Detection of blaKPC gene among carbapenemase producing Klebsiella pneumoniae isolated from different clinical specimens at tertiary care hospital of Nepal.

BACKGROUND: Klebsiella pneumoniae infections have become a major cause of hospital acquired infection worldwide with the increased rate of acquisition of resistance to antibiotics. Carbapenem resistance mainly among Gram negative is an ongoing problem which causes serious outbreaks dramatically limiting treatment options. This prospective cross-sectional study was designed to detect blaKPC gene from carbapenem resistant K. pneumoniae. MATERIALS AND METHODS: A totally of 1118 different clinical specimens were screened and confirmed for KPC producing K. pneumoniae phenotypically using Meropenem (10 µg) disc. The blaKPC gene was amplified from the isolates of K. pneumoniae to detect the presence of this gene. RESULT: Of the total samples processed, 18.6% (n = 36) were K. pneumoniae and among 36 K. pneumoniae, 61.1% (n = 22/36) were meropenem resistant. This study demonstrated the higher level of MDR 91.7% (n = 33) and KPC production 47.2% (n = 17) among K. pneumoniae isolates. The blaKPC gene was detected in 8.3% (n = 3) of meropenem resistant isolates. CONCLUSION: Since the study demonstrates the higher level of MDR and KPC producing K. pneumoniae isolates that has challenged the use of antimicrobial agents, continuous microbiology, and molecular surveillance to assist early detection and minimize the further dissemination of blaKPC should be initiated. We anticipate that the findings of this study will be useful in understanding the prevalence of KPC-producing K. pneumoniae in Nepal.

Application of wastewater-based epidemiology for monitoring COVID-19 in hospital and housing wastewaters.

An alternative and complementary diagnostic method of surveillance is provided by wastewater-based surveillance (WBS), particularly in low-income nations like Nepal with scant wastewater treatment facilities and clinical testing infrastructure. In this study, a total of 146 water samples collected from two hospitals (n = 63) and three housing wastewaters (n = 83) from the Kathmandu Valley over the period of March 2021-Febraury 2022 were investigated for severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) using quantitative reverse transcription TaqMan PCR assays targeting the N and E genes. Of the total, 67 % (98/146) samples were positive for SARS-CoV-2 RNA either by using N- or E-gene assay, with concentrations ranging from 3.6 to 9.1 log10 copies/L. There was a significant difference found between positive ratio (Chi-square test, p < 0.05) and concentration (t-test, p = 0.009) of SARS-CoV-2 RNA detected from hospital wastewater and housing waters. Wastewater data are correlated with COVID-19 active cases, indicating significance in specific areas like the Hospital (APFH) (p < 0.05). According to the application of a bivariate linear regression model (p < 0.05), the concentrations of N gene may be used to predict the COVID-19 cases in the APFH. Remarkably, SARS-CoV-2 RNA was detected prior to, during, and following clinical case surges, implying that wastewater surveillance could serve as an early warning system for public health decisions. The significance of WBS in tracking and managing pandemics is emphasized by this study, especially in resource-constrained settings.

Quantification of multiple respiratory viruses in wastewater in the Kathmandu Valley, Nepal: Potential implications of wastewater-based epidemiology for community disease surveillance in developing countries.

Despite being the major cause of death, clinical surveillance of respiratory viruses at the community level is very passive, especially in developing countries. This study focused on the surveillance of three respiratory viruses [severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), influenza A virus (IFV-A), and respiratory syncytial virus (RSV)] in the Kathmandu Valley, Nepal, by implication of wastewater-based epidemiology (WBE). Fifty-one untreated wastewater samples were from two wastewater treatment plants (WWTPs) between April and October 2022. Among eight combinations of the pre-evaluated methods, the combination of concentration by simple centrifugation, pretreatment by DNA/RNA Shield (Zymo Research), and extraction by the QIAamp Viral RNA Mini Kit (QIAGEN) showed the best performance for detecting respiratory viruses. Using this method with a one-step reverse transcription-quantitative polymerase chain reaction (RT-qPCR), SARS-CoV-2 RNA was successfully detected from both WWTPs (positive ratio, 100 % and 81 %) at concentrations of 5.6 ± 0.6 log10 copies/L from each WWTP. Forty-six SARS-CoV-2 RNA-positive samples were further tested for three mutation site-specific one-step RT-qPCR (L452R, T478K, and E484A/G339D), where G339D/E484A mutations were frequently detected in both WWTPs (96 %). IFV-A RNA was more frequently detected in WWTP A (84 %) compared to WWTP B (38 %). RSV RNA was also detected in both WWTPs (28 % and 8 %, respectively). This is the first study on detecting IFV-A and RSV in wastewater in Nepal, showing the applicability and importance of WBE for respiratory viruses in developing countries where clinical data are lacking.

