Phylogenetic characterization of canine distemper virus from stray dogs in Kathmandu Valley.

Canine distemper is a highly contagious, often fatal disease caused by canine distemper virus (CDV) in domestic dogs and wild carnivores. The virus has caused mass epidemics in both wild and captive carnivores of high conservation value such as tigers, lions and leopards. Hence, understanding and managing CDV outbreaks is particularly important in Nepal, which is home to many species of threatened wild carnivores including tigers, leopards, snow leopards, dholes and wolves, and also contains a large population of stray dogs. Previous studies have suggested that CDV may pose a threat to wild carnivores, but there have not been any studies characterizing the genetic strains of the virus circulating in Nepal's carnivores. We collected invasive and non-invasive biological samples from stray dogs in Kathmandu Valley and genetically characterized the strains of CDV in the dogs to belong to the Asia-5 lineage by using phylogenetic analysis. The same lineage also contained CDV strains sequenced from dogs, civets, red panda and lions in India. Based on our phylogenetic analysis, we think it is likely that CDV is maintained through sylvatic cycle among sympatric carnivores allowing the recurring spillovers and outbreaks. It is crucial to prevent the virus transmission from reservoir hosts to other species, especially threatened populations of large carnivores in Nepal. Hence, we recommend for regular surveillance of CDV targeting wild carnivores in addition to the domestic dogs.

Natural Clerodendrum-derived tick repellent: learning from Nepali culture.

Ticks attaching to ear canals of humans and animals are the cause of otoacariasis, common in rural areas of Nepal. The plant Clerodendrum viscosum is used in multiple indigenous systems of medicine by ethnic communities in the Indo-Nepali-Malaysian region. Visiting the Chitwan National Park, we learned that in indigenous medicine, flower extract of C. viscosum is utilized to treat digestive disorders and extracts from leaves as tick repellent to prevent ticks from invading or to remove them from the ear canal. The objective of our study was to provide support to indigenous medicine by characterizing the in vivo effect of leave extracts on ticks under laboratory conditions and its phytochemical composition. We collected plant parts of C. viscosum (leaves and flowers) and mango (Mangifera indica) leaves at the Chitwan National Park, previously associated with repellent activity to characterize their effect on Ixodes ricinus ticks by in vivo bioassays. A Q-ToF high-resolution analysis (HPLC-ESI-QToF) was conducted to elucidate phenolic compounds with potential repellent activity. Clerodendrum viscosum and M. indica leaf extracts had the highest tick repellent efficacy (%E = 80-100%) with significant differences when compared to C. viscosum flowers extracts (%E = 20-60%) and phosphate-buffered saline. Phytochemicals with tick repellent function as caffeic acid, fumaric acid and p-coumaric acid glucoside were identified in C. viscosum leaf extracts by HPLC-ESI-QToF, but not in non-repellent flower extracts. These results support the Nepali indigenous medicine application of C. viscosum leaf extracts to repel ticks. Additional research is needed for the development of natural and green repellent formulations to reduce the risks associated with ticks resistant to acaricides.

Understanding Mycobacterium tuberculosis complex in elephants through a One Health approach: a systematic review.

BACKGROUND: Mycobacterium tuberculosis complex (MTC) that causes the chronic infectious disease- tuberculosis (TB), often presents with a complicated epidemiological pattern where the transmission chain may include humans, domestic animals and wildlife, including elephants. TB has been reported globally in both captive and wild elephants. The One Health approach might be the most effective way of understanding the shared MTC infection dynamics in captive and wild animals like Asian elephants. This systematic review accumulates evidence on occurrence, transmission pathways, and preventive measures of TB in elephants from a One Health perspective. RESULTS: The prevalence of TB reported in elephant populations ranges from 0 to 23.33% and high prevalence's are reported for elephants that are in close proximity to infected humans. The risk of elephant to human infection transmission increased significantly with exposure duration and contact with infected elephants. Some studies described the plausible TB transmission to captive elephants from other animals (wild and domestic), suggesting inter- and intra-species transmission. The results of this systematic review based on 27 relevant published works, suggest three overarching interrelated transmission pathways for M. tuberculosis infections in Asian elephants- i) humans and elephants, ii) other animals (wild or domestic) and elephants and iii) unclear sources of infection. CONCLUSIONS: The progress made with new TB diagnostic tools provides multiple methods to choose from. However, lack of harmonization of TB testing in elephants and their human contacts remains a challenge to prevent TB in those animals. Routine TB screening among elephants and caretakers by setting up an occupational health program for early diagnosis of infection through combined efforts of public health, veterinary medicine, and occupational health experts is suggested. This implies the need for a One Health approach to elephant TB control. This review reveals the need for more research on Mycobacterium tuberculosis complex transmission pathways at the human-animal interface.

