A Computational Approach Applied to the Study of Potential Allosteric Inhibitors Protease NS2B/NS3 from Dengue Virus.

Dengue virus (DENV) is a danger to more than 400 million people in the world, and there is no specific treatment. Thus, there is an urgent need to develop an effective method to combat this pathology. NS2B/NS3 protease is an important biological target due it being necessary for viral replication and the fact that it promotes the spread of the infection. Thus, this study aimed to design DENV NS2B/NS3pro allosteric inhibitors from a matrix compound. The search was conducted using the Swiss Similarity tool. The compounds were subjected to molecular docking calculations, molecular dynamics simulations (MD) and free energy calculations. The molecular docking results showed that two compounds, ZINC000001680989 and ZINC000001679427, were promising and performed important hydrogen interactions with the Asn152, Leu149 and Ala164 residues, showing the same interactions obtained in the literature. In the MD, the results indicated that five residues, Lys74, Leu76, Asn152, Leu149 and Ala166, contribute to the stability of the ligand at the allosteric site for all of the simulated systems. Hydrophobic, electrostatic and van der Waals interactions had significant effects on binding affinity. Physicochemical properties, lipophilicity, water solubility, pharmacokinetics, druglikeness and medicinal chemistry were evaluated for four compounds that were more promising, showed negative indices for the potential penetration of the Blood Brain Barrier and expressed high human intestinal absorption, indicating a low risk of central nervous system depression or drowsiness as the the side effects. The compound ZINC000006694490 exhibited an alert with a plausible level of toxicity for the purine base chemical moiety, indicating hepatotoxicity and chromosome damage in vivo in mouse, rat and human organisms. All of the compounds selected in this study showed a synthetic accessibility (SA) score lower than 4, suggesting the ease of new syntheses. The results corroborate with other studies in the literature, and the computational approach used here can contribute to the discovery of new and potent anti-dengue agents.

Natural Products-Based Drug Design against SARS-CoV-2 Mpro 3CLpro.

Coronavirus disease 2019 (COVID-19), caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), has received global attention due to the serious threat it poses to public health. Since the outbreak in December 2019, millions of people have been affected and its rapid global spread has led to an upsurge in the search for treatment. To discover hit compounds that can be used alone or in combination with repositioned drugs, we first analyzed the pharmacokinetic and toxicological properties of natural products from Brazil's semiarid region. After, we analyzed the site prediction and druggability of the SARS-CoV-2 main protease (Mpro), followed by docking and molecular dynamics simulation. The best SARS-CoV-2 Mpro complexes revealed that other sites were accessed, confirming that our approach could be employed as a suitable starting protocol for ligand prioritization, reinforcing the importance of catalytic cysteine-histidine residues and providing new structural data that could increase the antiviral development mainly against SARS-CoV-2. Here, we selected 10 molecules that could be in vitro assayed in response to COVID-19. Two compounds (b01 and b02) suggest a better potential for interaction with SARS-CoV-2 Mpro and could be further studied.

Revisiting the Proposition of Binding Pockets and Bioactive Poses for GSK-3ß Allosteric Modulators Addressed to Neurodegenerative Diseases.

Glycogen synthase kinase-3 beta (GSK-3ß) is an enzyme pertinently linked to neurodegenerative diseases since it is associated with the regulation of key neuropathological features in the central nervous system. Among the different kinds of inhibitors of this kinase, the allosteric ones stand out due to their selective and subtle modulation, lowering the chance of producing side effects. The mechanism of GSK-3ß allosteric modulators may be considered still vague in terms of elucidating a well-defined binding pocket and a bioactive pose for them. In this context, we propose to reinvestigate and reinforce such knowledge by the application of an extensive set of in silico methodologies, such as cavity detection, ligand 3D shape analysis and docking (with robust validation of corresponding protocols), and molecular dynamics. The results here obtained were consensually consistent in furnishing new structural data, in particular by providing a solid bioactive pose of one of the most representative GSK-3ß allosteric modulators. We further applied this to the prospect for new compounds by ligand-based virtual screening and analyzed the potential of the two obtained virtual hits by quantum chemical calculations. All potential hits achieved will be subsequently tested by in vitro assays in order to validate our approaches as well as to unveil novel chemical entities as GSK-3ß allosteric modulators.

