Contaminated Water and an Indication of Risk: Examining Microbial Contamination in the Water Used by Consumers and Commercial Growers in Fresh Produce Systems in Nepal.

There has been limited research and understanding of the water quality in developing countries. Fresh produce consumed raw is nutrient-dense but is more susceptible to causing foodborne illness when contaminated water is used in production and consumption. There have been increasing reported incidences of foodborne outbreaks in Nepal linked to fresh produce contamination. However, water used in washing fresh produce by consumers and water used by growers or vendors is rarely tested. This research examines the source water used by consumers and growers in fresh produce systems in Nepal. To examine Escherichia coli (E. coli) detection as an indicator of contamination risk in water, we selected five major metropolitan cities for consumer households and ten districts representing commercial growers of vegetable growing areas of all seven provinces of Nepal. Altogether, we collected 394 water samples from randomly selected individual households: 156 from consumer households and 238 from growers or vendors. Results suggest that 59% of the water used in fresh produce systems is contaminated with E. coli in Nepal. On the water source used by consumers to wash fresh produce before consumption, we found that the dominant sources are the stored water in tanks or containers (46%) and municipal or communal supply water (39%)-which have E. coli prevalence rate of 66% and 57%, respectively. On the dominant sources of water used in fresh produce by growers or vendors, we found up to 88% of E. coli prevalence in the water they use. We also discussed the location or regional differences in contamination risks. This nationally represented study has implications for intervention policies and programs for safer food production and consumption practices in countries like Nepal where food safety is an emerging priority.

Antimicrobial Susceptibility Profile of Pathogenic and Commensal Bacteria Recovered from Cattle and Goat Farms.

The use of antibiotics in food animals results to antimicrobial resistant bacteria that complicates the ability to treat infections. The purpose of this study was to investigate the prevalence of pathogenic and commensal bacteria in soil, water, manure, and milk from cattle and goat farms. A total of 285 environmental and 81 milk samples were analyzed for Enterobacteriaceae by using biochemical and PCR techniques. Susceptibility to antibiotics was determined by the Kirby-Bauer disk diffusion technique. A total of 15 different Enterobacteriaceae species were identified from goat and cattle farms. Manure had significantly higher (p < 0.05) Enterobacteriaceae (52.0%) than soil (37.2%), trough water (5.4%), and runoff water (5.4%). There was a significant difference (p < 0.05) in Enterobacteriaceae in goat milk (53.9%) and cow milk (46.2%). Enterobacteriaceae from environment showed 100% resistance to novobiocin, erythromycin, and vancomycin E. coli O157:H7, Salmonella spp., Enterococcus spp., and Listeria monocytogenes displayed three, five, six, and ten. AMR patterns, respectively. NOV-TET-ERY-VAN was the most common phenotype observed in all isolates. Our study suggest that cattle and goat farms are reservoirs of multidrug-resistant bacteria. Food animal producers should be informed on the prudent use of antimicrobials, good agricultural practices, and biosecurity measures.

Clinicopathological characteristics of carcinoma penis over 10 years in a tertiary-level oncology center in Nepal: a retrospective study of 380 cases.

PURPOSE: This study was done to find out the clinicopathological characteristics of carcinoma penis in Nepali population and to evaluate various risk factors that predict its inguinal lymph node metastasis. METHODS: A retrospective cross-sectional study was carried out at the Urology Unit, Department of Surgical Oncology at BP Koirala Memorial Cancer Hospital, Bharatpur, Nepal. Case notes of biopsy-proven penile cancer, from January 2012 to December 2021, who underwent some form of surgical intervention were included. RESULTS: A total of 380 patients were included in the study. The mean age of the patients was 55.92 ± 13.81 years. At presentation, 78.5% had clinically node-positive disease. The most common treatment for the primary tumor was partial amputation of the penis (74.2%). Bilateral inguinal lymph node dissections were done in 370 cases. The most common histology was the usual SCC in 94.2% of cases and 69% were well differentiated. T3 was the most common staging in 49.4% cases. Pathologically nodal negative status was found in 58% cases. In univariate analysis, factors like duration of symptoms (≥ 6 months), high-risk histopathology (basaloid/sarcomatoid variant), increased T-stage, poorly differentiated tumor, and the presence of PNI or LVI were significantly associated with lymph node metastasis. CONCLUSIONS: Penile cancer is a common cancer in developing countries such as Nepal. The majority of the patients present late. Early recognition and prompt treatment are required to improve the overall outcome.