The Expectations and Challenges of Wildlife Disease Research in the Era of Genomics: Forecasting with a Horizon Scan-like Exercise.

The outbreak and transmission of disease-causing pathogens are contributing to the unprecedented rate of biodiversity decline. Recent advances in genomics have coalesced into powerful tools to monitor, detect, and reconstruct the role of pathogens impacting wildlife populations. Wildlife researchers are thus uniquely positioned to merge ecological and evolutionary studies with genomic technologies to exploit unprecedented "Big Data" tools in disease research; however, many researchers lack the training and expertise required to use these computationally intensive methodologies. To address this disparity, the inaugural "Genomics of Disease in Wildlife" workshop assembled early to mid-career professionals with expertise across scientific disciplines (e.g., genomics, wildlife biology, veterinary sciences, and conservation management) for training in the application of genomic tools to wildlife disease research. A horizon scanning-like exercise, an activity to identify forthcoming trends and challenges, performed by the workshop participants identified and discussed 5 themes considered to be the most pressing to the application of genomics in wildlife disease research: 1) "Improving communication," 2) "Methodological and analytical advancements," 3) "Translation into practice," 4) "Integrating landscape ecology and genomics," and 5) "Emerging new questions." Wide-ranging solutions from the horizon scan were international in scope, itemized both deficiencies and strengths in wildlife genomic initiatives, promoted the use of genomic technologies to unite wildlife and human disease research, and advocated best practices for optimal use of genomic tools in wildlife disease projects. The results offer a glimpse of the potential revolution in human and wildlife disease research possible through multi-disciplinary collaborations at local, regional, and global scales.

Association Between Earthquake Exposures and Mental Health Outcomes in Phulpingdanda Village After the 2015 Nepal Earthquakes.

In 2015, two strong earthquakes, as well as continuous, high magnitude aftershocks, struck Nepal. Phulpingdanda village was greatly impacted due to its lack of infrastructure and environmental remoteness. Adults from sampled households were surveyed 1-year later to examine the association between earthquake exposures and indicators of depression, post-traumatic stress disorder (PTSD), and resilience. Results showed 33% of surveyed residents screened positive for depression, 9% screened positive for severe PTSD, and 46% displayed moderate to high resilience. Additionally, participants experienced resource loss (100%), damaged home and goods (99%), and exposure to the grotesque (82%). Traumatic earthquake experiences related to personal harm were associated with symptoms of depression and PTSD and resource loss was associated with depressive symptoms. Earthquake experiences associated with less damage to home and goods, but greater exposure to the grotesque were associated with increased resilience. This research adds to our knowledge of the relationship between traumatic exposures and indicators of psychological distress and resilience following a disaster.

Range-Wide Snow Leopard Phylogeography Supports Three Subspecies.