Toward of Safer Phenylbutazone Derivatives by Exploration of Toxicity Mechanism.

A drug design for safer phenylbutazone was been explored by reactivity and docking studies involving single electron transfer mechanism, as well as toxicological predictions. Several approaches about its structural properties were performed through quantum chemistry calculations at the B3LYP level of theory, together with the 6-31+G(d,p) basis sets. Molecular orbital and ionization potential were associated to electron donation capacity. The spin densities contribution showed a preferential hydroxylation at the para-positions of phenyl ring when compared to other positions. In addition, on electron abstractions the aromatic hydroxylation has more impact than alkyl hydroxylation. Docking studies indicate that six structures 1, 7, 8 and 13⁻15 have potential for inhibiting human as well as murine COX-2, due to regions showing similar intermolecular interactions to the observed for the control compounds (indomethacin and refecoxib). Toxicity can be related to aromatic hydroxylation. In accordance to our calculations, the derivatives here proposed are potentially more active as well safer than phenylbutazone and only structures 8 and 13⁻15 were the most promising. Such results can explain the biological properties of phenylbutazone and support the design of potentially safer candidates.

In Silico Evaluation of Ibuprofen and Two Benzoylpropionic Acid Derivatives with Potential Anti-Inflammatory Activity.

Inflammation is a complex reaction involving cellular and molecular components and an unspecific response to a specific aggression. The use of scientific and technological innovations as a research tool combining multidisciplinary knowledge in informatics, biotechnology, chemistry and biology are essential for optimizing time and reducing costs in the drug design. Thus, the integration of these in silico techniques makes it possible to search for new anti-inflammatory drugs with better pharmacokinetic and toxicological profiles compared to commercially used drugs. This in silico study evaluated the anti-inflammatory potential of two benzoylpropionic acid derivatives (MBPA and DHBPA) using molecular docking and their thermodynamic profiles by molecular dynamics, in addition to predicting oral bioavailability, bioactivity and toxicity. In accordance to our predictions the derivatives proposed here had the potential capacity for COX-2 inhibition in the human and mice enzyme, due to containing similar interactions with the control compound (ibuprofen). Ibuprofen showed toxic predictions of hepatotoxicity (in human, mouse and rat; toxicophoric group 2-arylacetic or 3-arylpropionic acid) and irritation of the gastrointestinal tract (in human, mouse and rat; toxicophoric group alpha-substituted propionic acid or ester) confirming the literature data, as well as the efficiency of the DEREK 10.0.2 program. Moreover, the proposed compounds are predicted to have a good oral bioavailability profile and low toxicity (LD50 < 700 mg/kg) and safety when compared to the commercial compound. Therefore, future studies are necessary to confirm the anti-inflammatory potential of these compounds.

Influence of mechanical disintegration on the microbial growth of aerobic sludge biomass: A comparative study of ultrasonic and shear gap homogenizers by oxygen uptake measurements.

Wastewater treatment plant incorporates physical, chemical and biological processes to treat and remove the contaminants. The main drawback of conventional activated sludge process is the huge production of excess sludge, which is an unavoidable byproduct. The treatment and disposal of excess sludge costs about 60% of the total operating cost. The ideal way to reduce excess sludge production during wastewater treatment is by preventing biomass formation within the aerobic treatment train rather than post treatment of the generated sludge. In the present investigation two different mechanical devices namely, Ultrasonic and Shear Gap homogenizers have been employed to disintegrate the aerobic biomass. This study is intended to restrict the multiplication of microbial biomass and at the same time degrade the organics present in wastewater by increasing the oxidative capacity of microorganisms. The disintegrability on biomass was determined by biochemical methods. Degree of inactivation provides the information on inability of microorganisms to consume oxygen upon disruption. The soluble COD quantifies the extent of release of intra cellular compounds. The participation of disintegrated microorganism in wastewater treatment process was carried out in two identical respirometeric reactors. The results show that Ultrasonic homogenizer is very effective in the disruption of microorganisms leading to a maximum microbial growth reduction of 27%. On the other hand, Shear gap homogenizer does not favor the sludge growth reduction rather it facilitates the growth. This study also shows that for better microbial growth reduction, floc size reduction alone is not sufficient but also microbial disruption is essential.

