Multidrug-resistant Klebsiella pneumoniae clinical isolates producing NDM- and OXA-type carbapenemase in Nepal.

OBJECTIVES: The emergence of multidrug-resistant Klebsiella pneumoniae has become a serious problem in medical settings worldwide. METHODS: A total of 46 isolates of multidrug-resistant K. pneumoniae were obtained from 2 hospitals in Nepal from October 2018 to April 2019. RESULTS: Most of these isolates were highly resistant to carbapenems, aminoglycosides and fluoroquinolones with the minimum inhibitory concentrations (MICs) of more than 64 µg/mL. These isolates harbored carbapenemase-encoding genes, including blaNDM-1, blaNDM-5, blaOXA-181 and blaOXA-232, and 16S rRNA methyltransferase-encoding genes, including armA, rmtB, rmtC and rmtF. Multilocus sequence typing revealed that 44 of 46 isolates were high-risk clones such as ST11 (2%), ST14 (4%), ST15 (11%), ST37 (2%), ST101 (2%), ST147 (28%), ST231 (13%), ST340 (4%) and ST395 (28%). In particular, ST395 isolates, which spread across medical settings in Nepal, co-harbored blaNDM-5 and rmtB on IncFII plasmids and co-harbored blaOXA-181/-232 and rmtF on ColKP3 plasmids. Several isolates harbored blaOXA-181 or blaNDM-5 on their chromosomes and multi-copies of blaNDM-1 or genes encoding 16S rRNA methyltransferases on their plasmids. CONCLUSIONS: The presented study demonstrates that the high-risk clones of multidrug-resistant K. pneumoniae spread in a clonal manner across hospitals in Nepal.

Impact of topical applications of sunflower seed oil on neonatal mortality and morbidity in southern Nepal: a community-based, cluster-randomised trial.

INTRODUCTION: Hospital-based studies have demonstrated topical applications of sunflower seed oil (SSO) to skin of preterm infants can reduce nosocomial infections and improve survival. In South Asia, replacing traditional mustard with SSO might have similar benefits. METHODS: 340 communities in Sarlahi, Nepal were randomised to use mustard oil (MO) or SSO for community practice of daily newborn massage. Women were provided oil in late pregnancy and the first month post partum, and visited daily through the first week of life to encourage massage practice. A separate data collection team visited on days 1, 3, 7, 10, 14, 21 and 28 to record vital status and assess serious bacterial infection. RESULTS: Between November 2010 and January 2017, we enrolled 39 479 pregnancies. 32 114 live births were analysed. Neonatal mortality rates (NMRs) were 31.8/1000 (520 deaths, 16 327 births) and 30.5/1000 (478 deaths, 15 676 births) in control and intervention, respectively (relative risk (RR)=0.95, 95% CI: 0.84, 1.08). Among preterm births, NMR was 90.4/1000 (229 deaths, 2533 births) and 79.2/1000 (188 deaths, 2373 births) in control and intervention, respectively (RR=0.88; 95% CI: 0.74, 1.05). Among preterm births <34 weeks, the RR was 0.83 (95% CI: 0.67, 1.02). No statistically significant differences were observed in incidence of serious bacterial infection. CONCLUSIONS: We did not find any neonatal mortality or morbidity benefit of using SSO instead of MO as emollient therapy in the early neonatal period. Further studies examining whether very preterm babies may benefit are warranted. TRIAL REGISTRATION NUMBER: ClinicalTrials.gov Registry (NCT01177111).

Evaluating acute gastroenteritis-causing pathogen reduction in wastewater and the applicability of river water for wastewater-based epidemiology in the Kathmandu Valley, Nepal.