The snow leopard, Panthera uncia, is an elusive high-altitude specialist that inhabits vast, inaccessible habitat across Asia. We conducted the first range-wide genetic assessment of snow leopards based on noninvasive scat surveys. Thirty-three microsatellites were genotyped and a total of 683 bp of mitochondrial DNA sequenced in 70 individuals. Snow leopards exhibited low genetic diversity at microsatellites (AN = 5.8, HO = 0.433, HE = 0.568), virtually no mtDNA variation, and underwent a bottleneck in the Holocene (∼8000 years ago) coinciding with increased temperatures, precipitation, and upward treeline shift in the Tibetan Plateau. Multiple analyses supported 3 primary genetic clusters: (1) Northern (the Altai region), (2) Central (core Himalaya and Tibetan Plateau), and (3) Western (Tian Shan, Pamir, trans-Himalaya regions). Accordingly, we recognize 3 subspecies, Panthera uncia irbis (Northern group), Panthera uncia uncia (Western group), and Panthera uncia uncioides (Central group) based upon genetic distinctness, low levels of admixture, unambiguous population assignment, and geographic separation. The patterns of variation were consistent with desert-basin "barrier effects" of the Gobi isolating the northern subspecies (Mongolia), and the trans-Himalaya dividing the central (Qinghai, Tibet, Bhutan, and Nepal) and western subspecies (India, Pakistan, Tajikistan, and Kyrgyzstan). Hierarchical Bayesian clustering analysis revealed additional subdivision into a minimum of 6 proposed management units: western Mongolia, southern Mongolia, Tian Shan, Pamir-Himalaya, Tibet-Himalaya, and Qinghai, with spatial autocorrelation suggesting potential connectivity by dispersing individuals up to ∼400 km. We provide a foundation for global conservation of snow leopard subspecies, and set the stage for in-depth landscape genetics and genomic studies.

Online surveillance of media health event reporting in Nepal: digital disease detection from a One Health perspective.

BACKGROUND: Traditional media and the internet are crucial sources of health information. Media can significantly shape public opinion, knowledge and understanding of emerging and endemic health threats. As digital communication rapidly progresses, local access and dissemination of health information contribute significantly to global disease detection and reporting. METHODS: Health event reports in Nepal (October 2013-December 2014) were used to characterize Nepal's media environment from a One Health perspective using HealthMap - a global online disease surveillance and mapping tool. Event variables (location, media source type, disease or risk factor of interest, and affected species) were extracted from HealthMap. RESULTS: A total of 179 health reports were captured from various sources including newspapers, inter-government agency bulletins, individual reports, and trade websites, yielding 108 (60%) unique articles. Human health events were reported most often (n = 85; 79%), followed by animal health events (n = 23; 21%), with no reports focused solely on environmental health. CONCLUSIONS: By expanding event coverage across all of the health sectors, media in developing countries could play a crucial role in national risk communication efforts and could enhance early warning systems for disasters and disease outbreaks.

Strengthening adherence to Anti Retroviral Therapy (ART) monitoring and support: operation research to identify barriers and facilitators in Nepal.

BACKGROUND: Anti Retroviral Therapy (ART) is the cornerstone for comprehensive health sector response to Human Immunodeficiency Virus (HIV) treatment, care and support. Adherence of at least 95% is needed to keep HIV under control, as per World Health Organization (WHO) guidelines. This study was aimed at identifying the overall adherence level of, and barriers and facilitators to adherence for patients taking ART in different clinics in all five development regions of Nepal. METHODS: A descriptive cross-sectional study was conducted among ART clients receiving free ART from Government of Nepal ART clinics. A total of 435 clients taking ART from twelve ART clinics in different regions of Nepal, aged fifteen years and above were interviewed on one-and-one basis using questionnaires developed in reference to Adult AIDS Clinical Trial Group (AACTG) toolkit among them data from 404 were analyzed after cleaning. Data was entered and analyzed using Statistical Package for Social Sciences (SPSS) software where the P value of < 0.05 was accepted as being statistically significant. RESULTS: The overall adherence in the last month (missed less than three pills total) was 94.8% (383 out of 404). The main barrier to ART adherence was the fear of side effects (among 61.9% of the non adherent population) which included dizziness (18.1%) and headaches (15.4%). The standard wristwatch (39%) was found to be the most useful aid in enabling timely consumption of medication. Educational status (P = 0.018), drug using habits (P = 0.039) and the conducive environment at ART clinics (P = 0.004) were significantly associated with ART adherence. CONCLUSION: Improving better adherence may require a more holistic approach to treatment regimen and adapting it to patient daily routines. This study identifies issues such as pill count for assessing adherence, better access to health care facilities by clients, better access to medication, as well as improved nutritional support issues for better adherence by the population in the future.

Cholera outbreaks (2012) in three districts of Nepal reveal clonal transmission of multi-drug resistant Vibrio cholerae O1.