Whole-genomic DNA amplifications from individually isolated sweet sorghum microspores.

Premise: Sorghum is a multi-use crop, the efficient breeding of which requires the development of new genetic tools. One such tool could be the genetic assessment of free microspores, which are released just after the tetrad stage of pollen development. Microspores are ideal for DNA isolation as they have underdeveloped cell walls and can be readily lysed as natural protoplasts. Methods: Four cultivars of Sorghum bicolor ('Achi Turi', 'Dale', 'Local', and 'Topper 76-6') were grown in a greenhouse until flowering (7.7-11.5 cm flag leaf internode length), after which 30 immature microspores were isolated from each line. Plant height, time to flowering, boot radius, and spikelet maturation were recorded for each cultivar. The exine development of the microspores was observed under an inverted Nikon microscope, and those with underdeveloped exine were subjected to whole-genome amplification and sequencing. Results: Microspores in the early uninucleate to early binucleate stages had underdeveloped exine, and were therefore ideal for DNA extraction. High-quality DNA was obtained from these single-cell gametophytes. The average DNA concentration was 2902 ng/µL, with fragment sizes comparable to those obtained from leaf tissue extractions. Discussion: Harvesting panicles with immature microspores means the entire gametic population is accessible for DNA analyses. This is the first amplification of whole-genome DNA fragments from sorghum single-cell microspores isolated during gametogenesis.

Antibiotic Resistant Enterobacteriaceae in Milk Alternatives.

The consumption of non-dairy milk is on the rise due to health benefits. Although there is increasing inclination towards milk alternatives (MA), there is limited data on antibiotic resistant bacteria in these substitutes. The aim of this study was to investigate antimicrobial resistance of bacteria isolated from MA. A total of 138 extracts from almonds (n = 63), cashew nuts (n = 36), and soybeans (n = 39) were analyzed for Enterobacteriaceae. The identification of the bacteria was based on biochemical and PCR methods. Antibiotic sensitivity was determined by using the Kirby-Bauer disk diffusion technique. Overall, 31% (43 of 138) of extracts were positive for Enterobacteriaceae. Ten bacterial species were identified, of which Enterobacter cloacae (42.7%) and Enterobacter cancerogenus (35.4%) were the most predominant species (p < 0.05). Antibiotic resistance was exhibited to vancomycin (88.3%), novobiocin (83.8%), erythromycin (81.1%), which was significantly higher (p < 0.05) than in tetracycline (59.5%), cefpodoxime (30.6%), and nalidixic acid (6.3%). There was no resistance displayed to kanamycin and imipenem. ERY-NOV-VAN-TET and ERY-NOV-CEP-VAN-TET were the most common resistant patterns displayed by Enterobacter cloacae. The findings of this study suggest that MAs, though considered healthy, may be a reservoir of multidrug resistant opportunist pathogens.

Genomic Modeling as an Approach to Identify Surrogates for Use in Experimental Validation of SARS-CoV-2 and HuNoV Inactivation by UV-C Treatment.

Severe Acute Respiratory Syndrome coronavirus-2 (SARS-CoV-2) is responsible for the COVID-19 pandemic that continues to pose significant public health concerns. While research to deliver vaccines and antivirals are being pursued, various effective technologies to control its environmental spread are also being targeted. Ultraviolet light (UV-C) technologies are effective against a broad spectrum of microorganisms when used even on large surface areas. In this study, we developed a pyrimidine dinucleotide frequency based genomic model to predict the sensitivity of select enveloped and non-enveloped viruses to UV-C treatments in order to identify potential SARS-CoV-2 and human norovirus surrogates. The results revealed that this model was best fitted using linear regression with r 2 = 0.90. The predicted UV-C sensitivity (D 90 - dose for 90% inactivation) for SARS-CoV-2 and MERS-CoV was found to be 21.5 and 28 J/m2, respectively (with an estimated 18 J/m2 obtained from published experimental data for SARS-CoV-1), suggesting that coronaviruses are highly sensitive to UV-C light compared to other ssRNA viruses used in this modeling study. Murine hepatitis virus (MHV) A59 strain with a D 90 of 21 J/m2 close to that of SARS-CoV-2 was identified as a suitable surrogate to validate SARS-CoV-2 inactivation by UV-C treatment. Furthermore, the non-enveloped human noroviruses (HuNoVs), had predicted D 90 values of 69.1, 89, and 77.6 J/m2 for genogroups GI, GII, and GIV, respectively. Murine norovirus (MNV-1) of GV with a D 90 = 100 J/m2 was identified as a potential conservative surrogate for UV-C inactivation of these HuNoVs. This study provides useful insights for the identification of potential non-pathogenic (to humans) surrogates to understand inactivation kinetics and their use in experimental validation of UV-C disinfection systems. This approach can be used to narrow the number of surrogates used in testing UV-C inactivation of other human and animal ssRNA viral pathogens for experimental validation that can save cost, labor and time.

