between the microwave, control, and water bath groups. Microwave warming to 37°C was associated with increases of C12 (median increased by +0.40%, p = 0.0063), C14 (+0.05%, p = 0.0091), and C4 content (+6.90%, p = 0.0131). Microwaving up to 50°C slightly decreased C16:1trans (-5.00%, p = 0.0463) and C18:2trans1 (-5.13%, p = 0.0231). A paired comparison of pooled control and microwaved samples revealed increases in C12 (+0.18%, p = 0.0490) as well as a loss of C18:2trans1 (also -5.13%, p = 0.0073) and C18:2trans3 (-5.56%, p = 0.0042) after microwave treatment. C18:2trans1 (-2.63%, p = 0.0132) and C18:3trans1 (-2.26%, p = 0.0434) were lower in microwaved samples compared with the water bath. A slightly lower C18:2 content was found in the water bath samples than in the control groups (-0.11%, p = 0.0430). None of these differences would remain significant after a correction for multiple comparisons. Microwave heating of infant formula to 37°C or 50°C might marginally alter its total FA profile. Further studies are required to determine whether it alters the rate of free radical formation. [ABSTRACT FROM AUTHOR]

Lake Kivu, East Africa, is well known for its huge reservoir of dissolved methane (CH4) and carbon dioxide (CO2) in the stratified deep waters (below 250 m). The methane concentrations of up to ~ 20 mmol/l are sufficiently high for commercial gas extraction and power production. In view of the projected extraction capacity of up to several hundred MW in the next decades, reliable and accurate gas measurement techniques are required to closely monitor the evolution of gas concentrations. For this purpose, an intercomparison campaign for dissolved gas measurements was planned and conducted in March 2018. The applied measurement techniques included on-site mass spectrometry of continuously pumped sample water, gas chromatography of in-situ filled gas bags, an in-situ membrane inlet laser spectrometer sensor and a prototype sensor for total dissolved gas pressure (TDGP). We present the results of three datasets for CH4, two for CO2 and one for TDGP. The resulting methane profiles show a good agreement within a range of around 5–10% in the deep water. We also observe that TDGP measurements in the deep waters are systematically around 5 to 10% lower than TDGP computed from gas concentrations. Part of this difference may be attributed to the non-trivial conversion of concentration to partial pressure in gas-rich Lake Kivu. When comparing our data to past measurements, we cannot verify the previously suggested increase in methane concentrations since 1974. We therefore conclude that the methane and carbon dioxide concentrations in Lake Kivu are currently close to a steady state. [ABSTRACT FROM AUTHOR]

Background: Schistosomiasis is a water-based disease acquired through contact with cercaria-infested water. Communities living in endemic regions often rely on parasite-contaminated freshwater bodies for their daily water contact activities, resulting in recurring schistosomiasis infection. In such instances, water treatment can provide safe water on a household or community scale. However, to-date there are no water treatment guidelines that provide information on how to treat water containing schistosome cercariae. Here, we rigorously test the effectiveness of chlorine against Schistosoma mansoni cercariae. Method: S. mansoni cercariae were chlorinated using sodium hypochlorite under lab and field condition. The water pH was controlled at 6.5, 7.0 or 7.5, the water temperature at 20°C or 27°C, and the chlorine dose at 1, 2 or 3 mg/l. Experiments were conducted up to contact times of 45 minutes. 100 cercariae were used per experiment, thereby achieving up to 2-log10 inactivations of cercariae. Experiments were replicated under field conditions at Lake Victoria, Tanzania. Conclusion: A CT (residual chlorine concentration x chlorine contact time) value of 26±4 mg·min/l is required to achieve a 2-log10 inactivation of S. mansoni cercariae under the most conservative condition tested (pH 7.5, 20°C). Field and lab-cultivated cercariae show similar chlorine sensitivities. A CT value of 30 mg·min/l is therefore recommended to disinfect cercaria-infested water, though safety factors may be required, depending on water quality and operating conditions. This CT value can be achieved with a chlorine residual of 1 mg/l after a contact time of 30 minutes, for example. This recommendation can be used to provide safe water for household and recreational water activities in communities that lack safe alternative water sources. Author summary: Schistosomiasis is a preventable yet serious disease in many developing countries. Although schistosomiasis control strategies include the provision of water and sanitation facilities, specific WASH solutions are often lacking. Control programs generally focus on preventive chemotherapy with praziquantel which is effective in killing adult worms in humans but does not prevent re-infection. Communities lacking safe alternative water sources therefore have no choice but to rely on unsafe water for their daily water contact activities, even if this exposes them to high risk of infection. Water treatment can provide communities with safe water on a household or community scale. However, to-date there are no guidelines for treating cercaria-contaminated water. We rigorously tested the effectiveness of chlorine against S. mansoni cercariae in lab and field settings. The resulting chlorination recommendation can be used to treat water and provide schistosomiasis-endemic communities with safe water facilities (e.g. laundry or bathing stations). [ABSTRACT FROM AUTHOR]