BACKGROUND: Although endemic cholera causes significant morbidity and mortality each year in Nepal, lack of information about the causal bacterium often hinders cholera intervention and prevention. In 2012, diarrheal outbreaks affected three districts of Nepal with confirmed cases of mortality. This study was designed to understand the drug response patterns, source, and transmission of Vibrio cholerae associated with 2012 cholera outbreaks in Nepal. METHODS: V. cholerae (n = 28) isolated from 2012 diarrhea outbreaks {n = 22; Kathmandu (n = 12), Doti (n = 9), Bajhang (n = 1)}, and surface water (n = 6; Kathmandu) were tested for antimicrobial response. Virulence properties and DNA fingerprinting of the strains were determined by multi-locus genetic screening employing polymerase chain reaction, DNA sequencing, and pulsed-field gel electrophoresis (PFGE). RESULTS: All V. cholerae strains isolated from patients and surface water were confirmed to be toxigenic, belonging to serogroup O1, Ogawa serotype, biotype El Tor, and possessed classical biotype cholera toxin (CTX). Double-mismatch amplification mutation assay (DMAMA)-PCR revealed the V. cholerae strains to possess the B-7 allele of ctx subunit B. DNA sequencing of tcpA revealed a point mutation at amino acid position 64 (N → S) while the ctxAB promoter revealed four copies of the tandem heptamer repeat sequence 5'-TTTTGAT-3'. V. cholerae possessed all the ORFs of the Vibrio seventh pandemic island (VSP)-I but lacked the ORFs 498-511 of VSP-II. All strains were multidrug resistant with resistance to trimethoprim-sulfamethoxazole (SXT), nalidixic acid (NA), and streptomycin (S); all carried the SXT genetic element. DNA sequencing and deduced amino acid sequence of gyrA and parC of the NAR strains (n = 4) revealed point mutations at amino acid positions 83 (S → I), and 85 (S → L), respectively. Similar PFGE (NotI) pattern revealed the Nepalese V. cholerae to be clonal, and related closely with V. cholerae associated with cholera in Bangladesh and Haiti. CONCLUSIONS: In 2012, diarrhea outbreaks in three districts of Nepal were due to transmission of multidrug resistant V. cholerae El Tor possessing cholera toxin (ctx) B-7 allele, which is clonal and related closely with V. cholerae associated with cholera in Bangladesh and Haiti.

Antimicrobial stewardship hindered by inadequate biosecurity and biosafety practices, and inappropriate antibiotics usage in poultry farms of Nepal-A pilot study.

Nepal's poultry industry has experienced remarkable growth in the last decade, but farm biosafety and biosecurity measures are often overlooked by farmers. As a result, farms often suffer from sporadic and regular outbreaks of many diseases, impacting production and creating public health challenges. Poor management practices, including overuse of antibiotics for prophylaxis and therapeutics, can enhance the spread of poultry diseases by propagating antimicrobial resistance (AMR) that is threatening poultry and human health. We assessed biosafety, biosecurity risks and AMR stewardship in sixteen poultry farms located in four districts: Ramechhap, Nuwakot, Sindhupalchowk, and Kavre. Risk assessment and AMR stewardship evaluation questionnaires were administered to formulate biosafety and biosecurity compliance matrix (BBCM). Risk assessment checklist assessed facility operations, personnel and standard operating procedures, water supply, cleaning and maintenance, rodent/pest control and record keeping. Oral and cloacal samples from the poultry were collected, pooled, and screened for eight poultry pathogens using Polymerase Chain Reaction (PCR) tests. Based on BBCM, we identified the highest BBCM score of 67% obtained by Sindhupalchowk farm 4 and the lowest of 12% by Kavre farm 3. Most of the farms (61.6%) followed general poultry farming practices, only half had clean and well-maintained farms. Lowest scores were obtained for personnel safety standard (42.4%) and rodent control (3.1%). At least one of the screened pathogens were detected in all farms. Mycoplasma gallisepticum was the most common pathogen detected in all but three farms, followed by Mycoplasma synoviae. More than half of the farmers considered AMR a threat, over 26% of them used antibiotics as a preventive measure and 81% did not consider withdrawal period for antibiotics prior to processing of their meat products. Additionally, antibiotics classified as "Watch" and "Restrict" by the WHO were frequently used by the farmers to treat bacterial infections in their farms.