Prevalence of Multidrug-Resistant Foodborne Pathogens and Indicator Bacteria from Edible Offal and Muscle Meats in Nashville, Tennessee.

This study investigated the prevalence of antimicrobial-resistant bacteria in retail edible offal and muscle meats in Nashville, Tennessee. A total of 348 retail meats (160 edible offal and 188 muscle) were analyzed for Salmonella enterica serovar, Campylobacter, Escherichia coli, E. coli O157:H7, and enterococci. Bacteria was identified using biochemical and PCR methods. Salmonella enterica serovar (4.4% and 4.3%), Campylobacter (1.9% and 1.1%), E. coli (79.4% and 89.4%), and enterococci (88.1% and 95.7%) was detected in offal and muscle meats, respectively. Chicken liver (9.7%) was most frequently contaminated with Salmonella enterica serovar, followed by ground chicken (6.9%) and chicken wings (4.2%). No Salmonella enterica serovar was detected in beef liver, beef tripe, and ground beef. The prevalence of Campylobacter was 6.9%, 2.3%, and 1.4% in beef liver, ground beef, and ground chicken, respectively. None of the meats were positive for E. coli O157:H7. Resistance of isolates was significantly (p < 0.05) highest in erythromycin (98.3%; 99.1%), followed by tetracycline (94%; 98.3%), vancomycin (88.8%; 92.2%) as compared to chloramphenicol (43.1%; 53.9%), amoxicillin/clavulanic (43.5%; 45.7%), and ciprofloxacin (45.7%; 55.7%) in offal and muscle meats, respectively. Imipenem showed the lowest resistance (0%; 0.9%). A total of 41 multidrug-resistant patterns were displayed. Edible offal could be a source of antibiotic-resistant bacteria.

Disseminated infection with Nocardia otitidiscaviarum in a patient under steroid therapy.

Corticosteroid recipients with lung infections should be suspected of having nocardiosis; however, nocardiosis can easily mimic malignancy, tuberculosis, or fungal infection. Though cultural identification is possible, it might be missed due to its slow growth pattern.. Therefore, if filamentous bacteria are seen during staining, plate incubation time should be extended.

Carriage of Extended-Spectrum-ß-Lactamase- and AmpC-ß-Lactamase-Producing Enterobacteriaceae (ESBL-PE) in Healthy Community and Outpatient Department (OPD) Patients in Nepal.