Antibiotic resistance determinants among carbapenemase producing bacteria isolated from wastewaters of Kathmandu, Nepal.

The emergence of carbapenem resistant bacteria (CRB) possesses a remarkable threat to the health of humans. CRB and carbapenem resistance genes (CRGs) have frequently been reported in clinical isolates from hospitals, however, their occurrence and distribution in wastewaters from various sources and river water have not been emphasized in Nepal. So, this study aimed to detect carbapenem resistant bacterial isolates and their resistance determinants in river water and different types of wastewaters. River water and both untreated and treated wastewater samples from hospitals, pharmaceutical industries, and municipal sewage were collected in summer and winter seasons. From 68 grab wastewater samples, CRB were detected only in 16 samples, which included eight hospital wastewater, and four each from untreated municipal sewage and river water. A total of 25 CRB isolates were detected with dominance of E. coli (44.0%) and K. pneumoniae (24.0%). The majority of the isolates harbored blaNDM-1 (76.0%), followed by blaOXA (36.0%) and blaKPC (20.0%) genes. Hospital wastewater majorly contributed to the presence of blaNDM-1, blaKPC, and blaOXA along with intI1 genes compared to river water and untreated municipal sewage, especially during the winter season. However, CRB were not detected in treated effluents of hospitals and municipal sewage, and both influents and effluents from pharmaceutical industries. The combined presence of each blaNDM-1 & blaOXA and blaKPC & blaOXA occurred in 16.0% of the bacterial isolates. The increased minimum inhibitory concentration (MIC) of meropenem was significantly associated with the presence of CRGs. The results of this study highlight the significance of carbapenem resistance in bacteria isolated from wastewater and river water, and underscore the necessity for efficient monitoring and control strategies to prevent the dispersion of carbapenem resistance in the environment and its potential consequences on human health.

Extended-spectrum ß-lactamases producing Enterobacteriaceae (ESBL-PE) prevalence in Nepal: A systematic review and meta-analysis.

An alarming increase in the occurrence of extended-spectrum ß-lactamase-producing Enterobacteriaceae (ESBL-PE) has threatened the treatment and management of bacterial infections. This systematic review and meta-analysis aimed to provide a quantitative estimate of the prevalence of ESBL among the members of the Enterobacteriaceae family by analyzing the community-based and clinical studies published between 2011 and 2021 from Nepal and determine if ESBL-PE correlates with multidrug resistance (MDR). The Preferred Reporting Items for Systematic Reviews and Meta-Analysis (PRISMA) guidelines were followed for systematic review and meta-analysis and the articles' quality was assessed using the Newcastle-Ottawa scale. Of the 2529 articles screened, 65 articles were systematically reviewed, data extracted, and included in in-depth meta-analysis. The overall pooled prevalence of ESBL-producers in Enterobacteriaceae was 29 % (95 % CI: 26-32 %) with high heterogeneity (I2 = 96 %, p < 0.001). Escherichia coli was the predominant ESBL-producing member of the Enterobacteriaceae family, followed by Citrobacter spp. and Klebsiella spp. The prevalence of ESBL-PE increased from 18.7 % in 2011 to 29.5 % in 2021. A strong positive correlation (r = 0.98) was observed between ESBL production and MDR in Enterobacteriaceae. ESBL-PE isolates showed high resistance to ampicillin, cephalosporins, and amoxicillin-clavulanic acid, and blaCTX-M type was the most reported gene variant among ESBL-PE. In conclusion, this study demonstrated an increased prevalence of ESBL-PE in Nepal over the last decade, and such isolates showed a high level of MDR against the ß-lactams and non-ß-lactam antibiotics. Tackling the rising antibiotic resistance (AR) and MDR in ESBL-PE would require concerted efforts from all stakeholders to institute effective infection control programs in the community and clinical settings.

Antibiotic Resistance, Biofilm Formation and Sub-Inhibitory Hydrogen Peroxide Stimulation in Uropathogenic Escherichia coli.

