Adeno-Associated Virus 2 and Human Adenovirus F41 in Wastewater during Outbreak of Severe Acute Hepatitis in Children, Ireland.

During April-July 2022, outbreaks of severe acute hepatitis of unknown etiology (SAHUE) were reported in 35 countries. Five percent of cases required liver transplantation, and 22 patients died. Viral metagenomic studies of clinical samples from SAHUE cases showed a correlation with human adenovirus F type 41 (HAdV-F41) and adeno-associated virus type 2 (AAV2). To explore the association between those DNA viruses and SAHUE in children in Ireland, we quantified HAdV-F41 and AAV2 in samples collected from a wastewater treatment plant serving 40% of Ireland's population. We noted a high correlation between HAdV-F41 and AAV2 circulation in the community and SAHUE clinical cases. Next-generation sequencing of the adenovirus hexon in wastewater demonstrated HAdV-F41 was the predominant HAdV type circulating. Our environmental analysis showed increased HAdV-F41 and AAV2 prevalence in the community during the SAHUE outbreak. Our findings highlight how wastewater sampling could aid in surveillance for respiratory adenovirus species.

SARS-CoV-2 variant trends in Ireland: Wastewater-based epidemiology and clinical surveillance.

SARS-CoV-2 RNA quantification in wastewater is an important tool for monitoring the prevalence of COVID-19 disease on a community scale which complements case-based surveillance systems. As novel variants of concern (VOCs) emerge there is also a need to identify the primary circulating variants in a community, accomplished to date by sequencing clinical samples. Quantifying variants in wastewater offers a cost-effective means to augment these sequencing efforts. In this study, SARS-CoV-2 N1 RNA concentrations and daily loadings were determined and compared to case-based data collected as part of a national surveillance programme to determine the validity of wastewater surveillance to monitor infection spread in the greater Dublin area. Further, sequencing of clinical samples was conducted to determine the primary SARS-CoV-2 lineages circulating in Dublin. Finally, digital PCR was employed to determine whether SARS-CoV-2 VOCs, Alpha and Delta, were quantifiable from wastewater. No lead or lag time was observed between SARS-CoV-2 wastewater and case-based data and SARS-CoV-2 trends in Dublin wastewater significantly correlated with the notification of confirmed cases through case-based surveillance preceding collection with a 5-day average. This demonstrates that viral RNA in Dublin's wastewater mirrors the spread of infection in the community. Clinical sequence data demonstrated that increased COVID-19 cases during Ireland's third wave coincided with the introduction of the Alpha variant, while the fourth wave coincided with increased prevalence of the Delta variant. Interestingly, the Alpha variant was detected in Dublin wastewater prior to the first genome being sequenced from clinical samples, while the Delta variant was identified at the same time in clinical and wastewater samples. This work demonstrates the validity of wastewater surveillance for monitoring SARS-CoV-2 infections and also highlights its effectiveness in identifying circulating variants which may prove useful when sequencing capacity is limited.

An assessment of the performance of municipal constructed wetlands in Ireland.

While performance assessments of constructed wetlands sites around the world have appraised their capacity for effective removal of organics, a large variance remains in these sites' reported ability to retain nutrients, which appears to depend on differences in design, operation and climate factors. Nutrient retention is a very important objective for constructed wetlands, to avoid eutrophication of aquatic environments receiving their effluents. This study assessed the performance of constructed wetlands in terms of nutrient retention and associated parameters under the humid conditions of Ireland's temperate maritime climate. A review of the performance of 52 constructed wetland sites from 17 local authorities aimed to identify the best performing types of constructed wetlands and the treatment factors determining successful compliance with environmental standards. Data analysis compared effluent results from constructed wetlands with secondary free surface flow or tertiary horizontal subsurface flow, hybrid systems and integrated constructed wetlands with those from small-scale mechanical wastewater treatment plants of the same size class. Nutrient concentrations in effluents of constructed wetlands were negatively correlated (p < .01) with specific area, i.e. the ratio of surface area and population equivalents. The latest generation of integrated constructed wetlands, which had applied design guidelines issued by the Department of the Environment, performed best. Storm management design features improved treatment performance of constructed wetlands significantly (p < .05) for total suspended solids concentrations and exceedance frequency of limit values for total nitrogen. Mechanical wastewater treatment plants, secondary free surface water and tertiary horizontal subsurface flow wetlands showed a very large variance in effluent concentrations for organic and nutrient parameters. E. coli numbers in effluents were lowest for integrated constructed wetlands with an arithmetic mean of 89 MPN/100 ml. Despite Ireland's humid climate, some constructed wetland sites achieved long or frequent periods of zero effluent discharge and thus did not transfer any waterborne pollution to their receptors during these periods.

