Molecular Identification and Antioxidant Activity Determination among Coffee Varieties Cultivated in Nepal.

Coffee is the most popular beverage containing numerous phytochemical components that have antioxidant activity capable of scavenging free radicals. Antioxidant and phenolic contents have considerable benefits for human health. The aim of this study was the molecular identification of 9 coffee samples from the Nepal Agricultural Research Council, Lalitpur, Nepal, and the determination of the antioxidant activity and total phenolic content of green and roasted coffee beans. Molecular identification was performed using ITS-specific PCR followed by sequencing and phylogenetic tree construction using the maximum parsimony method. The DPPH assay was used to determine the antioxidant activity, and the Folin-Ciocalteu (F-C) assay was used to determine the total phenolic content. All the samples belonged to the taxa Coffea arabica. The antioxidant activity in roasted beans varied from 2.49 to 4.62 AAE mg/g and from 1.4 to 3.9 AAE mg/g in green beans. The total phenolic content varied from 2.58 to 3.38 GAE mg/g and from 4.16 to 5.36 GAE mg/g for the roasted beans and green beans, respectively. The data revealed that the highest antioxidant content (4.62 AAE mg/g) was found in roasted coffee and that the highest phenolic content (5.36 GAE mg/g) was found in green coffee. The study concludes that roasting increases the antioxidant activity but decreases the phenolic content of coffee.

Hysteroscopy among Patients Attending the Outpatient Department of Gynaecology in a Tertiary Care Centre: A Descriptive Cross-sectional Study.

Introduction: Hysteroscopy is a procedure used widely for diagnostic and therapeutic purposes. Hysteroscopy enables visualisation of the endometrial cavity and if possible treatment in the same setting avoiding invasive procedure. The objective of the study was to find out the prevalence of hysteroscopy among gynaecological patients attending the outpatient department of Obstetrics and Gynaecology in a tertiary care centre. Methods: A descriptive cross-sectional study was done among gynaecological patients from 1 January 2016 to 1 January 2020 visiting the outpatient Department of Obstetrics and Gynaecology in a tertiary care centre after ethical approval from the Institutional Review Committee (Registration number: 029/2021). Convenience sampling was used. Data regarding demographic parameters, hysteroscopy findings, procedures performed, histopathological findings and complications were retrieved from the electronic database of the hospital. Point estimate and 95% Confidence Interval were calculated. Results: Among 319 gynaecological patients, hysteroscopy was done in 72 (22.57%) (17.98-27.16, 95% Confidence Interval) patients. Conclusions: The prevalence of hysteroscopy among gynaecological patients was higher to the studies done in similar settings. Keywords: hysteroscopy; infertility; leiomyoma; polyps.

Preoperative Neutrophil Lymphocyte Ratio in Prediction of Adnexal Mass Torsion.

Aims: Adnexal torsion commonly affects reproductive age group females. Prompt diagnosis and early management help in the preservation of fertility. However, its diagnosis is challenging. Preoperative diagnosis can be suspected in only 23-66% of the cases and half of the patients operated for adnexal torsion have different diagnosis. This article thus aims to identify the diagnostic value of preoperative neutrophil lymphocyte ratio in adnexal torsion in comparison with other untwisted unruptured ovarian cysts. Methods: This was a retrospective study conducted in the duration of five years from 1st January 2016 to 1st January 2020. The data about demographic parameters, hematological parameters, operative approach, operative technique, and histopathological reports were derived from an electronic database and documented on proforma. SPSS was used for statistical analysis. Logistic regression analysis and influence of each factor on preoperative diagnosis of Adnexal torsion was evaluated. Results: A total of 125 patients were included in the article (adnexal torsion group n = 25, untwisted unruptured ovarian cyst group n = 100). There was no statistically significant difference in comparison to age, parity, and abortion between both groups. Most patients had undergone laparoscopic surgery which was based on surgeon's skill and preference. Nineteen (78%) patients in the adnexal torsion group underwent oophorectomy while infarcted ovary was seen in only 4 cases. Among the blood parameters, only neutrophil-lymphocyte ratio (NLR) >3 was found to be statistically significant under logistic regression analysis. Most common adnexal pathology to undergo torsion was serous cyst. Conclusion: Preoperative neutrophil-lymphocyte ratio can be a predictive marker for diagnosis of adnexal torsion and can differentiate it from untwisted unruptured ovarian cysts.

Screening and Identification of Thermotolerant and Osmotolerant Bacillus amyloliquefaciens BKHE Isolated from Kinema of Eastern Nepal for Alkaline Protease Production.