Uropathogenic Escherichia coli (UPEC) is the most prevalent cause of urinary tract infections (UTIs). Biofilm formation and antibiotic resistance could be high among the causative agent. The purpose of this study was to determine antibiotic resistance, biofilm production, and biofilm-associated genes, bcsA and csgD, and sub-inhibitory hydrogen peroxide (H2O2) stimulation in UPEC for biofilm formation. A total of 71 UPEC were collected from a tertiary care hospital in Kathmandu and subjected to identify antibiotic susceptibility using Kirby-Bauer disk diffusion. The biofilm formation was assessed using microtiter culture plate method while pellicle formation was tested by a tube method. In representative 15 isolates based on biofilm-forming ability, bcsA and csgD were screened by conventional polymerase chain reaction, and treated with sub-lethal H2O2. The UPEC were found the most susceptible to meropenem (90.2%), and the least to ampicillin (11.3%) in vitro and 90.1% of them were multi-drug resistant (MDR). Most UPEC harbored biofilm-producing ability (97.2%), and could form pellicle at 37°C. Among representative 15 isolates, csgD was detected only among 10 isolates (66.67%) while bcsA gene was present in 13 isolates (86.67%). This study revealed that level of biofilm production elevated after sub-lethal H2O2 treatment (P = .041). These findings suggested that the pathogens are emerging as MDR. The biofilm production is high and the majority of selected strains contained bcsA and csgD genes. Pellicle formation test was suggestive to be an alternative qualitative method to screen biofilm production in UPEC. The sub-inhibitory concentration of H2O2 may contribute in increasing biofilm formation in UPEC.

A barcoding pipeline for mosquito surveillance in Nepal, a biodiverse dengue-endemic country.

BACKGROUND: Vector-borne diseases are on the rise on a global scale, which is anticipated to further accelerate because of anthropogenic climate change. Resource-limited regions are especially hard hit by this increment with the currently implemented surveillance programs being inadequate for the observed expansion of potential vector species. Cost-effective methods that can be easily implemented in resource-limited settings, e.g. under field conditions, are thus urgently needed to function as an early warning system for vector-borne disease epidemics. Our aim was to enhance entomological capacity in Nepal, a country with endemicity of numerous vector-borne diseases and with frequent outbreaks of dengue fever. METHODS: We used a field barcoding pipeline based on DNA nanopore sequencing (Oxford Nanopore Technologies) and verified its use for different mosquito life stages and storage methods. We furthermore hosted an online workshop to facilitate knowledge transfer to Nepalese scientific experts from different disciplines. RESULTS: The use of the barcoding pipeline could be verified for adult mosquitos and eggs, as well as for homogenized samples, dried specimens, samples that were stored in ethanol and frozen tissue. The transfer of knowledge was successful, as reflected by feedback from the participants and their wish to implement the method. CONCLUSIONS: Cost effective strategies are urgently needed to assess the likelihood of disease outbreaks. We were able to show that field sequencing provides a solution that is cost-effective, undemanding in its implementation and easy to learn. The knowledge transfer to Nepalese scientific experts from different disciplines provides an opportunity for sustainable implementation of low-cost portable sequencing solutions in Nepal.

Methicillin-resistant Staphylococcus aureus nasal colonization in people living with HIV and healthy people in Kathmandu, Nepal.

AIM: This study aimed to compare methicillin-resistant Staphylococcus aureus (MRSA) nasal colonization in people living with HIV (PLHIV) and healthy people from Kathmandu. METHODS: MRSA isolated from 400 nasal swabs was screened using a cefoxitin disc and confirmed by the presence of the mecA gene. RESULTS: MRSA nasal carriers among the PLHIV and control cohorts were 3.5% (7 out of 200) and 5.0% (10 out of 200), respectively. All the MRSA from PLHIV and most of MRSA from healthy controls were PVL positive. Longer duration of antiretroviral therapy significantly reduces the risk of MRSA nasal colonization in PLHIV. CONCLUSION: There is no significant difference in MRSA nasal colonization in PLHIV and healthy controls in this study region.

Detection of SARS-CoV-2 and Other Viruses in Wastewater: Optimization and Automation of an Aluminum Hydroxide Adsorption-Precipitation Method for Virus Concentration.

