A 30-day follow-up study on the prevalence of SARS-COV-2 genetic markers in wastewater from the residence of COVID-19 patient and comparison with clinical positivity

Wastewater based epidemiology (WBE) is an important tool to fight against COVID-19 as it provides insights into the health status of the targeted population from a small single house to a large municipality in a cost-effective, rapid, and non-invasive way. The implementation of wastewater based surveillance (WBS) could reduce the burden on the public health system, management of pandemics, help to make informed decisions, and protect public health. In this study, a house with COVID-19 patients was targeted for monitoring the prevalence of SARS-CoV-2 genetic markers in wastewa-ter samples (WS) with clinical specimens (CS) for a period of 30 days. RT-qPCR technique was employed to target non-structural (ORF1ab) and structural-nucleocapsid (N) protein genes of SARS-CoV-2, according to a validated experimental protocol. Physiological, environmental, and biological parameters were also measured following the American Public Health Association (APHA) standard protocols. SARS-CoV-2 viral shedding in wastewater peaked when the highest number of COVID-19 cases were clinically diagnosed. Throughout the study period, 7450 to 23,000 gene copies/1000 mL were detected, where we identified 47 % (57/120) positive samples from WS and 35 % (128/360) from CS. When the COVID-19 patient number was the lowest (2), the highest CT value (39.4; i.e., lowest copy number) was identified from WS. On the other hand, when the COVID-19 patients were the highest (6), the lowest CT value (25.2 i.e., highest copy numbers) was obtained from WS. An advance signal of increased SARS-CoV-2 viral load from the COVID-19 patient was found in WS earlier than in the CS. Using customized primer sets in a traditional PCR approach, we confirmed that all SARS-CoV-2 variants identified in both CS and WS were Delta variants (B.1.617.2). To our knowledge, this is the first follow-up study to determine a temporal relationship be-tween COVID-19 patients and their discharge of SARS-CoV-2 RNA genetic markers in wastewater from a single house including all family members for clinical sampling from a developing country (Bangladesh), where a proper sewage system is lacking. The salient findings of the study indicate that monitoring the genetic markers of the SARS-CoV-2 virus in wastewater could identify COVID-19 cases, which reduces the burden on the public health system during COVID-19 pandemics.Peer reviewe

Wastewater-based epidemiological surveillance to monitor the prevalence of SARS-CoV-2 in developing countries with onsite sanitation facilities

Wastewater-based epidemiology (WBE) has emerged as a valuable approach for forecasting disease outbreaks in developed countries with a centralized sewage infrastructure. On the other hand, due to the absence of well-defined and systematic sewage networks, WBE is challenging to implement in developing countries like Bangladesh where most people live in rural areas. Identification of appropriate locations for rural Hotspot Based Sampling (HBS) and urban Drain Based Sampling (DBS) are critical to enable WBE based monitoring system. We investigated the best sampling locations from both urban and rural areas in Bangladesh after evaluating the sanitation infrastructure for forecasting COVID-19 prevalence. A total of 168 wastewater samples were collected from 14 districts of Bangladesh during each of the two peak pandemic seasons. RT-qPCR commercial kits were used to target ORF1ab and N genes. The presence of SARS-CoV-2 genetic materials was found in 98% (165/168) and 95% (160/168) wastewater samples in the first and second round sampling, respectively. Although wastewater effluents from both the marketplace and isolation center drains were found with the highest amount of genetic materials according to the mixed model, quantifiable SARS-CoV-2 RNAs were also identified in the other four sampling sites. Hence, wastewater samples of the marketplace in rural areas and isolation centers in urban areas can be considered the appropriate sampling sites to detect contagion hotspots. This is the first complete study to detect SARS-CoV-2 genetic components in wastewater samples collected from rural and urban areas for monitoring the COVID-19 pandemic. The results based on the study revealed a correlation between viral copy numbers in wastewater samples and SARS-CoV-2 positive cases reported by the Directorate General of Health Services (DGHS) as part of the national surveillance program for COVID-19 prevention. The findings of this study will help in setting strategies and guidelines for the selection of appropriate sampling sites, which will facilitate in development of comprehensive wastewater-based epidemiological systems for surveillance of rural and urban areas of low-income countries with inadequate sewage infrastructure.This research was supported by Water Aid Bangladesh, North South University, Dhaka, COVID-19 Diagnostic Lab, Department of Microbiology, Noakhali Science and Technology University (NSTU), Noakhali, Bangladesh, the International Training Network of Bangladesh University of Engineering and Technology (ITN-BUET) - Centre for Water Supply and Waste Management, and KTH Royal Institute of Technology, Sweden. We acknowledge the sincere help and support of the staff and volunteers of NSTU-COVID-19 Diagnostic Lab, Noakhali Science and Technology University, Bangladesh during the different phases of the study. PB and MTI acknowledge the Life Science Technology Platform, Science for Life Laboratory for the seed funding to initiate the wastewater-based epidemiological studies for SARS-CoV-2 in Bangladesh. We would also like to acknowledge the two anonymous reviewers for their critical comments as well as their thoughtful insights, which has significantly improved the manuscript.Peer reviewe