Analgesic Activity of 5-Acetamido-2-Hydroxy Benzoic Acid Derivatives and an In-Vivo and In-Silico Analysis of Their Target Interactions.

The design, synthesis, and evaluation of novel non-steroidal anti-inflammatory drugs (NSAIDs) with better activity and lower side effects are big challenges today. In this work, two 5-acetamido-2-hydroxy benzoic acid derivatives were proposed, increasing the alkyl position (methyl) in an acetamide moiety, and synthesized, and their structural elucidation was performed using 1H NMR and 13C NMR. The changes in methyl in larger groups such as phenyl and benzyl aim to increase their selectivity over cyclooxygenase 2 (COX-2). These 5-acetamido-2-hydroxy benzoic acid derivatives were prepared using classic methods of acylation reactions with anhydride or acyl chloride. Pharmacokinetics and toxicological properties were predicted using computational tools, and their binding affinity (kcal/mol) with COX-2 receptors (Mus musculus and Homo sapiens) was analyzed using docking studies (PDB ID 4PH9, 5KIR, 1PXX and 5F1A). An in-silico study showed that 5-acetamido-2-hydroxy benzoic acid derivates have a better bioavailability and binding affinity with the COX-2 receptor, and in-vivo anti-nociceptive activity was investigated by means of a writhing test induced by acetic acid and a hot plate. PS3, at doses of 20 and 50 mg/kg, reduced painful activity by 74% and 75%, respectively, when compared to the control group (20 mg/kg). Regarding the anti-nociceptive activity, the benzyl showed reductions in painful activity when compared to acetaminophen and 5-acetamido-2-hydroxy benzoic acid. However, the proposed derivatives are potentially more active than 5-acetamido-2-hydroxy benzoic acid and they support the design of novel and safer derivative candidates. Consequently, more studies need to be conducted to evaluate the different pharmacological actions, the toxicity of possible metabolites that can be generated, and their potential use in inflammation and pain therapy.

Identification of novel potential cyclooxygenase-2 inhibitors using ligand- and structure-based virtual screening approaches.

Cyclooxygenase 2 (COX-2) is a well-established target for the design of anti-inflammatory intermediates. Celecoxib was selected as a template molecule to perform ligand-based virtual screening, i.e. to search for structures with similarity in shape and electrostatic potential, with a gradual increase in accuracy through the combined fitting of several steps using eight commercial databases. The molecules ZINC408709 and ZINC2090319 reproduced values within the limits established in an initial study of absorption and distribution in the body. No alert was fired for possible toxic groups when these molecules were subjected to toxicity prediction. Molecular docking results with these compounds showed a higher binding affinity in comparison to rofecoxib for the COX-2 target. Additionally, ZINC408709 and ZINC2090319 were predicted to be potentially biologically active. In in silico prediction of endocrine disruption potential, it was established that the molecule ZINC2090319 binds strongly to the target related to cardiovascular risk in a desirable way as a non-steroidal antagonist and ZINC408709 binds strongly to the target that is associated with the treatment of inflammatory pathologies and similar to celecoxib. Metabolites generated from these compounds are less likely to have side effects. Simulations were used to evaluate the interaction of compounds with COX-1 and COX-2 during 200 ns. Despite the differences, ZINC408709 molecule showed better stability for COX-2 during molecular dynamics simulation. In the calculations of free energy MM/PBSA, the molecule ZINC408709 ΔGbind value has a higher affinity to celecoxib and rofecoxib COX-2. This demonstrates that the selected substances can be considered as promising COX-2 inhibitors. Communicated by Ramaswamy H. Sarma.