Cytotoxicity Study of Textile Fabrics Impregnated With CuO Nanoparticles in Mammalian Cells.

Copper and copper compounds have multifunctional properties (antibacterial, antiviral, and antifungal) with promising applications. Copper in its nanoparticle (Cu NPs) forms has been widely used in various industrial and commercial applications. In the current research, the cytotoxic effects of textile fabrics impregnated with copper oxide nanoparticles (CuO NPs) were studied in mammalian cell lines. CuO NPs were impregnated onto textile substrates using 2 different techniques: the sonochemical generation and impregnation of NPs from metal complexes ( insitu) and a "throwing the stones" technology using commercially prepared CuO NPs. The cytotoxicity of these 2 textile fabric types was assayed on human dermal fibroblast (HDF) cells and human hepatocellular carcinoma cells (HepG2) and was evaluated by indirect contact using an MTT assay. The impregnated fabrics were not exposed to the cells, rather their leachates were used to test cytotoxicity. The fabrics were soaked into the growth media for up to 7 days, and the leachates from day 1 and day 7 were incubated with the cell lines for 24 hours prior to the testing. The discharge or leaching from antimicrobial nanomaterials into the surroundings and surface waters is posing a serious environmental threat, which needs to be addressed. Hence, with regard to product safety, it is a good approach to study the fabric leachates rather than the intact material. The results showed that CuO NPs are not toxic to HDF cells. However, cytotoxicity was seen in HepG2 cells with cell viability decreasing by 20% to 25% for all the fabrics after 24 hours.

Effect of sonication frequency on the disruption of algae.

In this study, the efficiency of ultrasonic disruption of Chaetoceros gracilis, Chaetoceros calcitrans, and Nannochloropsis sp. was investigated by applying ultrasonic waves of 0.02, 0.4, 1.0, 2.2, 3.3, and 4.3 MHz to algal suspensions. The results showed that reduction in the number of algae was frequency dependent and that the highest efficiency was achieved at 2.2, 3.3, and 4.3MHz for C. gracilis, C. calcitrans, and Nannochloropsis sp., respectively. A review of the literature suggested that cavitation, rather than direct effects of ultrasonication, are required for ultrasonic algae disruption, and that chemical effects are likely not the main mechanism for algal cell disruption. The mechanical resonance frequencies estimated by a shell model, taking into account elastic properties, demonstrated that suitable disruption frequencies for each alga were associated with the cell's mechanical properties. Taken together, we consider here that physical effects of ultrasonication were responsible for algae disruption.

Combined Effect of Ultrasound and Ozone on Bacteria in Water.

The aim of this study is to assess the synergetic effect of combined ultrasound and ozone treatment on the biological disinfection of water on a large-scale application using viable plate counts and flow cytometry. Escherichia coli B bacteria in saline suspension was treated using a commercially available combined ultrasound and ozone system (USO3 (Ultrasonic Systems Gmbh)) for 16 min. Two analytical methods were used to assess the results in terms of live and dead cells in the bulk liquid: standard viable plate counting recorded in terms of colony forming units per milliliter and flow cytometry. In the latter case 1 mL of bacterial suspension was stained simultaneously with the fluorescent stains SYTO9 and propidium iodide (PI). Transmission electron microscopy was used to generate images identifying the biological effects of different treatments using ultrasound and ozone on bacterial cell walls. Results demonstrated that treatment with ozone alone (1 mg/L) resulted in a significant reduction (93%) in the number of live cells after 16 min treatment whereas ultrasound alone showed only a small reduction (24%). However, a combination of ozone and ultrasound showed a synergistic effect and enhanced the inactivation to 99% after 4 min. A combined ultrasound and ozone treatment of bacterial suspensions using a commercial system affords a promising method for water disinfection that is better than treatment using either method alone. Standard viable plate count analysis is normally used to assess the effectiveness of disinfection treatments; however flow cytometry proved to be a more sensitive method to determine the actual effects in terms of not only live and dead cells but also damaged cells. This type of analysis (cell damage) is difficult if not impossible to achieve using traditional plate counting methodology.

Effect of ultrasonic frequency and power on the disruption of algal cells.