A study on relapse/re-infection rate of Plasmodium vivax malaria and identification of the predominant genotypes of P. vivax in two endemic districts of Nepal.

BACKGROUND: Malaria is a major public health problem in Nepal inflicted primarily by the parasite Plasmodium vivax, - the only species responsible for relapse cases in Nepal. Knowledge on its relapse rate is important for successful malaria control, but is lacking in Nepal. The information on circulating predominant genotypes of P. vivax is equally relevant for high endemic districts of Nepal to understand the transmission dynamics of the parasite and to uncover the coverage and efficacy of potential vaccine beforehand. METHODS: A prospective observational study with a six months follow-up period was conducted from August 2010 to May 2011 in four health centres of Kailali and Kanchanpur districts of Nepal to access the relapse/re-infection rate of P. vivax. The prevalence and heterogeneity of its genotypes were identified by PCR-RFLP assay targeting central repeat region of circumsporozoite protein (Pvcsp). RESULTS: In total, 137 cases microscopically suspected to have P. vivax infection were enrolled in the study. Of these, 23 cases (17%) were detected for the relapse/ re-infection-during a six-month period, with a high proportion being male cases of age group 11-20 years. For genotyping, 100 whole blood samples were analysed, of which 95% of the parasite isolates were found to be of VK210 genotype. The minor genotype VK247 existed either in isolation or as mixed infection with VK210 in rest of the samples. CONCLUSIONS: The relapse/re-infection rate of 17% was determined for P. vivax in Kailali and Kanchanpur districts of Nepal. A heterogeneous Pvcsp genotypic distribution of P. vivax was detected with VK210 being a predominant type, suggesting a complex transmission dynamics of the parasite. Expanding such study in other endemic regions of Nepal would help provide a complete picture on relapse/re-infection rate and parasite genotypic variability that can help in effective control and management of malaria in Nepal.

Novel strains of Campylobacter cause diarrheal outbreak in Rhesus macaques (Macaca mulatta) of Kathmandu Valley.

Campylobacter spp. is often underreported and underrated bacteria that present real health risks to both humans and animals, including non-human primates. It is a commensal microorganism of gastrointestinal tract known to cause gastroenteritis in humans. Commonly found in many wild animals including non-human primates (monkeys- Rhesus macaques) these pathogens are known to be a common cause of diarrhea in humans in many parts of developing and under developed countries. Rhesus macaques from the two holy sites in Kathmandu (Pashupati and Swoyambhu) were included in this cross-sectional study. Diarrheal samples of monkeys were analyzed to detect and characterize the pathogen using 16S rRNA-based PCR screening, followed by DNA sequencing and phylogenetic analysis. Out of a total 67 collected diarrheal samples, Campylobacter spp. were detected in the majority of the samples (n = 64; 96%). DNA sequences of the amplified PCR products were successfully obtained from 13 samples. Phylogenetic analysis identified Candidatus Campylobacter infans (n = 10, Kimura-2 parameter (K2P) pairwise distance values of 0.002287). Remaining three sequences might potentially belong to a novel Campylobacter species/sub-species- closely relating to known species of C. helviticus (K2P pairwise distance of 0.0267). Both Candidatus Campylobacter infans and C. helvitucus are known to infect humans and animals. Additionally, we also detected the bacteria in water and soil samples from the sites. Campylobacter spp. caused the 2018 diarrhea outbreak in Rhesus macaques in the Kathmandu valley. Campylobacter might be one of the important contributing pathogens in diarrheal outbreaks-both in humans and animals (monkeys) in Nepal. Due to close interactions of these animals with humans and other animals, One Health approach might be the most effective way to prevent and mitigate the threat posed by this pathogen.

Phylogenomic analysis supports Mycobacterium tuberculosis transmission between humans and elephants.