A Novel Method for Differential Prognosis of Brain Degenerative Diseases Using Radiomics-Based Textural Analysis and Ensemble Learning Classifiers.

We propose a novel approach to develop a computer-aided decision support system for radiologists to help them classify brain degeneration process as physiological or pathological, aiding in early prognosis of brain degenerative diseases. Our approach applies computational and mathematical formulations to extract quantitative information from biomedical images. Our study explores the longitudinal OASIS-3 dataset, which consists of 4096 brain MRI scans collected over a period of 15 years. We perform feature extraction using Pyradiomics python package that quantizes brain MRI images using different texture analysis methods. Studies indicate that Radiomics has rarely been used for analysis of brain cognition; hence, our study is also a novel effort to determine the efficiency of Radiomics features extracted from structural MRI scans for classification of brain degenerative diseases and to create awareness about Radiomics. For classification tasks, we explore various ensemble learning classification algorithms such as random forests, bagging-based ensemble classifiers, and gradient-boosted ensemble classifiers such as XGBoost and AdaBoost. Such ensemble learning classifiers have not been used for biomedical image classification. We also propose a novel texture analysis matrix, Decreasing Gray-Level Matrix or DGLM. The features extracted from this filter helped to further improve the accuracy of our decision support system. The proposed system based on XGBoost ensemble learning classifiers achieves an accuracy of 97.38%, with sensitivity 99.82% and specificity 97.01%.

Identification of New Rofecoxib-Based Cyclooxygenase-2 Inhibitors: A Bioinformatics Approach.

The cyclooxygenase-2 receptor is a therapeutic target for planning potential drugs with anti-inflammatory activity. The selective cyclooxygenase-2 (COX-2) inhibitor rofecoxib was selected as a pivot molecule to perform virtual ligand-based screening from six commercial databases. We performed the search for similarly shaped Rapid Overlay of Chemical Structures (ROCS) and electrostatic (EON) compounds. After, we used pharmacokinetic and toxicological parameters to determine the best potential compounds, obtained through the softwares QikProp and Derek, respectively. Then, the compounds proceeded to the molecular anchorage study, which showed promising results of binding affinity with the hCOX-2 receptor: LMQC72 (∆G = -11.0 kcal/mol), LMQC36 (∆G = -10.6 kcal/mol), and LMQC50 (∆G = -10.2 kcal/mol). LMQC72 and LMQC36 showed higher binding affinity compared to rofecoxib (∆G = -10.4 kcal/mol). Finally, molecular dynamics (MD) simulations were used to evaluate the interaction of the compounds with the target hCOX-2 during 150 ns. In all MD simulation trajectories, the ligands remained interacting with the protein until the end of the simulation. The compounds were also complexing with hCOX-2 favorably. The compounds obtained the following affinity energy values: rofecoxib: ΔGbind = -45.31 kcal/mol; LMQC72: ΔGbind = -38.58 kcal/mol; LMQC36: ΔGbind = -36.10 kcal/mol; and LMQC50: ΔGbind = -39.40 kcal/mol. The selected LMQC72, LMQC50, and LMQC36 structures showed satisfactory pharmacokinetic results related to absorption and distribution. The toxicological predictions of these compounds did not display alerts for possible toxic groups and lower risk of cardiotoxicity compared to rofecoxib. Therefore, future in vitro and in vivo studies are needed to confirm the anti-inflammatory potential of the compounds selected here with bioinformatics approaches based on rofecoxib ligand.

In Silico Study to Identify New Antituberculosis Molecules from Natural Sources by Hierarchical Virtual Screening and Molecular Dynamics Simulations.