This study aimed to provide a low-cost technique for virus detection in wastewater by improving an aluminum hydroxide adsorption-precipitation method. The releasing efficiency of viruses trapped by the aluminum hydroxide precipitates was improved by adding ethylenediaminetetraacetic acid disodium salt (EDTA-2Na) to dissolve the precipitates at a Na2EDTA·2H2O:AlCl3 molar ratio of 1.8-3.6. The recovery rates of the improved method for seven viruses, including SARS-CoV-2-abEN pseudovirus and six animal viruses, were 5.9-22.3% in tap water and 4.9-35.1% in wastewater. Rotavirus A (9.0-4.5 × 103 copies/mL), porcine circovirus type 2 (5.8-6.4 × 105 copies/mL), and porcine parvovirus (5.6-2.7 × 104 copies/mL) were detected in China's pig farm wastewater, while rotavirus A (2.0 × 103 copies/mL) was detected in hospital wastewater. SARS-CoV-2 was detected in hospital wastewater (8.4 × 102 to 1.4 × 104 copies/mL), sewage (6.4 × 10 to 2.3 × 103 copies/mL), and river water (6.6 × 10 to 9.3 × 10 copies/mL) in Nepal. The method was automized, with a rate of recovery of 4.8 ± 1.4% at a virus concentration of 102 copies/mL. Thus, the established method could be used for wastewater-based epidemiology with sufficient sensitivity in coping with the COVID-19 epidemic and other virus epidemics.

Cost of wastewater-based environmental surveillance for SARS-CoV-2: Evidence from pilot sites in Blantyre, Malawi and Kathmandu, Nepal.

Environmental surveillance of rivers and wastewater for SARS-CoV-2 detection has been explored as an innovative way to surveil the pandemic. This study estimated the economic costs of conducting wastewater-based environmental surveillance for SARS-CoV-2 to inform decision making if countries consider continuing these efforts. We estimated the cost of two SARS-CoV-2 environmental surveillance pilot studies conducted in Blantyre, Malawi, and Kathmandu, Nepal. The cost estimation accounted for the consumables, equipment, and human resource time costs used for environmental surveillance from sample selection until pathogen detection and overhead costs for the projects. Costs are reported in 2021 US$ and reported as costs per month, per sample and person per year. The estimated costs for environmental surveillance range from $6,175 to $8,272 per month (Blantyre site) and $16,756 to $30,050 (Kathmandu site). The number of samples processed per month ranged from 84 to 336 at the Blantyre site and 96 to 250 at the Kathmandu site. Consumables costs are variable costs influenced by the number of samples processed and are a large share of the monthly costs for ES (ranging from 39% to 72%). The relatively higher costs per month for the Kathmandu site were attributable to the higher allocation of dedicated human resources and equipment to environmental surveillance for SARS-CoV-2 compared to the Blantyre site where these resources were shared with other activities. The average cost per sample ranged from $25 to $74 (Blantyre) and $120 to $175 (Kathmandu). There were associated economies of scale for human resources and equipment costs with increased sample processing and sharing of resources with other activities. The cost per person in the catchment area per year ranged from $0.07 to $0.10 in Blantyre and $0.07 to $0.13 in Kathmandu. Environmental surveillance may be a low-cost early warning signal for SARS-CoV-2 that can complement other SARS-CoV2 monitoring efforts.

Asymptomatic SARS-CoV-2 Carriers: A Systematic Review and Meta-Analysis.

Asymptomatic cases of SARS-CoV-2 can be unknown carriers magnifying the transmission of COVID-19. This study appraised the frequency of asymptomatic individuals and estimated occurrence by age group and gender by reviewing the existing published data on asymptomatic people with COVID-19. Three electronic databases, PubMed, Embase, and Web of Science (WoS), were used to search the literature following the guidelines of Preferred Reporting Items for Systematic Reviews and Meta-Analysis (PRISMA). The study population for this review included asymptomatic individuals infected with SARS-CoV-2 reported in original articles published up to 30 April 2020. A random effects model was applied to analyze pooled data on the prevalence of asymptomatic cases among all COVID-19 patients and also by age and gender. From the meta-analysis of 16 studies, comprising 2,788 SARS-CoV-2 infected patients, the pooled prevalence according to the random effect size of asymptomatic cases was 48.2% (95% CI, 30-67%). Of the asymptomatic cases, 55.5% (95% CI, 43.6-66.8%) were female and 49.6% (95% CI, 20.5-79.1%) were children. Children and females were more likely to present as asymptomatic COVID-19 cases and could act as unknown carriers of SARS-CoV-2. Symptom-based screening might fail to identify all SARS-CoV-2 infections escalating the threat of global spread and impeding containment. Therefore, a mass surveillance system to track asymptomatic cases is critical, with special attention to females and children.

MRSA Strains in Nepalese Rhesus Macaques (Macaca mulatta) and Their Environment.