BACKGROUND: Extended-spectrum ß-lactamase (ESBL)- and AmpC-ß-lactamase (ESBL)- and AmpC-Enterobacteriaceae have recently emerged as a public threat in the treatment of nosocomial as well as community-acquired infections. Very little information is currently available about its existence in Nepal. We, therefore, aim to determine the prevalence of ESBL and AmpC-ß-lactamase (ESBL)- and AmpC-Enterobacteriaceae have recently emerged as a public threat in the treatment of nosocomial as well as community-acquired infections. Very little information is currently available about its existence in Nepal. We, therefore, aim to determine the prevalence of ESBL and AmpC. METHODS: During a 6-month period (November 2014-April 2015), a total of 190 stool specimens from 190 participants were obtained from different population. Of the total 260 fecal isolates, 152 from outpatient department (OPD) and 108 from healthy volunteer were collected. Stool specimens were cultured and enterobacterial isolates were subjected to antimicrobial susceptibility tests according to the standard microbiologic guidelines. ESBL was screened using ceftazidime (CAZ, 30 µg) and cefotaxime (CTX, 30 µg) and cefotaxime (CTX, 30 ß-lactamase (ESBL)- and AmpC. RESULTS: The prevalence of ESBL, AmpC-ß-lactamase (ESBL)- and AmpC-ß-lactamase (ESBL)- and AmpC-Enterobacteriaceae have recently emerged as a public threat in the treatment of nosocomial as well as community-acquired infections. Very little information is currently available about its existence in Nepal. We, therefore, aim to determine the prevalence of ESBL and AmpC-E. coli was 70.2% followed by K. pneumoniae (12.7%), and among AmpC-ß-lactamase (ESBL)- and AmpC-E. coli was 70.2% followed by E. coli was 70.2% followed by K. pneumoniae (12.7%), and among AmpC-K. pneumoniae (12.7%), and among AmpC-C. freundii 2/7 (28.57%) were detected highest among AmpC-ß-lactamase (ESBL)- and AmpC. CONCLUSION: Our study revealed a high prevalence of ESBL- and AmpC-ß-lactamase-producing enteric pathogen in Nepalese OPD and healthy population. The significant increase of these isolates and increased rate of drug resistance indicates a serious threat that stress the need to implement the surveillance system and a proper control measure so as to limit the spread of ESBL-producing Enterobacteriaceae (ESBL-PE) in both OPD as well as in community. Therefore, healthcare providers need to be aware that ESBL- and AmpC-ß-lactamase-producing strains are not only circulating in hospital environments but also in the community and should be dealt with accordingly.ß-lactamase (ESBL)- and AmpC-Enterobacteriaceae have recently emerged as a public threat in the treatment of nosocomial as well as community-acquired infections. Very little information is currently available about its existence in Nepal. We, therefore, aim to determine the prevalence of ESBL and AmpC-ß-lactamase (ESBL)- and AmpC.

Ultraviolet inactivation of bacteria and model viruses in coconut water using a collimated beam system.

This study investigated the effect of ultraviolet-C irradiation on the inactivation of microorganisms in coconut water, a highly opaque liquid food (1.01 ± 0.018 absorption coefficient). Ultraviolet-C inactivation kinetics of two bacteriophages (MS2, T1UV) and three surrogate bacteria (Escherichia coli, Salmonella Typhimurium, Listeria monocytogenes) in 0.1% (w/v) peptone and coconut water were investigated. Ultraviolet-C irradiation at 254 nm was applied to stirred samples, using a collimated beam device. A series of known ultraviolet-C doses (0-40 mJ cm-2) were applied for ultraviolet-C treatment except for MS2 where higher doses were delivered (100 mJ cm-2). Inactivation levels of all organisms were proportional to ultraviolet-C dose. At the highest dose of 40 mJ cm-2, three surrogates of pathogenic bacteria were inactivated by more than 5-log10 (p < 0.05) in 0.1% (w/v) peptone and coconut water. Results showed that ultraviolet-C irradiation effectively inactivated bacteriophage and surrogate bacteria in highly opaque coconut water. The log reduction kinetics of microorganisms followed log-linear and exponential models with higher R2 (>0.95) and low root mean square error values. The D10 values of 3, 5.48, and 4.58 mJ cm-2 were obtained from the inactivation of E. coli, S. Typhimurium, and L. monocytogenes, respectively. Models for predicting log reduction as a function of ultraviolet-C irradiation dose were found to be significant (p < 0.05). Fluid optics were the key controlling parameters for efficient microbial inactivation. Therefore, the ultraviolet-C dose must be calculated not only from the incident ultraviolet-C intensity but must also consider the attenuation in the samples. The results from this study imply that adequate log reduction of vegetative cells and model viruses is achievable in coconut water and suggested significant potential for ultraviolet-C treatment of other liquid foods.

Toxoplasma gondii bradyzoites and tachyzoites isolation from vitreous of atypical necrotizing retinitis.