Tuberculosis (TB) is an infection caused by Mycobacterium tuberculosis, responsible for 1.5 million documented deaths in 2016. The increase in reported cases of M. tuberculosis resistance to the main drugs show the need for the development of new and efficient drugs for better TB control. Based on these facts, this work aimed to use combined in silico techniques for the discovery of potential inhibitors to ß-ketoacyl-ACP synthase (MtKasA). Initially compounds from natural sources present in the ZINC database were selected, then filters were sequentially applied by virtual screening, initially with pharmacophoric modeling, and later the selected compounds (based on QFIT scores) were submitted to the DOCK 6.5 program. After recategorization of the variables (QFIT score and GRID score), compounds ZINC35465970 and ZINC31170017 were selected. These compounds showed great hydrophobic contributions and for each established system 100 ns of molecular dynamics simulations were performed and the binding free energy was calculated. ZINC35465970 demonstrated a greater capacity for the KasA enzyme inhibition, with a ΔGbind = -30.90 kcal/mol and ZINC31170017 presented a ΔGbind = -27.49 kcal/mol. These data can be used in other studies that aim at the inhibition of the same biological targets through drugs with a dual action.

Analysis of Health Facility Based Perinatal Verbal Autopsy of Electoral Constituency 2 of Arghakhanchi District, Nepal.

BACKGROUND: Verbal autopsy is a method to diagnose possible cause of death by analyzing factors associated with death through detailed questioning. This study is a part of the operational research program in electoral constituency no. 2 (EC 2) of Arghakhanchi district by MIRA and HealthRight International. METHODS: Two day essential newborn care training followed by one day perinatal verbal autopsy training and later one day refresher verbal autopsy training was given for health staff of EC 2 of Arghakhanchi district in two groups. Stillbirths of >22wks or > 500 gms and Early neonatal deaths (newborns died within7 days of life) were included in this study. The Nepal Government approved verbal autopsy forms were used for performing autopsies. Perinatal deaths were classified according to Wigglesworth's Classification. Causes of Perinatal deaths were analyzed. Data were analyzed in the form of frequencies and tabulation in SPSS 16 . RESULTS: There were 41 cases of perinatal deaths (PND) were identified. Among them, 37 PNDs were from Arghakhanchi district hospital, 2 PNDs from Thada PHC, and one PND each from Subarnakhal and Pokharathok HPs. Among the 41 PNDs, 26 were stillbirths (SB) and 15 were early neonatal deaths (ENND). The perinatal mortality rate (PMR) of Arghakhanchi district hospital was 32.2 per 1,000 births and neonatal mortality rate (NMR) was 9.8 per 1,000 live births. Out of 26 stillbirths, 54% (14) were fresh SBs and 46% (12) were macerated stillbirths. The most common cause of stillbirth was obstetric complications (47%) where as birth asphyxia (53%) was the commonest cause of ENND. According to Wigglesworth's classification of perinatal deaths, Group IV (40%) was the commonest cause in the health facilities. CONCLUSIONS: Obstetric complication was the commonest cause of stillbirth and birth asphyxia was the commonest cause of early neonatal death. This study highlighted the need for regular antenatal check-ups and proper intrapartum fetal monitoring with timely and appropriate intervention to reduce the incidence of stillbirths and intrauterine asphyxia.

Maternal and Neonatal Health Knowledge, Service Quality and Utilization: Findings from a Community Based Quasi-experimental Trial in Arghakhanchi District of Nepal.