This study looked at 227 saliva samples from Rhesus macaques (Macaca mulatta) and 218 samples from the surrounding environments. From these samples, MRSA isolates were collected from Rhesus saliva samples (n = 13) and environmental samples (n = 19) near temple areas in Kathmandu, Nepal. For comparison, selected MRSA isolates (n = 5) were obtained from patients with wound infections from a Kathmandu hospital. All isolates were characterized using Abbott StaphyType® DNA microarrays. Eighteen isolates (62%) from monkeys (n = 4; 31%) and environmental samples (n = 14; 74%), were CC22-MRSA-IV. Most (n = 16) of them carried both, the PVL locus and toxic shock toxin gene (tst1), an unusual combination which is the same as in previously characterized strain from Nepalese macaques and pigs. The five human isolates also belonged to that strain type. Eight monkey MRSA isolates were CC361-MRSA-IV. One MRSA from a monkey and one from an environmental sample, were CC88-MRSA-V. Other environmental MRSA included one each, CC121-MRSA-VT, and CC772 -MRSA-V. Two were CC779-MRSA-VT, potentially a novel clone. All MRSA carried the blaZ gene. The aacA-aphD, dfrA, and erm (C) genes were very common in isolates from all sources. One macaque MRSA carried the resistance genes aphA3 and sat, neither previously identified in primate MRSA isolates. This current study suggests that humans could be a potential source of the MRSA in the macaques/environment and transmission may be linked to humans feeding the primates and/or living in close proximity to each other.

Climatic factors influencing dengue incidence in an epidemic area of Nepal.

OBJECTIVE: Geographic expansion of dengue incidence has drawn a global interest to identify the influential factors that instigate the spread of this disease. The objective of this study was to find the environmental factors linked to dengue incidence in a dengue epidemic area of Nepal by negative binomial models using climatic factors from 2010 to 2017. RESULTS: Minimum temperature at lag 2 months, maximum temperature and relative humidity without lag period significantly affected dengue incidence. Rainfall was not associated with dengue incidence in Chitwan district of Nepal. The incident rate ratio (IRR) of dengue case rise by more than 1% for every unit increase in minimum temperature at lag 2 months, maximum temperature and relative humidity, but decrease by .759% for maximum temperature at lag 3 months. Considering the effect of minimum temperature of previous months on dengue incidence, the vector control and dengue management program should be implemented at least 2 months ahead of dengue outbreak season.

Effect of meteorological factors on the seasonal prevalence of dengue vectors in upland hilly and lowland Terai regions of Nepal.

BACKGROUND: The expansion of dengue vectors from lowland plains to the upland hilly regions of Nepal suggests the likelihood of increased risk of dengue. Our objective was to assess the effects of meteorological variables on vector indices and populations of dengue vectors in two different ecological regions of Nepal. An entomological survey was conducted in Kathmandu and Lalitpur (upland) and Chitwan (lowland) of Nepal in three different seasons from July 2015 to May 2016. The effect of meteorological variables on vector indices (house index, container index and Breteau index) and Aedes spp. population abundance was analyzed. A gamma regression was used to fit the models for vector indices and a negative binomial regression was used to model Aedes spp. population abundance. RESULTS: Monsoon season showed higher values for vector indices and vector populations compared to post-monsoon and pre-monsoon. Overall, the factor temperature-rainfall effect had a more significant influence on vector indices compared to relative humidity. The regression models showed that relative humidity has a greater impact in Chitwan than in Kathmandu. Variation was observed in the effect of predictor variables on Aedes aegypti and Ae. albopictus abundance. CONCLUSIONS: Temperature and rainfall contribute to the vector indices in the upland hilly region while relative humidity contributes in the lowland plains. Since vector prevalence is not only linked to meteorological factors, other factors such as water storage practices, waste disposal, sanitary conditions and vector control strategy should also be considered. We recommend strengthening and scaling up dengue vector surveillance and control programmes for monsoon season in both upland and lowland regions in Nepal.

Dengue periodic outbreaks and epidemiological trends in Nepal.

Dengue is a global health problem and expansion of its endemics towards new territories in the hilly regions in Nepal is a serious concern. It appeared as a new disease in Nepal in 2004 from Japanese traveler with sporadic cases every year and massive outbreaks in 2010, 2013 and 2016. The serotype was responsible for outbreak in particular year was dengue virus serotype-1 (DENV-1) in 2010, 2016; and DENV-2 in 2013. Nepal lacks basic health related infrastructure in rural areas and does not have a stringent health care policy. With severances of epidemic like dengue, a new surveillance or an upgrading of existing one are direly needed to better challenge the possible outbreaks. This review paper aims to explain the dengue trend in last one decade in Nepal and warrants concerted and timely public health interventions to minimize the deleterious effects of the disease.