BACKGROUND: Detection of Toxoplasma gondii cysts in vitreous of immunocompetent patient with necrotizing retinitis is extremely rare. We herein report the isolation of Toxoplasma bradyzoites and tachyzoites from the vitreous of healthy person. RESULTS: A 19-year-old immunocompetent female presented with sudden loss of vision in left eye since 1 week. The BCVA was 6/6 and HM in right and left eye. The left eye finding was suggestive of diffuse necrotizing retinitis with retinal detachment. The IgM and IgG for TORCH infection were negative and HIV, HCV and HBsAg tests were also non reactive. The patient underwent diagnostic and therapeutic vitrectomy with silicon oil installation. The vitreous toxoplasma IgG titre was found to be significantly raised to 1:16. Bradyzoites of toxoplasma were identified in H&E staining and tachyzoites of Toxoplasma were identified in Giemsa staining of vitreous sample. She received oral clindamycin and oral corticosteroid but the vision could not be restored in left eye. CONCLUSION: Hence, atypical toxoplasmosis with necrotizing retinitis is a fulminant condition with the diagnostic and therapeutic challenge.

Microbial inactivation and cytotoxicity evaluation of UV irradiated coconut water in a novel continuous flow spiral reactor.

A continuous-flow UV reactor operating at 254nm wave-length was used to investigate inactivation of microorganisms including bacteriophage in coconut water, a highly opaque liquid food. UV-C inactivation kinetics of two surrogate viruses (MS2, T1UV) and three bacteria (E. coli ATCC 25922, Salmonella Typhimurium ATCC 13311, Listeria monocytogenes ATCC 19115) in buffer and coconut water were investigated (D10 values ranging from 2.82 to 4.54mJ·cm-2). A series of known UV-C doses were delivered to the samples. Inactivation levels of all organisms were linearly proportional to UV-C dose (r2>0.97). At the highest dose of 30mJ·cm-2, the three pathogenic organisms were inactivated by >5 log10 (p<0.05). Results clearly demonstrated that UV-C irradiation effectively inactivated bacteriophage and pathogenic microbes in coconut water. The inactivation kinetics of microorganisms were best described by log linear model with a low root mean square error (RMSE) and high coefficient of determination (r2>0.97). Models for predicting log reduction as a function of UV-C irradiation dose were found to be significant (p<0.05) with low RMSE and high r2. The irradiated coconut water showed no cytotoxic effects on normal human intestinal cells and normal mouse liver cells. Overall, these results indicated that UV-C treatment did not generate cytotoxic compounds in the coconut water. This study clearly demonstrated that high levels of inactivation of pathogens can be achieved in coconut water, and suggested potential method for UV-C treatment of other liquid foods. INDUSTRIAL RELEVANCE: This research paper provides scientific evidence of the potential benefits of UV-C irradiation in inactivating bacterial and viral surrogates at commercially relevant doses of 0-120mJ·cm-2. The irradiated coconut water showed no cytotoxic effects on normal intestinal and healthy mice liver cells. UV-C irradiation is an attractive food preservation technology and offers opportunities for horticultural and food processing industries to meet the growing demand from consumers for healthier and safe food products. This study would provide technical support for commercialization of UV-C treatment of beverages.

Human fascioliasis by Fasciola hepatica: the first case report in Nepal.

BACKGROUND: Fascioliasis is a zoonotic disease caused by Fasciola species. Patient may be asymptomatic or presents with jaundice and biliary colic or right hypochondriac pain due to bile duct obstruction with gastrointestinal symptoms. CASE PRESENTATION: We report a case of human fascioliasis in a 45 years old female presented to Tribhuvan University Teaching Hospital (TUTH), Kathmandu, Nepal on August, 2015 with fever, right hypochondriac pain, jaundice and occasional vomiting with anorexia for 4 months whose alkaline phosphatase was elevated and peripheral blood smear revealed eosinophilia. The patient also gives the history of consumption of water-cress. Endoscopic Retrograde Cholagiopancretography (ERCP) showed the presence of a flat worm resembling Fasciola hepatica and stool routine examination revealed ova of F. hepatica. The patient was treated with nitazoxanide by which she got improved. Repeat stool examination 2 weeks after treatment revealed no ova of F. hepatica. CONCLUSIONS: Patient with fascioliasis can be simply diagnosed with stool routine microscopy and treated with nitazoxanide. So patient with right hypochondriac pain, sign and symptoms of obstructive jaundice, eosinophilia and history of water-cress consumption should be suspected for fascioliasis and investigated and treated accordingly.