BACKGROUND: As part of the Partnership for Maternal and Newborn Health Project (PMNH), HealthRight International collaborated with Mother and Infant Research Activities (MIRA) to conduct operations research in Arghakhanchi district of Nepal to explore the intervention impact of strengthening health facility, improving community facility linkages along with Community Based Newborn Care Program (CB-NCP) on Maternal Neonatal Care (MNC) service quality, utilization, knowledge and care seeking behavior. METHODS: This was a quasi-experimental study. Siddahara, Pokharathok, Subarnakhal,Narpani Health Posts (HPs) and Thada Primary Health Care Center(PHCC)in Electoral Constituency-2 were selected as intervention sites and Arghatosh, ,Argha, Khana, Hansapur HPs and Balkot PHCC in Electoral Constituency-1 were chosen as controls. The intervention started in February 2011 and was evaluated in August 2013. To compare MNC knowledge and practice in the community, mothers of children aged 0-23 months were selected from the corresponding Village Development Committees(VDCs) by a two stage cluster sampling design during both baseline (July 2010) and endline (August, 2013) assessments. The difference in difference analysis was used to understand the intervention impact. RESULTS: Local resource mobilization for MNC, knowledge about MNC and service utilization increased in intervention sites. Though there were improvements, many effects were not significant. CONCLUSIONS: Extensive trainings followed by reviews and quality monitoring visits increased the knowledge, improved skills and fostered motivation of health facility workers for better MNC service delivery. MNC indicators showed an upsurge in numbers due to the synergistic effects of many interventions.

Microbial Desulfurization of Coals in a Slurry Pipeline Reactor Using. Thiobacillus ferrooxidans.

Microbial desulfurization of Illinois #6 and Indiana #3 bituminous coal having a total sulfur content of 2 to 8% has been investigated using acidophilic microorganism, Thiobacillus ferrooxidans, in laboratory shake flash experiments and in a two-inch pipeline loop. The results indicate that about 80 to 85% pyritic sulfur removal was achieved with 10 to 25% coal/water slurry recirculated at 6-7 ft/sec at room temperature in 7 to 14 days. The experimental conditions have been optimized for maximum desulfurization. Results from this study show that the rates of bacterial desulfurization from coal samples are higher in the pipeline loop under turbulent flow conditions as compared to the shake-flask experiments for particle sizes ranging from 43 to 200 mum. It is visualized that the proposed coal slurry pipelines could be used as biological plug flow reactors under aerobic conditions. The laboratory corrosion studies under dynamic test conditions show that use of a corrosion inhibitor will limit the pipeline corrosion rate to acceptable levels.

Conditions in rural Nepal for which depot-medroxyprogesterone acetate initiation is not recommended: implications for community-based service delivery.

The presence of medical conditions that might affect the use of depot-medroxyprogesterone acetate (DMPA) as a contraceptive method was assessed in a rural district in Nepal. A general health survey was conducted in nonpregnant and noncontracepting women aged 15-44 years to determine the presence of any health problems. The survey included a general assessment interview by nonphysicians, followed by formal medical histories and physical exams by female gynecologists. Possible pregnancy (nine cases) and abnormal uterine bleeding (one case) were the only conditions identified in which DMPA should not be used, based on the World Health Organization (WHO) Medical Eligibility Criteria for Contraceptive Use. Five additional cases of cardiovascular problems, in which DMPA initiation is not usually recommended, were also detected. Recently developed checklists based on the WHO criteria for DMPA use would have identified all of these health conditions. This checklist would allow the safe delivery of DMPA at the community health worker level, and increase the availability and accessibility of DMPA in rural Nepal.

PIP: A general health survey was conducted in nonpregnant and noncontracepting women aged 15-44 years to determine the presence of any health problems that might affect the use of depot-medroxyprogesterone acetate (DMPA) as a contraceptive method in rural district in Nepal. The survey included a general assessment interview by nonphysicians, followed by formal medical histories and physical exams by female gynecologists. Findings revealed that a possible pregnancy (9 cases) and abnormal uterine bleeding (1 case) were the only conditions identified in which DMPA should not be used based on the WHO Medical Eligibility Criteria for Contraceptive Use. 5 additional cases of cardiovascular problems, in which DMPA initiation was not usually recommended, were also detected. The reports included heart disease (2 cases), past history of hypertension (1 case), current hypertension (1 case), and headache and hypertension (1 case).

Effect of intestinal helminth infection on some nutritional parameters among rural villagers in Nepal.