Neuropharmacological and antibacterial effects of the ethyl acetate extract of Diospyros malabarica (Ebenaceae) seeds

Abstract Background Diospyros malabarica is a well known flowering plant indigenous to Indian subcontinent which is used in folklore medicine for several purposes. Our study is designed to assess the neuropharmacological and antibacterial efficacy of the ethyl acetate extract of D. malabarica seeds. Methods The behavioral anxiolytic activities of the extract were assessed by using open field (OFT), hole cross (HCT), elevated plus maze (EPZ), hole board (HBT), light dark test (LDT); and antidepressant activities through forced swimming (FST) and tail suspension test (TST). Antimicrobial potential was assessed through disc diffusion method. Results In OFT and HCT, the extract treated groups significantly (*p < 0.05) decrease the movement of animals when compared to vehicle-treated group. Higher dose (400 mg/kg b.w.) of extract greatly increased the spending time in open arm of EPZ, which endorses anxiolytic-like behavior of extract. The observed effect may be due to binding of any phytoconstituent with GABAA receptor. HBT and LDT results support the exploratory behavior of mice. The extract significantly decreased the immobility time in FST (20.71% for 200 mg/kg, and 31.59% for 400 mg/kg extract) and TST, which indicates the occupancy of antidepressant-active constituents. Gram negative bacteria were susceptible to extract than Gram positive strains; however the antimicrobial effect is not significant, hence trivial to declare. Conclusion Our study demonstrates the possession of significant anxiolytic and antidepressant effects of D. malabarica extract which could be helpful for drug development program. Before potential therapeutic use, finding of the exact phytoconstituents with their mechanisms, and clinical trial are recommended

Evaluation of antioxidant and antibacterial activities of Crotalaria pallida stem extract

Abstract Background Antioxidants play an important role in protecting cellular damage by reactive oxygen species. The aim of the present study is to assess the phytochemical nature, antioxidant and antibacterial activities of petroleum ether, chloroform, methanol, and aqueous extracts of Crotalaria pallida stem. Methods The preliminary screening of the various extracts was carried out using standard methods. Total phenolic content (TPC) was determined by the modified Folin-Ciocalteu method and antioxidant activities were assayed using 2,2-diphenyl-1-picrylhydrazyl (DPPH) radical scavenging activity, reducing power assay, total antioxidant capacity (TAC) and reduction of ferric ions. Antibacterial activity of different extracts were assayed against Gram-positive and Gram-negative bacterial strains by observing the zone of inhibition using disc diffusion method, where Staphylococcus aureus, Bacillus cereus, Escherichia coli and Pseudomonas aeruginosa were used as bacterial strains. Results Petroleum ether, chloroform, methanol and aqueous extracts of stem exhibited remarkable antioxidant activity in terms of all the assays tested. Petroleum ether extract (PEE) showed DPPH radical scavenging activity with IC50 value of 126.96 µg/ml. In antibacterial assay, the PEE and chloroform extract (CE) showed mild to moderate antibacterial activity against all the bacterial strains except B. cereus while methanol extract (ME) showed no inhibitory effect against any of the tested bacteria. Conclusion PEE, CE and ME of C. pallida stem were found to be the most effective free radical quencher and a potent source of natural antioxidants as well as antibacterial activity. Thus justifying their traditional use in green therapeutics

Characterization of phytoconstituents and evaluation of antimicrobial activity of silver-extract nanoparticles synthesized from Momordica charantia fruit extract

