As part of a comprehensive health care programme for people who use drugs in Nepal, HIV and viral hepatitis B and C status—including risk factors, HCV-genotypes and co-infections—as well as two IL28B Single-nucleotide polymorphisms (SNPs) were assessed for a random sample of 401 people who inject drugs in three regions of Nepal: mid-western Terrai (Nepalgunj), the eastern region (Dharan, Biratnagar) and the central region (Kathmandu, Lalitpur and Chitwan). Individuals were included who showed at least a minimum of health care seeking behaviour. This latter criterion was defined by being registered with any organisation offering health services. The average age of the participants was 30.5 yrs, and the average length of intravenous drug use was 8.5 yrs. The prevalence rates of HBsAg, anti-HIV antibodies and HCV-RNA were 3.5%, 13.8% and 41.9%, respectively. Spontaneous HCV clearance was evident in 16% of all of those who tested positive for anti-HCV antibodies. Independent risk factors for HCV-RNA positivity were age, gender, geographical region, duration of injecting drug use, history of imprisonment and HIV co-infection. In the age group ≤24 yrs, the rate of spontaneous HCV clearance was 43.5%. Overall, 59.8% of HCV infections were caused by HCV genotype 3 and 40.2% by HCV genotype 1. No other HCV genotypes were identified in this study. The IL28B SNP rs12979860 and rs8099917 were identified in 122 patients, and 75.4% of all participants had both favourable genotypes rs12979860 C/C and rs8099917 T/T.

BackgroundAlthough 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.MethodsV. 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).ResultsAll 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.ConclusionsIn 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.

With fewer than 200 tigers (Panthera tigris tigris) left in Nepal, that are generally confined to five protected areas across the Terai Arc Landscape, genetic studies are needed to provide crucial information on diversity and connectivity for devising an effective country-wide tiger conservation strategy. As part of the Nepal Tiger Genome Project, we studied landscape change, genetic variation, population structure, and gene flow of tigers across the Terai Arc Landscape by conducting Nepal’s first comprehensive and systematic scat-based, non-invasive genetic survey. Of the 770 scat samples collected opportunistically from five protected areas and six presumed corridors, 412 were tiger (57%). Out of ten microsatellite loci, we retain eight markers that were used in identifying 78 individual tigers. We used this dataset to examine population structure, genetic variation, contemporary gene flow, and potential population bottlenecks of tigers in Nepal. We detected three genetic clusters consistent with three demographic sub-populations and found moderate levels of genetic variation (He = 0.61, AR = 3.51) and genetic differentiation (FST = 0.14) across the landscape. We detected 3–7 migrants, confirming the potential for dispersal-mediated gene flow across the landscape. We found evidence of a bottleneck signature likely caused by large-scale land-use change documented in the last two centuries in the Terai forest. Securing tiger habitat including functional forest corridors is essential to enhance gene flow across the landscape and ensure long-term tiger survival. This requires cooperation among multiple stakeholders and careful conservation planning to prevent detrimental effects of anthropogenic activities on tigers.

BackgroundThe 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.ResultsAmong 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.ConclusionsThis 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.

Background Multi-drug resistance (MDR) and extensive-drug resistance (XDR) associated with extended-spectrum beta-lactamases (ESBLs) and carbapenemases in Gram-negative bacteria are global public health concerns. Data on circulating antimicrobial resistance (AMR) genes in Gram-negative bacteria and their correlation with MDR and ESBL phenotypes from Nepal is scarce. Methods A retrospective study was performed investigating the distribution of ESBL and carbapenemase genes and their potential association with ESBL and MDR phenotypes in E. coli, Klebsiella spp., Enterobacter spp. and Acinetobacter spp. isolated in a major tertiary hospital in Kathmandu, Nepal, between 2012 and 2018. Results During this period, the hospital isolated 719 E. coli, 532 Klebsiella spp., 520 Enterobacter spp. and 382 Acinetobacter spp.; 1955/2153 (90.1%) of isolates were MDR and half (1080/2153) were ESBL producers. Upon PCR amplification, blaTEM (1281/1771; 72%), blaCTXM-1 (930/1771; 53%) and blaCTXM-8 (419/1771; 24%) were the most prevalent ESBL genes in the enteric bacilli. BlaOXA and blaOXA-51 were the most common blaOXA family genes in the enteric bacilli (918/1771; 25%) and Acinetobacter spp. (218/382; 57%) respectively. Sixteen percent (342/2153) of all isolates and 20% (357/1771) of enteric bacilli harboured blaNDM-1 and blaKPC carbapenemase genes respectively. Of enteric bacilli, Enterobacter spp. was the most frequently positive for blaKPC gene (201/337; 60%). The presence of each blaCTX-M and blaOXA were significantly associated with non-susceptibility to third generation cephalosporins (OR 14.7, p < 0.001 and OR 2.3, p < 0.05, respectively).The presence of each blaTEM, blaCTXM and blaOXA family genes were significantly associated with ESBL positivity (OR 2.96, p < 0.001; OR 14.2, p < 0.001 and OR 1.3, p < 0.05 respectively) and being MDR (OR 1.96, p < 0.001; OR 5.9, p < 0.001 and OR 2.3, p < 0.001 respectively). Conclusions This study documents an alarming level of AMR with high prevalence of MDR ESBL- and carbapenemase-positive ESKAPE microorganisms in our clinical setting. These data suggest a scenario where the clinical management of infected patients is increasingly difficult and requires the use of last-resort antimicrobials, which in turn is likely to intensify the magnitude of global AMR crisis.