Introduction: Tuberculosis is an infectious disease caused by a group of acid-fast bacilli known as Mycobacterium tuberculosis complex (MTC), which has a major impact on humans. Transmission of MTC across the human-animal interface has been demonstrated by several studies. However, the reverse zoonotic transmission from humans to animals (zooanthroponosis) has often been neglected. Methods: In this study, we used Nanopore MinION and Illumina MiSeq approaches to sequence the whole genome of M. tuberculosis strains isolated from two deceased Asian elephants (Elephas maximus) and one human in Chitwan, Nepal. The evolutionary relationships and drug resistance capacity of these strains were assessed using the whole genome data generated by the stand-alone tool Tb-Profiler. Phylogenomic trees were also constructed using a non-synonymous SNP alignment of 2,596 bp, including 94 whole genome sequences representative of the previously described M. tuberculosis lineages from elephants worldwide (lineages 1 and 4) and from humans in Nepal (lineages 1, 2 and 3). Results and Discussion: The new genomes achieved an average coverage of 99.6%, with an average depth of 55.67x. These M. tuberculosis strains belong to lineage 1 (elephant DG), lineage 2 (elephant PK) and lineage 4 (human), and none of them were found to have drug-resistant variants. The elephant-derived isolates were evolutionarily closely related to human-derived isolates previously described in Nepal, both in lineages 1 and 2, providing additional support for zooanthroponosis or bidirectional transmission between humans and elephants. The human-derived isolate clustered together with other published human isolates from Argentina, Russia and the United Kingdom in the lineage 4 clade. This complex multi-pathogen, multi-host system is challenging and highlights the need for a One Health approach to tuberculosis prevention and control at human-animal interface, particularly in regions where human tuberculosis is highly endemic.

Newcastle disease burden in Nepal and efficacy of Tablet I2 vaccine in commercial and backyard poultry production.

Poultry (Gallus domesticus) farming plays an important role as an income generating enterprise in a developing country like Nepal, contributing more than 4% to the national Gross Domestic Product (GDP). Newcastle Disease (ND) is a major poultry disease affecting both commercial and backyard poultry production worldwide. There were more than 90 reported ND outbreaks in Nepal in 2018 with over 74,986 birds being affected. ND is responsible for over 7% of total poultry mortality in the country. Recent outbreaks of ND in 2021 affected many farms throughout Nepal and caused massive loss in poultry production. ND is caused by a single-stranded ribonucleic acid (RNA) virus that presents very similar clinical symptoms as Influenza A (commonly known as bird flu) adding much complexity to clinical disease identification and intervention. We conducted a nationwide ND and Influenza A (IA) prevalence study, collecting samples from representative commercial and backyard poultry farms from across the major poultry production hubs of Nepal. We used both serological and molecular assessments to determine disease exposure history and identification of strains of ND Virus (NDV). Of the 40 commercial farms tested, both NDV (n = 28, 70%) and IAV (n = 11, 27.5%) antibodies were detected in majority of the samples. In the backyard farms (n = 36), sero-prevalence of NDV and IAV were 17.5% (n = 7) and 7.5% (n = 3) respectively. Genotype II NDV was present in most of the commercial farms, which was likely due to live vaccine usage. We detected never reported Genotype I NDV in two backyard farm samples. Our investigation into 2021 ND outbreak implicated Genotype VII.2 NDV strain as the causative pathogen. Additionally, we developed a Tablet formulation of the thermostable I2-NDV vaccine (Ranigoldunga™) and assessed its efficacy on various (mixed) breeds of chicken (Gallus domesticus). Ranigoldunga™ demonstrated an overall efficacy >85% with a stability of 30 days at room temperature (25°C). The intraocularly administered vaccine was highly effective in preventing ND, including Genotype VII.2 NDV strain.

Identification and characterization of probiotics isolated from indigenous chicken (Gallus domesticus) of Nepal.