Abstract Background Our present study was conducted to characterize the phytoconstituents present in the aqueous extract of Momordica charantia and evaluate the antimicrobial efficacy of silver-extract nanoparticles (Ag-Extract-NPs). Methods Silver nanoparticles (AgNPs) were prepared by reducing AgNO3; and NaBH4 served as reducing agent. After screening of phytochemicals; AgNPs and aqueous extract were mixed thoroughly and then coated by polyaniline. These NPs were characterized by using Visual inspection, UV spectroscopy, FTIR, SEM and TEM techniques. Antimicrobial activities were assessed against Staphylococcus aureus, Salmonella typhi, Escherichia coli and Pseudomonas aeruginosa. Results Aqueous extract of M. charantia fruits contain alkaloid, phenol, saponin etc. UV–Vis spectrum showed strong absorption peak around 408 nm. The presence of –CH, −NH, −COOH etc. stretching in FTIR spectrum of Ag-Extract-NPs endorsed that AgNPs were successfully capped by bio-compounds. SEM and TEM result revealed that synthesized NPs had particle size 78.5–220 nm. Ag-Extract-NPs showed 34.6 ± 0.8 mm zone of inhibition against E. coli compared to 25.6 ± 0.5 mm for ciprofloxacin. Maximum zone of inhibition for Ag-Extract-NPs were 24.8 ± 0.7 mm, 26.4 ± 0.4 mm, 7.4 ± 0.4 mm for S. aureus, P. aeruginosa and S. typhi. We found that Ag-Extract-NPs have much better antibacterial efficacy than AgNPs and M. charantia extract has individually. It is also noticed that gram negative bacteria (except S. typhi) are more susceptible to Ag-Extract-NPs than gram positive bacteria. Conclusion Ag-Extract-NPs showed strong antibacterial activity. In order to make a reliable stand for mankind, further study is needed to consider determining the actual biochemical pathway by which AgNPs-extracts exert their antimicrobial effect

Additional file 1: Table S1. of Characterization of phytoconstituents and evaluation of antimicrobial activity of silver-extract nanoparticles synthesized from Momordica charantia fruit extract

Antibacterial efficacy of M. charantia, AgNPs and Ag-Extract-NPs. (XLSX 13 kb

Pharmacophore-based virtual screening, quantum mechanics calculations, and molecular dynamics simulation approaches identified potential natural antiviral drug candidates against MERS-CoV S1-NTD

Middle East respiratory syndrome coronavirus (MERS-CoV) is a highly infectious zoonotic virus first reported into the human population in September 2012 on the Arabian Peninsula. The virus causes severe and often lethal respiratory illness in humans with an unusually high fatality rate. The N-terminal domain (NTD) of receptor-binding S1 subunit of coronavirus spike (S) proteins can recognize a variety of host protein and mediates entry into human host cells. Blocking the entry by targeting the S1-NTD of the virus can facilitate the development of effective antiviral drug candidates against the pathogen. Therefore, the study has been designed to identify effective antiviral drug candidates against the MERS-CoV by targeting S1-NTD. Initially, a structure-based pharmacophore model (SBPM) to the active site (AS) cavity of the S1-NTD has been generated, followed by pharmacophore-based virtual screening of 11,295 natural compounds. Hits generated through the pharmacophore-based virtual screening have re-ranked by molecular docking and further evaluated through the ADMET properties. The compounds with the best ADME and toxicity properties have been retrieved, and a quantum mechanical (QM) based density-functional theory (DFT) has been performed to optimize the geometry of the selected compounds. Three optimized natural compounds, namely Taiwanhomoflavone B (Amb23604132), 2,3-Dihydrohinokiflavone (Amb23604659), and Sophoricoside (Amb1153724), have exhibited substantial docking energy >−9.00 kcal/mol, where analysis of frontier molecular orbital (FMO) theory found the low chemical reactivity correspondence to the bioactivity of the compounds. Molecular dynamics (MD) simulation confirmed the stability of the selected natural compound to the binding site of the protein. Additionally, molecular mechanics generalized born surface area (MM/GBSA) predicted the good value of binding free energies (ΔG bind) of the compounds to the desired protein. Convincingly, all the results support the potentiality of the selected compounds as natural antiviral candidates against the MERS-CoV S1-NTD

Additional file 1 of Emergence of multidrug-resistant Bacillus spp. derived from animal feed, food and human diarrhea in South-Eastern Bangladesh