Alkaline protease is one of the most important industrial enzymes which are excessively used in the detergent industry, food industry, feed industry, pharmaceutical industry, leather industry, etc. 60% of the produced alkaline protease is consumed by the detergent industry alone. In the present study, bacterial isolates that can produce alkaline protease for purpose of bio-detergent were screened among the isolates isolated from kinema (an alkaline fermented food of eastern Nepal). Selected bacterial isolates were further screened for hemolysis activity and the production of other hydrolytic enzymes. Four bacterial isolates selected were tested for their capacity to produce alkaline protease in five different fermentation mediums. Isolate BKHE produces a high amount of alkaline protease (0.4705 ± 0.035 U/mL/min) in fermentation medium M2 (sucrose, 11 g/L; yeast extract, 5 g/L; and KNO3, 5.2 g/l, pH 9). The selected isolate was identified as Bacillus amyloliquefaciens BKHE based on 16S rRNA sequencing and phenotypic features. This bacterial strain was also found to be thermotolerant (confluent growth at 50°C) and salt tolerant up to 10% NaCl concentration. With its versatile ability, bacterial isolate or purified enzymes have potential applications in the food and detergent industry.

In Vitro and In Silico Studies for the Identification of Potent Metabolites of Some High-Altitude Medicinal Plants from Nepal Inhibiting SARS-CoV-2 Spike Protein.

Despite ongoing vaccination programs against COVID-19 around the world, cases of infection are still rising with new variants. This infers that an effective antiviral drug against COVID-19 is crucial along with vaccinations to decrease cases. A potential target of such antivirals could be the membrane components of the causative pathogen, SARS-CoV-2, for instance spike (S) protein. In our research, we have deployed in vitro screening of crude extracts of seven ethnomedicinal plants against the spike receptor-binding domain (S1-RBD) of SARS-CoV-2 using an enzyme-linked immunosorbent assay (ELISA). Following encouraging in vitro results for Tinospora cordifolia, in silico studies were conducted for the 14 reported antiviral secondary metabolites isolated from T. cordifolia-a species widely cultivated and used as an antiviral drug in the Himalayan country of Nepal-using Genetic Optimization for Ligand Docking (GOLD), Molecular Operating Environment (MOE), and BIOVIA Discovery Studio. The molecular docking and binding energy study revealed that cordifolioside-A had a higher binding affinity and was the most effective in binding to the competitive site of the spike protein. Molecular dynamics (MD) simulation studies using GROMACS 5.4.1 further assayed the interaction between the potent compound and binding sites of the spike protein. It revealed that cordifolioside-A demonstrated better binding affinity and stability, and resulted in a conformational change in S1-RBD, hence hindering the activities of the protein. In addition, ADMET analysis of the secondary metabolites from T. cordifolia revealed promising pharmacokinetic properties. Our study thus recommends that certain secondary metabolites of T. cordifolia are possible medicinal candidates against SARS-CoV-2.

Molecular Docking and Dynamics Simulation of Several Flavonoids Predict Cyanidin as an Effective Drug Candidate against SARS-CoV-2 Spike Protein.

The in silico method has provided a versatile process of developing lead compounds from a large database in a short duration. Therefore, it is imperative to look for vaccinations and medications that can stop the havoc caused by SARS-CoV-2. The spike protein of SARS-CoV-2 is required for the viral entry into the host cells, hence inhibiting the virus from fusing and infecting the host. This study determined the binding interactions of 36 flavonoids along with two FDA-approved drugs against the spike protein receptor-binding domain of SARS-CoV-2 through molecular docking and molecular dynamics (MD) simulations. In addition, the molecular mechanics generalized Born surface area (MM/GBSA) approach was used to calculate the binding-free energy (BFE). Flavonoids were selected based on their in vitro assays on SARS-CoV and SARS-CoV-2. Our pharmacokinetics study revealed that cyanidin showed good drug-likeness, fulfilled Lipinski's rule of five, and conferred favorable toxicity parameters. Furthermore, MD simulations showed that cyanidin interacts with spike protein and alters the conformation and binding-free energy suited. Finally, an in vitro assay indicated that about 50% reduction in the binding of hACE2 with S1-RBD in the presence of cyanidin-containing red grapes crude extract was achieved at approximately 1.25 mg/mL. Hence, cyanidin may be a promising adjuvant medication for the SARS-CoV-2 spike protein based on in silico and in vitro research.

Prevalence of methicillin-resistant Staphylococcus aureus and pattern of antimicrobial resistance in mastitis milk of cattle in Chitwan, Nepal.