Rapid urbanization and population growth without the implementation of proper waste management are capable of contaminating water sources, which can lead to acute gastroenteritis. This study examined the detection and reduction of five gastroenteritis-causing enteropathogens, Salmonella, Campylobacter coli, Campylobacter jejuni, Clostridium perfringens, and genogroup IV norovirus, and one respiratory pathogen, influenza A virus, in two municipal wastewater treatment plants (WWTP) using an oxidation ditch system (WWTP A; n = 20) and a stabilization pond system (WWTP B; n = 18) in the Kathmandu Valley, Nepal, collected between August 2017 and August 2019. All enteropathogens were detected in wastewater via quantitative PCR. The concentrations of the pathogens ranged from 5.7 to 7.9 log10 copies/L in WWTP A and from 4.9 to 8.1 log10 copies/L in WWTP B. The log10 reduction values of the pathogens ranged from 0.3 to 1.0 in WWTP A and from -0.1 to 0.2 in WWTP B. The association between the pathogen concentrations and the number of clinical cases in the corresponding week could not be evaluated; however, the consistent detection of pathogens in the wastewater despite low number of case reports suggested the use of wastewater-based epidemiology (WBE) for early warning of acute gastroenteritis (AGE) in the Kathmandu Valley. The pathogens were also detected in river water at approximately 7.0 log10 copies/L and exhibited no significant difference in concentration compared to wastewater, suggesting the applicability of river water for WBE of AGE. Insufficient treatment of all pathogens in the wastewater was observed, suggesting the need for full rehabilitation of the treatment plants. However, the influent may be utilized for early detection of AGE-causing pathogens in the city, whereas the river water may serve as an alternative in areas without connection to the WWTPs.

Knowledge, attitudes and practices (KAP) on COVID-19 among the general population in most affected districts of Nepal.

The COVID-19 pandemic has become one of the global health challenges in the current context. In Nepal, the first confirmed case was reported on 23 January 2020, and since then, it has resulted in several negative impacts, including economic disruption and deterioration of physical and mental health. In such a pandemic, it is indispensable to understand the knowledge and behavioral patterns of the general population regarding COVID-19. Therefore, our study aimed to assess the knowledge, attitudes and practices on COVID-19 among the general population in most affected districts and its relationship with socio-demographic conditions. The cross-sectional study was conducted among the general population above the age of 18 years from eight districts of Nepal including Kathmandu, Bhaktapur, Lalitpur, Morang, Sunsari, Rupandehi, Chitwan, and Kaski. A convenient non-probability sampling method was considered with total sample size of 702. The questionnaire survey was conducted to collect data. Descriptive statistics, non-parametric statistical tests, and a logistic regression model were used for analysis. The study showed that 93.3% of respondents had knowledge of overall preventive practices, whereas only 32% had knowledge of overall symptoms of COVID-19. Regarding attitudes, only 14.3% strongly believed that the COVID-19 pandemic would end soon. The preventive practice was reduced after the lockdown compared to that of during the lockdown. The respondents with white-collar occupations, high-income, and unmarried were good at KAP. Similarly, highly educated and those residing in urban areas had good knowledge and practice. The study findings will help in the development of targeted programs to improve the general population's knowledge, attitudes and practices on COVID-19, which is paramount to dealing with the existing pandemic and also such possible future waves of the pandemic.

Prevalence of antibiotic resistance genes in drinking and environmental water sources of the Kathmandu Valley, Nepal.

Antibiotic-resistant bacteria-associated infections are responsible for more than 1.2 million annual deaths worldwide. In low- and middle-income countries (LMICs), the consumption of antibiotics for human and veterinary uses is not regulated effectively. Overused and misused antibiotics can end up in aquatic environments, which may act as a conduit for antibiotic resistance dissemination. However, data on the prevalence of antibiotic resistance determinants in aquatic environments are still limited for LMICs. In this study, we evaluated the prevalence and concentration of antibiotic resistance genes (ARGs) in different drinking and environmental water sources collected from the Kathmandu Valley, Nepal, using droplet digital polymerase chain reaction to understand the current situation of ARG contamination. River water and shallow dug well water sources were the most contaminated with ARGs. Almost all samples contained sul1 (94%), and intI1 and tet(A) were detected in 83 and 60% of the samples, respectively. Maximum ARG concentration varied between 4.2 log10 copies/100 ml for mecA and 9.3 log10 copies/100 ml for sul1. Significant positive correlations were found between ARGs (r > 0.5, p < 0.01), except for mecA, qnrS, and vanA. As sul1 and intI1 were detected in almost all samples, the presence of these genes in a given sample may need to be considered as background antibiotic resistance in LMICs. Therefore, monitoring of ARGs, such as ß-lactam ARGs, quinolone resistance genes, and vancomycin resistance genes, may provide a better picture of the antibiotic resistance determinants in aquatic environments of LMICs.