In this work the effect of ultrasonic waves on suspensions of Chlamydomonas concordia and Dunaliella salina have been investigated at frequencies of 20, 585, 864 and 1146 kHz and at different acoustic powers. Results showed that the reduction in algal numbers was dependent on both frequency and acoustic power. The order of efficiency of the ultrasonic disruption of C. concordia at different frequencies was 20 < 580 < 864 < 1146 kHz, and for D. salina was 20< 580 ≅ 864 ⩽ 1146 kHz. It is clear that high-frequency sonication is more effective than conventional low-frequency sonication for the disruption of cells for both species. Results showed that suitable disruption frequencies for each algae were associated with the mechanical properties of the cell. The frequency dependence of the efficiency of algae disruption on the mechanical resonances of both the algae cell is discussed in terms of bubble oscillation in an ultrasonic field.

Chitosan and chitosan-ZnO-based complex nanoparticles: formation, characterization, and antibacterial activity.

Nanostructured chitosan (CS) and a chitosan-Zn based (Zn-CS) complex have been synthesized and simultaneously deposited on cotton fabrics using ultrasound. SEM measurements revealed that the coating consists of nanoparticles (NPs) of ca. 40 nm in diameter, homogeneously dispersed along the yarns. XANES studies pointed out that the complex consisted of a less than 2.1 nm ZnO core to which the chitosan was bonded. Additionally, FTIR measurements indicated the in situ formation of a Zn-CS complex which is the only deposited material on the cotton surface. The antibacterial properties of the CS and Zn-CS coated textiles were tested against Gram positive and Gram negative bacteria species. More than two-fold increase of the antibacterial activity of Zn-CS NP coated textiles was detected as compared to the fabrics treated with the sonochemically synthesized CS NPs alone. The sonochemical technique appears to be a suitable method for producing organic NPs of soluble compounds, without loss of their intrinsic properties, i.e. the antimicrobial activity of chitosan. Moreover, hybrid nanoorganometallic particles were simultaneously synthesized and deposited on cotton sonochemically.

Evaluation of the mechanisms of the effect of ultrasound on Microcystis aeruginosa at different ultrasonic frequencies.

Blooms of cyanobacteria are now considered to be a common environmental issue. They are hazardous to both domestic and wild animals and humans. Current treatments are unable to effectively control such blooms as they become tolerant to biocides and it is difficult to degrade cyanobacterial toxins in water. Alternative methods for control are currently under investigation. One potential effective method is ultrasonic irradiation. Ultrasound inactivates algal and cyanobacteria cells through cavitation by generating extreme conditions, resulting in a number of physical, mechanical and chemical effects. The aim of this study was to investigate the effect of ultrasound at different frequencies on Microcystis aeruginosa. Flow cytometry was used to measure cyanobacterial metabolic cell viability in addition to the more commonly used haemocytometry, optical density and fluorimetry. Results indicate low frequency 20 kHz ultrasound with high intensity (0.0403 W cm(-3)) is effective for the inactivation of cyanobacterial cells. Higher frequencies of 580 kHz (0.0041 W cm(-3)) also resulted in an inactivation effect, but 1146 kHz (0.0018 W cm(-3)) showed a declumping effect as evidenced by flow cytometry. Ultrasonic treatment over time under different sonication conditions demonstrates the following: 1. Acoustic cavitation via mechanical effects can induce sufficient shear forces to directly rupture cyanobacteria cells. 2. At higher ultrasonic frequencies the mechanical energy of cavitation is less but a larger proportion of free radicals are produced from the ultrasonic degradation of water, which chemically attacks and weakens the cyanobacteria cell walls. 3. At higher frequencies free radicals also damage chlorophyll a leading to a loss in photosynthetic cell viability. 4. At low powers ultrasonic energy results in declumping of cyanobacteria.

Effect of ultrasonic frequency and power on algae suspensions.

Cyanobacteria are photosynthetic bacteria with some characteristics of algae. Some cyanobacteria produce toxins that have been shown to be hazardous to both animals and humans. Previous research has demonstrated power ultrasound can provide a suitable method to control algae blooms although the optimum ultrasonic parameter settings have not been determined to ensure an effective and energy efficient treatment. In this work the effect of ultrasound on suspensions of Microcystis aeruginosa has been investigated at the following frequencies 20, 40, 580, 864 and 1146 kHz. Results showed that the reduction in algal numbers is dependent on both frequency and intensity. In order to quantify the effect we have defined the efficiency of the ultrasonic control of algae at a specific frequency as: (% inactivation of the algae) / (ultrasonic intensity applied). When this is applied to the results at different frequencies the order of efficiency for algae reduction is 20 < 1146 < 864 < 580 kHz. This suggests that ultrasound can offer a suitable method for algae inactivation or control but the sonication conditions must be taken into account.