BACKGROUND: The threat of methicillin-resistant Staphylococcus aureus (MRSA) exists globally and has been listed as a priority pathogen by the World Health Organization. One of the sources of MRSA emergence is livestock and its products, often raised in poor husbandry conditions. There are limited studies in Nepal to understand the prevalence of MRSA in dairy animals and its antimicrobial resistance (AMR) profile. A cross-sectional study was conducted in Chitwan, one of the major milk-producing districts of Nepal, from February 2018 to September 2019 to estimate the prevalence of MRSA in milk samples and its AMR profile. The collected milk samples (n = 460) were screened using the California Mastitis Test (CMT) and positive samples were subjected to microbiological analysis to isolate and identify S. aureus. Polymerase Chain Reaction (PCR) was used to identify the presence of the mecA gene and screen for MRSA. RESULTS: In total, 41.5% (191/460) of milk samples were positive in the CMT test. Out of 191 CMT positive milk samples, the biochemical tests showed that the prevalence of S. aureus was 15.2% (29/191). Among the 29 S. aureus isolates, 6.9% (2/29) were identified as MRSA based on the detection of a mecA gene. This indicates that that 1.05% (2/191) of mastitis milk samples had MRSA. The antibiotic sensitivity test showed that 75.9% (22/29) and 48.3% (14/29) S. aureus isolates were found to be sensitive to Cefazolin and Tetracycline respectively (48.3%), whereas 100% of the isolates were resistant to Ampicillin. In total 96.6% (28/29) of S. aureus isolates were multidrug-resistant (MDR). CONCLUSIONS: This study revealed a high prevalence of S. aureus-mediated subclinical mastitis in dairy herds in Chitwan, Nepal, with a small proportion of it being MRSA carrying a mecA gene. This S. aureus, CoNS, and MRSA contaminated milk poses a public health risk due to the presence of a phenotype that is resistant to very commonly used antibiotics. It is suggested that dairy herds be screened for subclinical mastitis and treatments for the animals be based on antibiotic susceptibility tests to reduce the prevalence of AMR. Furthermore, future studies should focus on the Staphylococcus spp. to explore the antibiotic resistance genes in addition to the mecA gene to ensure public health.

Purification and characterization of a noble thermostable algal starch liquefying alpha-amylase from Aeribacillus pallidus BTPS-2 isolated from geothermal spring of Nepal.

A thermophilic strain, Aeribacillus pallidus BTPS-2 was isolated from Bhurung geothermal spring of Nepal. The 16 s rRNA sequence showed 99.8 % similarity with the type strain Aeribacillus pallidus DSM 3670. The morphological, physiological and biochemical properties were similar to the type strain. Alpha-amylase from A. pallidus BTPS-2 was purified to 19-fold purification by DEAE-Cellulose ion exchange chromatography. The Km value of amylase on starch was 0.51 ±â€¯0.05 mg/mL. The optimum pH and temperature were 7.0 and 70 °C. SDS-PAGE analysis showed a single band at 100 kDa. The half-life of the enzyme at 80 °C was 2.81 h. The enzyme showed an inhibitory effect in the presence of Fe2+, Pb2+, Sn2+ and Hg2+ at 10 mM concentrations. TLC analysis showed that the enzyme is a liquifying alpha-amylase. The enzyme reduced the viscosity of algal biomass suspension up to 74.2 ±â€¯0.17 % which was more efficient than Bacillus amyloliquefaciens alpha-amylase (80.5 ±â€¯0.2 %).

Risk factors of tuberculosis in human and its association with cattle TB in Nepal: A one health approach.

Tuberculosis (TB) in humans is primarily caused by Mycobacterium tuberculosis (M. tuberculosis), with millions of infections and hundreds of thousands of deaths worldwide. It creates a substantial economic burden on the community. Unlike M. tuberculosis, Mycobacterium bovis infects cattle and causes bovine TB, also known as zoonotic TB. People can contract zoonotic TB after consumption of unpasteurized dairy products, handling the sick animals, and via occupational exposures. The association between the zoonotic TB in humans and cattle is not well known in Nepal. The study examined the associated risk factors, including exposure to infected cattle, that contribute to TB's development in human beings in Nepal. The study consists of human and animal subjects. Firstly, a retrospective matched case-control study was conducted at the National Tuberculosis Center (NTC), Bhaktapur, Nepal. A total of 290 people (equal numbers of TB cases and control subjects) were interviewed to obtain information on socio-demographic, behavioral, and occupational risks, including the history of cattle related exposures. Secondly, a cross-sectional study was performed among the cattle owned by the TB-confirmed patients. Comparative tuberculin skin test, rapid antibody test, and ELISA were used in parallel to detect M. bovis infection in cattle. The risk factors for the development of TB in humans were smokers (OR = 4.6, 95% CI: 2.1-10.0, p < 0.001), previous history of TB (OR = 7.9, 95% CI: 3.0-20.6, p < 0.001) and history of cattle exposures (OR = 3.9, 95% CI: 2.1-7.4, p = 0.001). Out of 123 cattle sampled, 12 cattle (9.76%, 95% CI: 5.37-16.76, p < 0.0001) were positive by the tuberculin test, 46 (37.4%, 95% CI: 28.97-46.62, p = 0.007) were tested positive by the rapid test, and 7 (5.7%, 95% CI: 2.52-11.80, p < 0.0001) by ELISA test. The inter-test agreement between the tuberculin and ELISA was very strong (κ = 0.72, 95% CI: 0.48-0.95, p < 0.01). This study indicates that exposure to infected cattle and socio-demographic risk factors can contribute to the development of TB in human beings.