Stenotrophomonas maltophilia from Nepal Producing Two Novel Antibiotic Inactivating Enzymes, a Class A ß-Lactamase KBL-1 and an Aminoglycoside 6'-N-Acetyltransferase AAC(6')-Iap.

Seven drug-resistant strains of Stenotrophomonas maltophilia were isolated from patients at two university hospitals in Nepal. S. maltophilia JUNP497 was found to encode a novel class A ß-lactamase, KBL-1 (Kathmandu ß-lactamase), consisting of 286 amino acids with 52.98% identity to PSV-1. Escherichia coli transformants expressing blaKBL-1 were less susceptible to penicillins. The recombinant KBL-1 protein efficiently hydrolyzed penicillins. The genomic environment surrounding blaKBL-1 was a unique structure, with the upstream region derived from strains in China and the downstream region from strains in India. S. maltophilia JUNP350 was found to encode a novel 6'-N-aminoglycoside acetyltransferase, AAC(6')-Iap, consisting of 155 amino acids with 85.0% identity to AAC(6')-Iz. E. coli transformants expressing aac(6')-Iap were less susceptible to arbekacin, amikacin, dibekacin, isepamicin, neomycin, netilmicin, sisomicin and tobramycin. The recombinant AAC(6')-Iap protein acetylated all aminoglycosides tested, except for apramycin and paromomycin. The genomic environment surrounding aac(6')-Iap was 90.99% identical to that of S. maltophilia JV3 obtained from a rhizosphere in Brazil. Phylogenetic analysis based on whole genome sequences showed that most S. maltophilia isolates in Nepal were similar to those isolates in European countries, including Germany and Spain. IMPORTANCE The emergence of drug-resistant S. maltophilia has become a serious problem in medical settings worldwide. The present study demonstrated that drug-resistant S. maltophilia strains in Nepal harbored novel genes encoding a class A ß-lactamase, KBL-1, or a 6'-N-aminoglycoside acetyltransferase, AAC(6')-Iap. Genetic backgrounds of most S. maltophilia strains in Nepal were similar to those in European countries. Surveillance of drug-resistant S. maltophilia in medical settings in Nepal is necessary.

Detection of SARS-CoV-2 RNA in wastewater, river water, and hospital wastewater of Nepal.

The applicability of wastewater-based epidemiology (WBE) has been extensively studied throughout the world with remarkable findings. This study reports the presence and reduction of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) at two wastewater treatment plants (WWTPs) of Nepal, along with river water, hospital wastewater (HWW), and wastewater from sewer lines collected between July 2020 and February 2021. SARS-CoV-2 RNA was detected in 50%, 54%, 100%, and 100% of water samples from WWTPs, river hospitals, and sewer lines, respectively, by at least one of four quantitative PCR assays tested (CDC-N1, CDC-N2, NIID_2019-nCOV_N, and N_Sarbeco). The CDC-N2 assay detected SARS-CoV-2 RNA in the highest number of raw influent samples of both WWTPs. The highest concentration was observed for an influent sample of WWTP A (5.5 ± 1.0 log10 genome copies/L) by the N_Sarbeco assay. SARS-CoV-2 was detected in 47% (16/34) of the total treated effluents of WWTPs, indicating that biological treatments installed at the tested WWTPs are not enough to eliminate SARS-CoV-2 RNA. One influent sample was positive for N501Y mutation using the mutation-specific qPCR, highlighting a need for further typing of water samples to detect Variants of Concern. Furthermore, crAssphage-normalized SARS-CoV-2 RNA concentrations in raw wastewater did not show any significant association with the number of new coronavirus disease 2019 (COVID-19) cases in the whole district where the WWTPs were located, suggesting a need for further studies focusing on suitability of viral as well as biochemical markers as a population normalizing factor. Detection of SARS-CoV-2 RNA before, after, and during the peaking in number of COVID-19 cases suggests that WBE is a useful tool for COVID-19 case estimation in developing countries.