Kathmandu, Nepal has been classified as a high-risk area for highly pathogenic avian influenza (HPAI) by the Nepali Government. While ducks have an important role in the transmission of avian influenza viruses (AIV), including HPAI, seroprevalence of antibodies to AIV in domestic ducks of Kathmandu has never been assessed. The objectives of this study were (i) to estimate the prevalence of seroconversion to AIV in domestic ducks in major duck-raising areas of Kathmandu and (ii) to assess the effect of age, sex, presence of swine and the number of ducks on the farm on the carriage of antibodies to AIV in these ducks. From April through July of 2011, a cross-sectional study was conducted and a total of 310 ducks in the major duck-raising areas of Kathmandu were sampled. The estimated prevalence of AIV antibodies was 27.2% [95% confidence interval (CI): 24.6-29.5]. Of 62 enrolled farms, 42% had at least one seropositive duck. Half of the enrolled farms also kept pigs of which 52% had at least one seropositive duck. Bivariate analysis indicated association between ducks' seroconversion to AIV and their age, sex and farm size. However, the final multivariable model, after controlling for clustering of ducks within farms, identified age as the only significant risk factor. Based on this model, ducks older than 1 year of age were more likely to be seropositive compared to ducks <6 months of age [odds ratio = 2.17 (1.07-4.39)]. These results provide baseline information about the AIV seroprevalence in domestic ducks in the major duck-raising areas of Kathmandu and identify a high-risk group that can be targeted in surveillance activities. Future studies should be conducted to differentiate the subtypes of AIV present among domestic ducks in Kathmandu, with particular interest in the presence of HPAI viruses.

BackgroundMalaria 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.MethodsA 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).ResultsIn 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.ConclusionsThe 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.

Tiger (Panthera tigris) populations are in danger across their entire range due to habitat loss, poaching and the demand for tiger parts. The Bengal tiger (Panthera tigris tigris) is an endangered apex predator with a population size estimated to be less than 200 in Nepal. In spite of strict wildlife protection laws, illegal trade of tiger parts is increasing; and Nepal has become one of the major sources and transit routes for poached wildlife parts. Identification of wildlife parts is often challenging for law enforcement officials due to inadequate training and lack of available tools. Here, we describe a molecular forensic approach to gain insight into illegally trafficked tiger parts seized across Nepal. We created Nepal’s first comprehensive reference genetic database of wild tigers through the Nepal Tiger Genome Project (2011–2013). This database has nuclear DNA microsatellite genotype and sex profiles, including geo-spatial information, of over 60% (n = 120) of the wild tigers of Nepal. We analyzed 15 putative cases of confiscated poached tiger parts and all were confirmed to be of tiger. Ten samples were identified as male and five were female. We determined probable geo-source location for 9 of the 14 samples with 6–8 nuclear DNA microsatellite loci using inferences from four different statistical assignment methods. Six samples were assigned to Bardia National Park and one of these was an exact match to a female tiger previously profiled in our fecal DNA reference database. Two tiger samples were assigned to Shuklaphanta Wildlife Reserve and one to Chitwan National Park. We are unable to definitively assign five tiger samples which could be offspring dispersers or might have come from tiger population outside of Nepal. Our study revealed that the western region, particularly Bardia National Park, is a poaching hotspot for illegal tiger trade in Nepal. We present feasibility of using molecular forensic based evidence to incriminate criminals in a court of law in the fight against wildlife crime.