Nutritional Status of Children and Its Associated Factors in Selected Earthquake-Affected VDCs of Gorkha District, Nepal.

BACKGROUND: Malnutrition is a major public health problem and most enveloping cause of morbidity and mortality among children and adolescents throughout the world. This study was aimed at assessing the nutritional status and associated factors among 6-10-year-old children in selected earthquake-affected areas of Gorkha district, Nepal. METHODS: A community-based cross-sectional study among 420 mothers having children of age groups 6-10 years (with anthropometric measurement among children) was conducted using a mixed method in selected earthquake-affected areas of Gorkha district, Nepal, from October 2015 to April 2016. Gorkha was selected purposively from 14 earthquake-affected districts. Two village development committees were selected randomly among 6 having severe impact. Randomly, 5 wards were selected from each of the 2 village development committees. As the sample was 420, 42 children were selected randomly from every ward. RESULT: Among the 420 children, 31.9% were underweight, 51.9% were stunted, and 2.9% were wasted after the earthquake. Children who were more prone to being underweight were the following: male children (RR = 1.34 95% CI: 1.01-1.78) and children from illiterate mothers (RR = 2.49, 95% CI: 1.85-3.36), illiterate fathers (RR = 1.73, 95% CI: 1.32-2.27), and homemaker mothers (RR = 0.28, 95% CI: 0.20-0.38); children whose families were using nonimproved sources of water (RR = 2.60, 95% CI: 1.07-6.60); and households having food insecurity (RR = 12.97, 95% CI: 3.29-51.18). Similarly, children of illiterate fathers (RR = 1.67, 95% CI: 1.41-1.97), children of illiterate mothers (RR = 2.32, 95% CI: 1.91-2.83), children of homemaker mothers (RR = 0.59, 95% CI: 0.49-0.70), children whose family were using treated water (RR = 0.32, 95% CI: 0.15-0.67), and children from food insecure households (RR = 10.52, CI: 4.05-27.33) were found to be stunted. After adjustment, children from households consuming nonimproved water were 6 times more likely (OR = 6.75; 95% CI: 1.59-28.62) to be wasted. CONCLUSION: Illiterate mothers, illiterate fathers, mothers engaged in occupation other than household work, and food insecure households were found to be independent predictors of underweight and stunting. Nonimproved source of drinking water was found to be independent predictors of wasting.

Translation and cross-cultural adaptation of the Nepali version of the Rowland universal dementia assessment scale (RUDAS).

BACKGROUND: Mild Cognitive Impairment (MCI) is a transitional state between normal cognition and dementia during ageing process. Early screening of elderly for MCI can help for early prevention and treatment of dementia. Rowland Universal Dementia Assessment Scale (RUDAS) is a brief cognitive assessment tool with excellent psychometric properties and is particularly useful in culturally and linguistically diverse (CALD) populations. The purpose of this study was to translate RUDAS into Nepali and to evaluate internal consistency of Nepali version of RUDAS. METHODS: RUDAS was translated to Nepali following recommended guidelines for translation and cross-cultural adaptation of patient reported measures. The pre-final Nepali version of RUDAS was tested on 30 elderly and appropriate changes were made by expert committee. The final Nepali version of RUDAS was developed and was administered on 100 elderly populations (mean age 67.70) to assess internal consistency. RESULTS: The rating of the participants upon their understanding of each 6 items of Nepali version of RUDAS on a 10-point Likert scale received a good score of more than 8 except 'cube drawing' which only received a mean score of 5.5. Additionally, only 8(27%) elderly have responded to the item 'cube drawing'. The preference test was done and cube drawing was replaced by stick design test. Nepali version of RUDAS showed acceptable internal consistency with Cronbach's alpha 0.7. CONCLUSION: The results of our study presents translated and cross-culturally adapted Nepali version of RUDAS that has been proved to be an appropriate assessment tool for screening cognitive impairment among elderly.