Emergence of a highly colistin-resistant Aeromonas jandaei clinical isolate harbouring four genes encoding phosphoethanolamine transferases in Nepal.

OBJECTIVES: This study aimed to describe a clinical isolate of Aeromonas jandaei (A. jandaei) in Nepal that harboured four types of genes encoding phosphoethanolamine transferases. METHODS: An isolate of colistin-resistant A. jandaei was obtained from a blood sample of an inpatient in a hospital in Nepal, and its complete genome sequence was determined. Escherichia coli (E. coli) and Aeromonas hydrophila (A. hydrophila) transformants expressing genes encoding novel phosphoethanolamine transferase variants were constructed and colistin-susceptibility profiles were determined. RESULTS: The isolate harboured four genes encoding phosphoethanolamine transferases on the chromosome, which were designated eptAv3.2, eptAv3.3, eptAv3.4 and eptAv7.2. The amino acid sequences of EptAv3.2, 3.3 and 3.4 were > 80% identical to MCR-3.1, and that of EptAv7.2 was > 79% identical to MCR-7.1. E. coli expressing eptAv3.2, 3.3 and 3.4 showed reduced susceptibility to colistin, whereas E. coli expressing eptAv7.2 did not. In contrast, A. hydrophila expressing eptAv7.2 showed reduced susceptibility to colistin, whereas A. hydrophila expressing eptAv3.2, 3.3 and 3.4 did not; eptAv3.3 and 3.4 formed a tandem structure. The genomic environments surrounding eptAv3.2, 3.3 and 3.4 were similar to Aeromonas veronii obtained from the effluent of a treatment plant in Japan in 2018. The genomic environment surrounding eptAv7.2 was similar to that of A. jandaei obtained from a chicken in the USA in 2019. CONCLUSIONS: The highly colistin-resistant A. jandaei clinical isolate harboured four chromosomal genes encoding phosphoethanolamine transferases, suggesting that Aeromonas spp. harbouring eptAv genes with strong similarities to mcr-3 and mcr-7 are emerging in medical settings as well as environments.

Molecular characterisation of carbapenem-resistant Pseudomonas aeruginosa clinical isolates in Nepal.

OBJECTIVES: The emergence of carbapenem-resistant Pseudomonas aeruginosa has become a serious worldwide medical problem. The aim of this study was to determine the genetic and epidemiological properties of carbapenem-resistant P. aeruginosa strains isolated from hospitals in Nepal. METHODS: A total of 43 carbapenem-resistant P. aeruginosa isolates obtained from patients in two hospitals in Nepal between 2018 and 2020 were analysed. Their whole genomes were sequenced by next-generation sequencing. A phylogenetic tree was constructed from single nucleotide polymorphism (SNP) concatemers. Multilocus sequence typing (MLST) was performed and antimicrobial resistance genes were identified. RESULTS: Of the 43 isolates, 17 harboured genes encoding carbapenemases, including IMP-1, IMP-26, KPC-2, NDM-1, VIM-2 and VIM-5, and 12 harboured genes encoding 16S rRNA methylases, including RmtB4 and RmtF2. The carbapenem-resistant P. aeruginosa isolated in Nepal belonged to various sequence types (STs), including ST235 (5 isolates), ST244 (7 isolates), ST274 (1 isolate), ST357 (10 isolates), ST654 (3 isolates), ST664 (1 isolate), ST773 (1 isolate), ST823 (3 isolates), ST1047 (8 isolates), ST1203 (2 isolates) and ST3453 (2 isolates). CONCLUSION: To the best of our knowledge, this is the first molecular epidemiological analysis of carbapenem-resistant P. aeruginosa clinical isolates from Nepal. The findings strongly suggest that P. aeruginosa isolates producing carbapenemases and 16S rRNA methylases have spread throughout medical settings in Nepal.

Highly multidrug-resistant Morganella morganii clinical isolates from Nepal co-producing NDM-type metallo-ß-lactamases and the 16S rRNA methylase ArmA.