Antimicrobial susceptibility pattern of Shigella spp. isolated from children under 5 years of age attending tertiary care hospitals, Nepal along with first finding of ESBL-production.

BACKGROUND: Shigella is an important cause of bacterial gastroenteritis in resource-poor countries. The treatment of shigellosis mostly requires antibiotics. However, the increase of multidrug resistance along with emergence of extended-spectrum ß-lactamase and ciprofloxacin resistance among Shigella spp. has challenged the situation. This study was conducted to determine the distribution of species and antibiotic susceptibility pattern of Shigella species isolated from stool specimen among children less than 5 years of age in Nepal. RESULTS: Out of total 717 stool samples collected, 15 cases of Shigella spp. was isolated which includes 12 S. flexneri and 3 S. sonnei. Multidrug resistance was found among 13(86%) of the isolates. One of the isolates of S. flexneri was found to be ESBL-producer with MIC >256 mg/L for cefixime. CONCLUSION: The high occurrence of multidrug resistance among Shigella spp. along with a case of ESBL-production for the first time in Nepal alarms the concerns about dissemination of the resistant isolates. So, systemic monitoring of the antimicrobial susceptibility pattern of Shigella spp. is becoming crucial to guide therapy.

Measuring impacts of community forestry program through repeat photography and satellite remote sensing in the Dolakha district of Nepal.

During the 1990's community-based forest management gained momentum in Nepal. This study systematically evaluates the impacts that this had on land cover change and other associated aspects during the period 1990-2010 using repeat photography and satellite imagery in combination with interviews with community members. The results of the study clearly reflect the success of community-based forest management in the Dolakha district of the mid-hills of Nepal: during the study period, the rate of conversion of sparse forest into dense forest under community-based management was found to be between 1.13% and 3.39% per year. Similarly, the rate of conversion of non-forest area into forest was found to be between 1.11% and 1.96% per year. Community-based forest management has resulted in more efficient use of forest resources, contributed to a decline in the use of slash-and-burn agricultural practices, reduced the incidence of forest fires, spurred tree plantation, and encouraged the conservation and protection of trees on both public and private land. The resulting reclamation of forest in landside areas and river banks and the overall improvement in forest cover in the area has reduced flash floods and associated landslides.

Multidrug-resistant and extended-spectrum beta-lactamase (ESBL)-producing Salmonella enterica (serotypes Typhi and Paratyphi A) from blood isolates in Nepal: surveillance of resistance and a search for newer alternatives.

OBJECTIVES: We evaluated the prevalence of multidrug resistance (MDR) and production of extended spectrum beta-lactamase (ESBL) by Salmonella enterica (serotypes Typhi and Paratyphi A) in a teaching hospital in Nepal. The MDR strains of S. enterica were also tested for susceptibility to newer antibiotics. METHODS: Blood cultures were obtained from 4105 patients with febrile illnesses. Isolates of S. enterica were serotyped and antibiotic susceptibility testing was carried out using disk diffusion (Kirby-Bauer) and E-tests. ESBL screening and phenotype confirmation were done following National Committee for Clinical Laboratory Standards (NCCLS) recommendations for Escherichia coli. RESULTS: A total of 541 isolates of S. enterica serotypes Typhi (47%) and Paratyphi A (53%) were grown. Twenty-eight isolates (5%) of S. enterica were resistant to two or more antibiotics (MDR isolates), with a greater prevalence among serotype Paratyphi A (7%). All ESBL producers (three isolates) were serotype Paratyphi A. Most of the MDR S. enterica showed reduced susceptibility to ampicillin, chloramphenicol, trimethoprim-sulfamethoxazole, ofloxacin, and ciprofloxacin, and had good susceptibility to extended-spectrum cephalosporins and carbapenems. Among the fluoroquinolones, gatifloxacin demonstrated better in vitro activity compared to levofloxacin, ciprofloxacin, and ofloxacin. CONCLUSIONS: A greater prevalence of S. enterica serotype Paratyphi A with higher rates of multidrug resistance and ESBL production is concerning for natives as well as travelers in Nepal since the current typhoid vaccines do not provide protection against this serotype.