Additional file 1: Figure S1. Cultural morphology and Grams staining  of isolated Bacillus spp. Figure S2.16srDNA gene of the isolated Bacillus spp. by PCR test. Figure S3. Toxin genes (nheA, nheB, nheC, cytK) of isolated Bacillus spp. by PCR test. Figure S4. Toxin genes (hblA, hblC, hblD, entFM) of isolated Bacillus spp. by PCR test. Figure S5. Antibiotic resistance genes of isolated Bacillus spp. by PCR test. Table S1. Strains and source of selected Bacillus spp. Table S2. Biochemical characteristics of Bacillus spp. Table S3. Primers of toxin gene and 16srRNA gene and PCR protocol used in this study. Table S4. Overall antimicrobial susceptibility profile of Bacillus spp. Table S5. Overall antimicrobial susceptibility profiles of 7 Bacillus species. Table S6. Distributions of antibiotic genes among animal feed, food and diarrhea. Table S7. Prevalence of ARGs of Bacillus spp. in animal feed, food and diarrhea. Table S8. Prevalence of ARGs of 7 Bacillus species. Table S9. Distributions of ARGs among 7 Bacillus species. Table S10. MDR profiles of B. cereus, B. subtilis, B. amyloliquefaciens, B. licheniformis, B. thuringiensis, B. megaterium and B. coagulans. Table S11. Species wise percentage of MAR index >0.2. Table S12. Sample wise percentage of MAR index >0.2. Table S13. MIC break point of Antibiotic used. Table S14. PCR protocol of antibiotic resistant gene primer used in this study

Genome-wide study of globally distributed respiratory syncytial virus (RSV) strains implicates diversification utilizing phylodynamics and mutational analysis

Abstract Respiratory syncytial virus (RSV) is a common respiratory pathogen that causes mild cold-like symptoms and severe lower respiratory tract infections, causing hospitalizations in children, the elderly and immunocompromised individuals. Due to genetic variability, this virus causes life-threatening pneumonia and bronchiolitis in young infants. Thus, we examined 3600 whole genome sequences submitted to GISAID by 31 December 2022 to examine the genetic variability of RSV. While RSVA and RSVB coexist throughout RSV seasons, RSVA is more prevalent, fatal, and epidemic-prone in several countries, including the United States, the United Kingdom, Australia, and China. Additionally, the virus's attachment glycoprotein and fusion protein were highly mutated, with RSVA having higher Shannon entropy than RSVB. The genetic makeup of these viruses contributes significantly to their prevalence and epidemic potential. Several strain-specific SNPs co-occurred with specific haplotypes of RSVA and RSVB, followed by different haplotypes of the viruses. RSVA and RSVB have the highest linkage probability at loci T12844A/T3483C and G13959T/C2198T, respectively. The results indicate that specific haplotypes and SNPs may significantly affect their spread. Overall, this analysis presents a promising strategy for tracking the evolving epidemic situation and genetic variants of RSV, which could aid in developing effective control, prophylactic, and treatment strategies

A 30-day follow-up study on the prevalence of SARS-COV-2 genetic markers in wastewater from the residence of COVID-19 patient and comparison with clinical positivity

Wastewater based epidemiology (WBE) is an important tool to fight against COVID-19 as it provides insights into the health status of the targeted population from a small single house to a large municipality in a cost-effective, rapid, and non-invasive way. The implementation of wastewater based surveillance (WBS) could reduce the burden on the public health system, management of pandemics, help to make informed decisions, and protect public health. In this study, a house with COVID-19 patients was targeted for monitoring the prevalence of SARS-CoV-2 genetic markers in wastewa-ter samples (WS) with clinical specimens (CS) for a period of 30 days. RT-qPCR technique was employed to target non-structural (ORF1ab) and structural-nucleocapsid (N) protein genes of SARS-CoV-2, according to a validated experimental protocol. Physiological, environmental, and biological parameters were also measured following the American Public Health Association (APHA) standard protocols. SARS-CoV-2 viral shedding in wastewater peaked when the highest number of COVID-19 cases were clinically diagnosed. Throughout the study period, 7450 to 23,000 gene copies/1000 mL were detected, where we identified 47 % (57/120) positive samples from WS and 35 % (128/360) from CS. When the COVID-19 patient number was the lowest (2), the highest CT value (39.4; i.e., lowest copy number) was identified from WS. On the other hand, when the COVID-19 patients were the highest (6), the lowest CT value (25.2 i.e., highest copy numbers) was obtained from WS. An advance signal of increased SARS-CoV-2 viral load from the COVID-19 patient was found in WS earlier than in the CS. Using customized primer sets in a traditional PCR approach, we confirmed that all SARS-CoV-2 variants identified in both CS and WS were Delta variants (B.1.617.2). To our knowledge, this is the first follow-up study to determine a temporal relationship be-tween COVID-19 patients and their discharge of SARS-CoV-2 RNA genetic markers in wastewater from a single house including all family members for clinical sampling from a developing country (Bangladesh), where a proper sewage system is lacking. The salient findings of the study indicate that monitoring the genetic markers of the SARS-CoV-2 virus in wastewater could identify COVID-19 cases, which reduces the burden on the public health system during COVID-19 pandemics.Peer reviewe