Morganella morganii can harbour extended-spectrum ß-lactamases and carbapenemases, resulting in increased resistance to multiple antibiotics and a high mortality rate. This study describes the emergence of highly multidrug-resistant clinical isolates of M. morganii from Nepal co-producing NDM-type metallo-ß-lactamases, including NDM-1 and NDM-5, and the 16S rRNA methylase ArmA. This is the first report of M. morganii clinical isolates from Nepal co-producing NDM-1/-5 and ArmA. It is important to establish infection control systems and effective treatments against multidrug-resistant M. morganii.

Arsenic disturbs the gut microbiome of individuals in a disadvantaged community in Nepal.

Arsenic is ubiquitous in nature, highly toxic, and is particularly abundant in Southern Asia. While many studies have focused on areas like Bangladesh and West Bengal, India, disadvantaged regions within Nepal have also suffered from arsenic contamination levels, with wells and other water sources possessing arsenic contamination over the recommended WHO and EPA limit of 10 µg/L, some wells reporting levels as high as 500 µg/L. Despite the region's pronounced arsenic concentrations within community water sources, few investigations have been conducted to understand the impact of arsenic contamination on host gut microbiota health. This study aims to examine differential arsenic exposure on the gut microbiome structure within two disadvantaged communities in southern Nepal. Fecal samples (n = 42) were collected from members of the Mahuawa (n = 20) and Ghanashyampur (n = 22) communities in southern Nepal. The 16S rRNA gene was amplified from fecal samples using Illumina-tag PCR and subject to high-throughput sequencing to generate the bacterial community structure of each sample. Bioinformatics analysis and multivariate statistics were conducted to identify if specific fecal bacterial assemblages and predicted functions were correlated with urine arsenic concentration. Our results revealed unique assemblages of arsenic volatilizing and pathogenic bacteria positively correlated with increased arsenic concentration in individuals within the two respective communities. Additionally, we observed that commensal gut bacteria negatively correlated with increased arsenic concentration in the two respective communities. Our study has revealed that arsenic poses a broader human health risk than was previously known. It is influential in shaping the gut microbiome through its enrichment of arsenic volatilizing and pathogenic bacteria and subsequent depletion of gut commensals. This aspect of arsenic has the potential to debilitate healthy humans by contributing to disorders like heart and liver cancers and diabetes, and it has already been shown to contribute to serious diseases and disorders, including skin lesions, gangrene and several types of skin, renal, lung, and liver cancers in disadvantaged areas of the world like Nepal.

Emergence of clinical isolates of highly carbapenem-resistant Klebsiella pneumoniae co-harboring blaNDM-5 and blaOXA-181 or -232 in Nepal.

OBJECTIVES: The aim of this study was to describe the emergence in Nepal of clinical isolates of Klebsiella pneumoniae harboring both blaNDM-5 and blaOXA-181/-232. METHODS: Six clinical isolates of K. pneumoniae highly resistant to carbapenems and aminoglycosides were obtained from inpatients in Nepal. Their whole genomes were sequenced by a next generation sequencer. RESULTS: The minimum inhibitory concentrations of meropenem, amikacin and ciprofloxacin were ≥128 µg/ml, >1024 µg/ml and ≥256 µg/ml, respectively. All six isolates co-harbored blaNDM-5, blaOXA-181 or -232 and rmtB. Of them, 1 also harbored rmtF. The blaNDM-5, blaOXA-232 and rmtB in all six isolates were located on plasmids. Of the six isolates tested, one isolate harbored two copies of blaOXA-181 and rmtF on the chromosome. CONCLUSIONS: This is the first report of clinical isolates of K. pneumoniae co-harboring blaNDM-5, blaOXA-181 or -232 and rmtB in Nepal. These strains were highly carbapenem- and aminoglycoside-resistant, and belonged to ST147 or ST395. Of them, ST147 isolate harbored two copies of blaOXA-181 on the chromosome.

Impact of sunflower seed oil versus mustard seed oil on skin barrier function in newborns: a community-based, cluster-randomized trial.