BACKGROUND: Excessive and irrational use of antibiotics as growth promoters in poultry has been one of key factors contributing to increased emergence of antibiotics resistant bacteria. Several alternatives for antibiotic growth promoters are being sought, and the search for effective probiotics to be used as feed additives is amongst the promising ones. Our study aimed to isolate and test potential probiotics bacteria from cloacal swabs of various indigenous chicken (Gallus domesticus) breeds from rural outskirts of the Kathmandu valley (Nepal). METHODS: Selective isolation of probiotics was conducted by micro-aerophilic enrichment of sample in MRS Broth at 37°C, followed by culturing on MRS agar supplemented with 5 g/L of CaCO3. Isolated bacterial colonies producing transparent halo were selected as potential lactic acid bacteria (LAB), and tested for their antibacterial activity, phenotypic and biochemical characteristics, acidic yield, and tolerance to acid and bile. RESULTS: A total of 90 potential LAB were isolated from cloacal samples collected from 41 free-ranging chickens of indigenous breeds. Of these, 52 LAB isolates (57%) showed variable antibacterial activity to at least one bacterial pathogen. Of 52 LAB, 46 isolates fulfilled phenotypic and biochemical criteria of Lactobacillus spp. Of these, 37 isolates produced varying percentage yields of lactic acid, 27 isolates showed survival at pH 3.0, and 17 isolates showed survival tolerances in the presence of 0.3% and 0.5% bile salts for 24 hours. Phylogenetic analysis of 16S rDNA sequencing of LAB isolates fulfilling in vitro probiotics properties showed that 3 isolates had genetic identity of 99.38% with Lactobacillus plantarum, while one isolate was genetically similar (99.85%) with the clade of L. reuteri, L. antri and L. panis. CONCLUSION: Our study identified four Lactobacillus spp. strains having potential probiotics properties. Further investigations are needed to evaluate these isolates to be used as poultry probiotics feed supplement.

Rapid genomic surveillance of SARS-CoV-2 in a dense urban community of Kathmandu Valley using sewage samples.

Understanding disease burden and transmission dynamics in resource-limited, low-income countries like Nepal are often challenging due to inadequate surveillance systems. These issues are exacerbated by limited access to diagnostic and research facilities throughout the country. Nepal has one of the highest COVID-19 case rates (915 cases per 100,000 people) in South Asia, with densely-populated Kathmandu experiencing the highest number of cases. Swiftly identifying case clusters (hotspots) and introducing effective intervention programs is crucial to mounting an effective containment strategy. The rapid identification of circulating SARS-CoV-2 variants can also provide important information on viral evolution and epidemiology. Genomic-based environmental surveillance can help in the early detection of outbreaks before clinical cases are recognized and identify viral micro-diversity that can be used for designing real-time risk-based interventions. This research aimed to develop a genomic-based environmental surveillance system by detecting and characterizing SARS-CoV-2 in sewage samples of Kathmandu using portable next-generation DNA sequencing devices. Out of 22 sites in the Kathmandu Valley from June to August 2020, sewage samples from 16 (80%) sites had detectable SARS-CoV-2. A heatmap was created to visualize the presence of SARS-CoV-2 infection in the community based on viral load intensity and corresponding geospatial data. Further, 47 mutations were observed in the SARS-CoV-2 genome. Some detected mutations (n = 9, 22%) were novel at the time of data analysis and yet to be reported in the global database, with one indicating a frameshift deletion in the spike gene. SNP analysis revealed possibility of assessing circulating major/minor variant diversity on environmental samples based on key mutations. Our study demonstrated the feasibility of rapidly obtaining vital information on community transmission and disease dynamics of SARS-CoV-2 using genomic-based environmental surveillance.

A study of the prevalence and risk factors leading to HIV infection among a sample of street children and youth of Kathmandu.

BACKGROUND: The true prevalence of HIV and other sexually transmitted diseases among street children in Nepal is virtually unknown while information on related behavioural risk factors in this population is non-existent. The risk of HIV infection among street children and adolescents may be especially high due to their marginalized social and economic conditions. This study was conducted to determine the prevalence of HIV infection among a sample of street children and youth of Kathmandu and to identify risk factors associated with HIV infection in this group.A sample of street children and youth was recruited based on the purposive sampling of ten streets in Kathmandu, Nepal, known to have a high density of street children and youth. A total of 251 street children (aged 11-16 years) and youth (aged 17-24 years) were enrolled, with informed consent, from November, 2008 through June, 2009. Most of the participants (95%) were male. Case status was determined by serological assessment of HIV status; data on risk factors were obtained using structured survey interviews. HIV prevalence and rates of a number of behavioural risk factors suspected to play a role in HIV transmission among street children and youth were determined, including unprotected sex, intravenous drug use, and other risky sex and substance use behaviours. RESULTS: Among the 251 children and youth, we found an overall HIV prevalence of 7.6%. As the sample size of females was small (n = 13) and the behavioural risk factors are likely to be quite different for boys and girls, we conducted separate analyses by gender. As our small sample of females is unlikely to be representative and lacks power for statistical testing, our report focuses on the results for the males surveyed.The strongest behavioural risk factor to emerge from this study was intravenous drug use; 30% of the male subjects were injecting drug users and 20% of those were HIV positive. Furthermore, frequency of drug injection was a highly significant predictor with a dose-response relationship; males reporting occasional injection drug use were nearly 9 times more likely to be HIV positive than never users, while weekly drug injectors had over 46 times the risk of non-users, controlling for exposure to group sex, the only other significant risk factor in the multivariate model. CONCLUSIONS: This sample of street children and youth of Kathmandu has a nearly 20-fold higher prevalence of HIV infection than the general population of Nepal (0.39%). The children and youth engage in number of high risk behaviours, including intravenous drug use, putting them at significant risk of contracting HIV and other sexually transmitted infections.

