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Introduction SARS-CoV-2 RNA is excreted in feces of most patients and therefore viral load in wastewater can be used as a surveillance tool to develop an early warning system to help and manage future pandemics. Methods We collected wastewater from 24 random locations at Bangkok city center and 26 nearby suburbs from July to December 2020. SARS-CoV-2 RNA copy numbers were measured using real-time polymerase chain reaction (PCR). Results SARS-CoV-2 RNA was detected in wastewater from both the city center and suburbs. Except for July, there were no significant differences in copy numbers between the city center and suburbs. Between October and November, a sharp rise in copy number was observed in both places followed by two to three times increase in December, related to SARS-CoV-2 cases reported for same month. Conclusions Our study provided the first dataset related to SARS-CoV-2 viral RNA in the wastewater of Bangkok. Our results suggest that wastewater could be used as a complementary source for detecting viral RNA and predicting upcoming outbreaks and waves.

Background/Aims: Intermedin (IMD) is a novel peptide with significant vasodilator and cardiac protective actions similar to the related peptide adrenomedullin (ADM). Unlike those of ADM the actions and expression of IMD in endothelial cells are poorly characterised. ADM expression can be increased during cardiovascular disease/stress in vitro and in vivo where it may have a role in several cardiovascular protective actions. To characterise IMD mRNA expression cultured human aortic endothelial cells (HAEC) were stressed by removing serum and bicarbonate, and the addition of hydrogen peroxide. The responses were compared to those of ADM mRNA. We also compared the effects of ADM and IMD on caspase activity and cell viability, and investigated if IMD actions could be altered by a CGRP receptor antagonist. Methods/Results: Using the cell immunoblot assay, immunoreactive IMD was shown to be secreted by HAEC. IMD mRNA expression was also detected in HAEC grown in endothelial growth media (but at markedly lower levels than that of ADM). Absence of bicarbonate, a redox-mediated regulator of endothelial response to various stresses, increased IMD mRNA and ADM mRNA expression. However IMD mRNA, but not ADM mRNA, was markedly increased over time in HAEC in conditions of cell stress including incubation with serum-free Dulbecco’s modified Eagle’s medium (DMEM) and in response to hydrogen peroxide (H₂O₂). These vigorous responses in IMD mRNA expression were further enhanced by incubation in 5% serum in DMEM without bicarbonate, but in a selective manner since ADM expression was suppressed by serum. We also observed that IMD mRNA was markedly increased and ADM mRNA suppressed in HAEC following a period of suspension and replating. Finally, we observed that IMD, like ADM, increased cell viability in HAEC in DMEM without serum but only IMD reduced caspase activity, perhaps via and a yet to be defined receptor system. Conclusion: HAECs express IMD mRNA and secrete IMD peptide. IMD mRNA expression is markedly dependent on metabolic conditions and is selectively regulated in a contrary fashion to ADM mRNA. IMD mRNA expression in endothelial cells is markedly sensitive to oxidative stress, and IMD peptide itself has antiapoptotic activity in human endothelial cells. Our data suggest that IMD has a different role to ADM and may perform a protective function in humans.

Peritoneal dialysis inevitability results in activation of inflammatory processes and its efficiency is highly variable between patients. An improved method to isolate biomarkers and study pathophysiological mechanisms in peritoneal dialysis effluent (PDE) is expected to be of much benefit for the development of this treatment approach and help with patient management. Extracellular vesicles (EVs) are released as part of normal cellular processes. Their proteome is expected to reflect both type and health of their cell of origin. Although there is a significant interest in using EVs for “liquid biopsies”, little is reported of their presence or composition in plentiful dialysis waste fluids, including peritoneal dialysis effluent (PDE). Here we determined the presence of EVs in PDE and subsequently characterized their proteome. EVs were first isolated from PDE using differential centrifugation, then a further enrichment using size exclusion chromatography (SEC) was performed. The presence of EVs was demonstrated using transmission electron microscopy, and their particle counts were investigated using nanoparticle tracking analysis and dynamic light scattering. Using tandem mass spectrometry, marker proteins from three types of EVs i.e. apoptotic bodies, ectosomes, and exosomes were identified. The proteomic results demonstrated that the isolation of EVs by differential centrifugation helped enrich for over 2,000 proteins normally masked by abundant proteins in PDE such as albumin and SEC markedly further improved the isolation of low abundant proteins. Gene ontology analysis of all identified proteins showed the marked enrichment of exosome and membrane-associated proteins. Over 3,700 proteins were identified in total, including many proteins with known roles in peritoneal pathophysiology. This study demonstrated the prominence of EVs in PDE and their potential value as a source of biomarkers for peritoneal dialysis patients.

Despite strengthened antimicrobial therapy, biofilm infections of Acinetobacter baumannii are associated with poor prognosis and limited therapeutic options. Assessing antibiotics on planktonic bacteria can result in failure against biofilm infections. Currently, antibiotics to treat biofilm infections are administered empirically, usually without considering the susceptibility of the biofilm objectively before beginning treatment. For effective therapy to resolve biofilm infections it is essential to assess the efficacy of commonly used antibiotics against biofilms. Here, we offer a robust and simple assay to assess the efficacy of antibiotics against biofilms. In the present work, we carefully optimized the incubation time, detection range, and fluorescence reading mode for resazurin-based viability staining of biofilms in 96-well-plates and determined minimal biofilm eradication concentrations (MBECs) for A. baumannii isolates from patients with chronic infection. By applying this assay, we demonstrated that antibiotic response patterns varied uniquely within the biofilm formation of various clinical samples. MBEC-50 and 75 have significant discriminatory power over minimum inhibitory concentrations for planktonic suspensions to differentiate the overall efficiency of an antibiotic to eradicate a biofilm. The present assay is an ideal platform on which to assess the efficacy of antibiotics against biofilms in vitro to pave the way for more effective therapy.

Treatment of infections by Pseudomonas aeruginosa forming biofilms after antimicrobial testing on planktonic bacteria can result in substantial failure. Therefore, we offer a robust and simple experimental platform to test the impact of antimicrobials on biofilms. Antibiotic response patterns varied uniquely within biofilm formation capacity and minimal biofilm eradication concentrations (MBECs) has a significantly better discriminatory power than minimum inhibitory concentrations (MICs) to differentiate the overall efficiency of antibiotics to eradicate biofilm. Our resazurinbased 96-well-plate platform is able to emulate bacterial responses to antibiotics under biofilm conditions in a fast, simple, and cost-effective screening method adaptable to automation, and warrants trials in the clinic.

Treatment of infections of Pseudomonas aeruginosa forming biofilms after antimicrobial testing on planktonic bacteria can result in substantial failure. Therefore, we offer a robust and simple experimental platform to test the impact of antimicrobials on biofilms. Antibiotic response patterns varied uniquely within biofilm formation capacity and minimal biofilm eradication concentrations (MBECs) has a significantly better discriminatory power than minimum inhibitory concentrations (MICs) to differentiate the overall efficiency of antibiotics to eradicate biofilm. Our resazurin-based 96-well-plate platform is able to emulate bacterial responses to antibiotics under biofilm conditions in a fast, simple, and cost-effective screening method adaptable to automation, and warrants trials in the clinic.

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