BACKGROUND: Natural vegetable oils are widely used for newborn massage in many low resource settings. Animal models indicated that sunflower seed oil (SSO) can accelerate skin barrier recovery following damage, while other oils, including mustard oil (MO), may cause further skin barrier damage. The objective was to compare the effects of two SSO and MO used for routine massage on skin integrity in premature and full-term neonates. METHODS: This community-based cluster randomized controlled trial included 995 neonates assigned to full body massage with sunflower seed oil (SSO, intervention) or mustard seed oil (MO, standard practice) from July 2012-May 2014 in Sarlahi, Nepal. Skin integrity measures were evaluated over 28 days, including skin condition (erythema, rash, dryness), skin surface pH, stratum corneum (SC) cohesion/protein concentration, and transepidermal water loss (TEWL). Overall means and rates of change in these skin measures were compared between oil groups using bivariate random-effects models. RESULTS: 500 and 495 live born neonates received repeated massage with MO and SSO, respectively. Skin pH decreased more quickly for SSO than MO in the first week of life, with a difference in mean daily reductions of 0.02 (95% CI: 0.002-0.040). Erythema, rash and dryness increased (worsened) over days 1-14 then decreased by day 28, with no significant oil group differences. TEWL increased over time, with no significant oil group differences. Gestational age did not modify the effect; the slightly faster decrease in skin pH among SSO infants was similar in magnitude between term and preterm infants. CONCLUSIONS: Oil type may contribute to differences in skin integrity when neonates are massaged regularly. The more rapid acid mantle development observed for SSO may be protective for neonates in lower resource settings. TRIAL REGISTRATION: ClinicalTrials.gov (NCT01177111); registered August 6th, 2010.

Identification of 16S rRNA and Virulence-Associated Genes of Arcobacter in Water Samples in the Kathmandu Valley, Nepal.

This study aimed to determine the prevalence of Arcobacter and five associated virulence genes (cadF, ciaB, mviN, pldA, and tlyA) in water samples in the Kathmandu Valley, Nepal. A total of 286 samples were collected from deep tube wells (n = 30), rivers (n = 14), a pond (n = 1), shallow dug wells (n = 166), shallow tube wells (n = 33), springs (n = 21), and stone spouts (n = 21) in February and March (dry season) and August (wet season), 2016. Bacterial DNA was extracted from the water samples and subjected to SYBR Green-based quantitative PCR for 16S rRNA and virulence genes of Arcobacter. The 16S rRNA gene of Arcobacter was detected in 36% (40/112) of samples collected in the dry season, at concentrations ranging from 5.7 to 10.2 log copies/100 mL, and 34% (59/174) of samples collected in the wet season, at concentrations of 5.4-10.8 log copies/100 mL. No significant difference in Arcobacter 16S rRNA gene-positive results was observed between samples collected in the two seasons (p > 0.05). Seventeen (17%), 84 (84%), 19 (19%), 23 (23%), and 17 (17%) of the 99 Arcobacter 16S rRNA gene-positive samples were also positive for cadF, ciaB, mviN, pldA, and tlyA, respectively. At least one virulence gene was detected in 87 (88%) of the 99 Arcobacter 16S rRNA gene-positive samples. The presence of Arcobacter and the virulence genes in these samples illustrates the persistence of pathogenic bacteria in the environment and highlights the importance of regular monitoring of water for pathogens.

Detection of Pathogenic Viruses, Pathogen Indicators, and Fecal-Source Markers within Tanker Water and Their Sources in the Kathmandu Valley, Nepal.

Tanker water is used extensively for drinking as well as domestic purposes in the Kathmandu Valley of Nepal. This study aimed to investigate water quality in terms of microbial contamination and determine sources of fecal pollution within these waters. Thirty-one samples from 17 tanker filling stations (TFSs) and 30 water tanker (WT) samples were collected during the dry and wet seasons of 2016. Escherichia coli was detected in 52% of the 31 TFS samples and even more frequently in WT samples. Of the six pathogenic viruses tested, enteroviruses, noroviruses of genogroup II (NoVs-GII), human adenoviruses (HAdVs), and group A rotaviruses were detected using quantitative PCR (qPCR) at 10, five, four, and two TFSs, respectively, whereas Aichi virus 1 and NoVs-GI were not detected at any sites. Index viruses, such as pepper mild mottle virus and tobacco mosaic virus, were detected using qPCR in 77% and 95% out of 22 samples, respectively, all of which were positive for at least one of the tested pathogenic viruses. At least one of the four human-associated markers tested (i.e., BacHum, HAdVs, and JC and BK polyomaviruses) was detected using qPCR in 39% of TFS samples. Ruminant-associated markers were detected at three stations, and pig- and chicken-associated markers were found at one station each of the suburbs. These findings indicate that water supplied by TFSs is generally of poor quality and should be improved, and proper management of WTs should be implemented.