Climate change and health vulnerability in Nepal: A systematic review of the literature since 2010.

The Nepali population is among those most vulnerable to the health impacts of climate change. We conducted a systematic literature review to document the health effects of climate change in Nepal and identify knowledge gaps by examining vulnerability categories related to health. Three databases were searched for journal articles that addressed health and vulnerability related to climate change in Nepal from 2010 onwards. Of the 1063 articles identified, 37 were eligible for inclusion. The findings suggested the health of the population was affected mostly by food insecurity, floods, droughts, and reduced water levels. Studies revealed both morbidity and mortality increased due to climate change, with the most impacted populations being women, children, and the elderly. At greatest risk for impacts from climate change were those from poor and marginal populations, especially impoverished women. The public health sector, healthcare, and potable water sources were some of the least mentioned vulnerability subcategories, indicating more research is needed to better understand their adaptation capacities. We propose that identifying vulnerabilities and areas of limited research are critical steps in the prioritization of health policy and interventions for the most vulnerable populations in Nepal.

Antibiotic resistance genes of public health importance in livestock and humans in an informal urban community in Nepal.

Efforts to mitigate the increasing emergence of antimicrobial resistance (AMR) will benefit from a One Health perspective, as over half of animal antimicrobials are also considered medically important in humans, and AMR can be maintained in the environment. This is especially pertinent to low- and middle-income countries and in community settings, where an estimated 80% of all antibiotics are used. This study features AMR genes found among humans, animals, and water at an urban informal settlement in Nepal with intensifying livestock production. We sampled humans, chickens, ducks, swine, and water clustered by household, as well as rodents and shrews near dwellings, concurrently in time in July 2017 in southeastern Kathmandu along the Manohara river. Real-time qualitative PCR was performed to screen for 88 genes. Our results characterize the animal-human-environmental interfaces related to the occurrence of specific resistance genes (blaSHV-1 (SHV(238G240E) strain), QnrS, ermC, tetA, tetB, aacC2, aadA1) associated with antibiotics of global health importance that comprise several drug classes, including aminoglycosides, beta-lactams, tetracyclines, macrolides, and fluoroquinolones. By characterizing risk factors across AMR genes of public health importance, this research highlights potential transmission pathways for further investigation and provides prioritization of community-based prevention and intervention efforts for disrupting AMR transmission of critically important antibiotics used in both humans and animals in Nepal.

Molecular and morphological identification of frog species collected at Rara Lake in Rara National Park, Nepal.

This study seeks to clarify the taxonomic identity of three adult frogs and two tadpoles from Rara Lake situated in Rara National Park, Mugu district, Western Nepal, using both phenotypic and mitochondrial sequence data (16S rRNA gene). Based on the molecular data, we determined our specimens belonged to two species; Paa cf. ercepeae (Dubois, 1974) and Paa rarica (Dubois, Matsui, and Ohler, 2001), and this assignment is also supported by morphology. Principal component analysis of phenotypic data clustered species into large-bodied (SVL > 55 mm) and small-bodied (SVL < 55 mm) groups. The molecular data suggest that Paa rarica belonged to a group that contains several undescribed lineages and that Paa liebigii (Gnther, 1860) hosts extensive cryptic diversity. Our study also documents that Paa species in the Himalayan region are more widely distributed than previously believed.