We report the prevalence of intestinal helminthiasis and its impact on health of Nepalese in two different rural Village Development Committees (VDC) in Nepal. A total of 261 school children from Okharpauwa VDC in Nuwakot district (Central Region) and 242 inhabitants of Boya VDC in Bhojpur district (Eastern Region) in Nepal were included in this study. The overall prevalence in these two different districts were 57.5% and 55.4%, respectively. Altogether four types of helminths were detected. In both districts, Ascaris lumbricoides was the commonest helminth. Mixed helminth infection in both study areas accounted for less than 5.0%. All helminth infected subjects were treated with a single dose of albendazole. Heavy Trichuris trichiura infection needed a second dose. Blood hemoglobin and serum total protein level in school children in Okharpauwa VDC increased significantly after chemotherapy (P < 0.05). Eosinophil count (10.1%; SD 5.0325) returned to normal (4.8%; SD 4.6997). Helminth infection in Boya VDC was more common in children than in adults. Serum total protein, albumin, total cholesterol and triglycerides levels were not influenced by the presence or absence of helminths among the inhabitants of Boya VDC. In both districts, more than 80.0% of the household had no latrine. In Boya VDC, 94.0% of inhabitants interviewed were not aware of other intestinal parasite other than A. lumbricoides.

Management of secondary pupillary membrane in aphakia (YAG discission vs parsplana membranectomy).

Forty aphakic eyes, with secondary pupillary membranes, underwent pars plana membranectomy and YAG laser discission randomly. Visual improvement was similar in both the groups. IOP remained low for a week in pars-plana membranectomy while it transiently increased following YAG laser discission Complications like anterior chamber reaction, corneal edema and CME were more after pars plana membranectomy than in YAG laser discission. In membranes thicker than 1.2 mm, only pars plana membranectomy is recommended.

A large-scale study of bacterial contamination of drinking water and its public health impact in Nepal.

Bacterial contamination of drinking water is a major public health in Nepal. A large scale study on contamination of drinking water was done covering all three ecological belts (mountain, hill and Terai) in all five (eastern, central, western, mid-western and far-western) development regions of Nepal during 2009-2011. Of the total 506 water samples studied, one-forth (25.1%; 127/506) were visually turbid. Bacteriologically, 88.5% (448/506) samples were positive for total coliform (TC) whereas 56.5% (286/506) were positive for fecal coliform (FC) (Esch. coli). The TC positive rate ranged from 53.8% in Damak (Jhapa) to 100.0% in different districts. The FC positive rate varied more widely ranging from 10.0% in Bharatpur City (Chitawan) to 100.0% in Baglung Township (Baglung) with over 50.0% in most of the districts (over 75.0% in eight districts). Both TC and FC positive rate were highest in Far-western Development Region (DR). High TC positive rate (96.7%) in Far-western DR was followed by Western DR (93.9%), Eastern DR (89.2%), Central DR (87.0%) and Mid-western DR (74.6%). Highest FC positive rate (65.5%) in Far-western DR was followed by Med-western DR (63.5%), Western DR (55.9%), Central DR (53.2%) and Eastern DR (52.0%). TC positive was highest (90.7%) in hills followed by mountain (89.7%) and Terai (plain) (84.1%) belt. In contrast, FC positive rate was highest (66.2%) in mountain, followed by hills (58.0%) and Terai (49.7%). Of the total 506, 335 were piped tap water, 129 were boring water, 16 natural tap (spout), 16 were well (sallow/deep well) and 10 were mineral/uroguard treated water. TC positive rate was very high (81.2% to 100.0%) in different type water samples (piped tap: 90.1%; boring water: 85.2%; natural spout/tap: 81.2%; well water 100.0% and mineral water/uroguard treated water: 80.0%). FC positive rate ranged from 0.0% in mineral water/uroguard treated water to 93.7% in well water samples. These findings are of serious public health concern with regard to both endemicity and outbreak of waterborne diseases in the country.