Performance Evaluation of Human-Specific Viral Markers and Application of Pepper Mild Mottle Virus and CrAssphage to Environmental Water Samples as Fecal Pollution Markers in the Kathmandu Valley, Nepal.

Monitoring of environmental water is crucial to protecting humans and animals from possible health risks. Although numerous human-specific viral markers have been designed to track the presence of human fecal contamination in water, they lack adequate sensitivity and specificity in different geographical regions. We evaluated the performances of six human-specific viral markers [Aichi virus 1 (AiV-1), human adenoviruses (HAdVs), BK and JC polyomaviruses (BKPyVs and JCPyVs), pepper mild mottle virus (PMMoV), and crAssphage] using 122 fecal-source samples collected from humans and five animal hosts in the Kathmandu Valley, Nepal. PMMoV and crAssphage showed high sensitivity (90-100%) with concentrations of 4.5-9.1 and 6.2-7.0 log10 copies/g wet feces (n = 10), respectively, whereas BKPyVs, JCPyVs, HAdVs, and AiV-1 showed poor performances with sensitivities of 30-40%. PMMoV and crAssphage were detected in 40-100% and 8-90%, respectively, of all types of animal fecal sources and showed no significantly different concentrations among most of the fecal sources (Kruskal-Wallis test, P > 0.05), suggesting their applicability as general fecal pollution markers. Furthermore, a total of 115 environmental water samples were tested for PMMoV and crAssphage to identify fecal pollution. PMMoV and crAssphage were successfully detected in 62% (71/115) and 73% (84/115) of water samples, respectively. The greater abundance and higher mean concentration of crAssphage (4.1 ± 0.9 log10 copies/L) compared with PMMoV (3.3 ± 1.4 log10 copies/L) indicated greater chance of detection of crAssphage in water samples, suggesting that crAssphage could be preferred to PMMoV as a marker of fecal pollution.

Co-Infection by Waterborne Enteric Viruses in Children with Gastroenteritis in Nepal.

Enteric viruses are highly contagious and a major cause of waterborne gastroenteritis in children younger than five years of age in developing world. This study examined the prevalence of enteric virus infection in children with gastroenteritis to identify risk factors for co-infections. In total, 107 stool samples were collected from patients with acute gastroenteritis along with samples of their household drinking water and other possible contamination sources, such as food and hand. The presence of major gastroenteritis-causing enteric virus species (group A rotaviruses, enteroviruses, adenoviruses, and noroviruses of genogroup I) in stool and water samples was examined using quantitative polymerase chain reaction. Among the 107 stool samples tested, 103 (96%) samples contained at least one of the four tested enteric viruses, and the combination of group A rotaviruses and enteroviruses was the most common co-infection (52%, n = 54/103). At least one viral agent was detected in 16 (16%) of 103 drinking water samples. Identical enteric viruses were detected in both the stool and water samples taken from the same patients in 13% of cases (n = 13/103). Group A rotaviruses were most frequently found in children suffering from acute diarrhea. No socio-demographic and clinical factors were associated with the risk of co-infection compared with mono-infection. These less commonly diagnosed viral etiological agents in hospitals are highly prevalent in patients with acute gastroenteritis.

Seasonal Hyperacute Panuveitis in Nepal: A Review over 40 Years of Surveillance.

Purpose: This review aims to provide a detailed update in Seasonal hypercute panuveitis (SHAPU) which is a blinding disease of unknown etiology reported only from Nepal, occurring every odd year since 1975 predominantly in children. Methods: Data sources were literature reviewed using PubMed, Medline, and ISI Databases (since 1975 to late 2017). Search items included SHAPU, seasonal endophthalmitis, hypopyon uveitis, caterpillar induced uveitis alone or in combination with white moth, panuveitis, and review. Results: Epidemics of SHAPU occur between August to December usually in odd years. Two thirds of the children present with unilateral blind eye which eventually becomes soft with shallowing of anterior chamber called as Malignant Hypotension. Hypopyon, fibrinous exudates in anterior chamber, difficult to dilate pupil and inability to visualize the retina because of massive exudation into vitreous producing a "White pupil in a Red eye" with little or no pain is its characteristic features. Contact with moths has been suspected. Because of presence of microbial agents in few cases, an infectious etiology has not been ruled out. Conclusions: Although SHAPU has been reported only from Nepal, lack of its report from ecologically similar terrain along Hindukush Mountains and possible association with moths remain intriguing.

Hospital based surveillance and molecular characterization of rotavirus in children less than 5 years of age with acute gastroenteritis in Nepal.

BACKGROUND: Rotavirus remains a significant causative agent of childhood acute gastroenteritis, particularly among children less than 5 years of age. Although precise data on childhood mortality associated with diarrheal disease in Nepal is not available, it is estimated that22% of all rotavirus deaths globally occurs in neighboring country of India. In spite of the substantial burden of rotavirus gastroenteritis in the Indian subcontinent, rotavirus vaccine has not been introduced in Nepal. Continuous surveillance for monitoring rotavirus disease burden and molecular characterization is needed prior to rotavirus vaccine introduction in Nepal. METHODS: A total of 3310 stool samples (2849 hospitalized cases and 461 non-hospitalized cases), were collected from patients <5 years of age from January 2013 to December 2016 and tested for rotavirus antigen by ELISA (ProSpecT, USA). A subset of ELISA positive stool samples was genotyped. Demographic data were collected. RESULTS: During the four-year surveillance period, the overall burden of rotavirus infection was 24% among hospitalized children which was much higher than among non-hospitalized children (12%). The majority of children hospitalized with rotavirus gastroenteritis were less than 2 years of age (86%). Rotavirus-associated gastroenteritis hospitalizations occur year-round in Nepal, but a distinct peak in winter (up to 40% among hospitalized) was observed. Of 735 ELISA positive samples, 492 were genotyped by RT-PCR. The most prevalent genotype was G12P[6] (45.3%), followed byG2P[4](12.2%), G1P[8] (9.6%), G9P[4](7.3%), and G9P[8](4.5%). Mixed infection accounted for 4.4% of cases, 6.2% were partially typed and 10.5% of the samples were G and P untypable. CONCLUSIONS: A high burden of rotavirus gastroenteritis and a diversity of circulating rotavirus strains in Nepal were observed. Recommendation to introduce a rotavirus vaccine with known vaccine effectiveness would help in reducing the severity of Rotavirus diarrheal disease in children less than 5 years of age.

Development of a Quantitative PCR Assay for Arcobacter spp. and its Application to Environmental Water Samples.

Arcobacter spp. are emerging pathogens associated with gastroenteritis in humans. The objective of this study was to develop a highly sensitive and broadly reactive quantitative PCR (qPCR) assay for Arcobacter spp. and to apply the developed assay to different water sources in the Kathmandu Valley, Nepal. Fifteen samples to be analyzed by next-generation sequencing were collected from 13 shallow dug wells, a deep tube well, and a river in the Kathmandu Valley in August 2015. Among the 86 potential pathogenic bacterial genera identified, Acinetobacter, Pseudomonas, Flavobacterium, and Arcobacter were detected with relatively high abundance in 15, 14, 12, and 8 samples, respectively. A primer pair was designed with maximal nucleotide homologies among Arcobacter spp. by comparing the sequences of 16S rRNA genes. These primers were highly specific to most of the known species of Arcobacter and quantified between 1.0×101 and 6.4×106 copies reaction-1 and sometimes detected as few as 3 copies reaction-1. The qPCR assay was used to quantify Arcobacter spp. in bacterial DNA in not only the above 15 water samples, but also in 33 other samples collected from 15 shallow dug wells, 6 shallow tube wells, 5 stone spouts, 4 deep tube wells, and 3 springs. Thirteen (27%) out of 48 samples tested were positive for Arcobacter spp., with concentrations of 5.3-9.1 log copies 100 mL-1. This qPCR assay represents a powerful new tool to assess the prevalence of Arcobacter spp. in environmental water samples.