Efficacy of Native Bacillus thuringiensis against Mosquito Vector.

BACKGROUND: Larval source management is an effective measure to control mosquito-borne diseases. Bacillus thuringiensis produces specific insecticidal crystal proteins toxic to mosquito larvae. In many parts of the South East Asian region, Bacillus thuringiensis is used for larval source management. In Nepal, larvicidal Bacillus thuringiensis is not available. The study aims to isolate larvicidal Bacillus thuringiensis from soil samples of Nepal to control mosquitoes. METHODS: Native Bacillus thuringiensis was obtained from soil samples by the acetate selection method. It was identified by observing crystal protein with Coomassie Brilliant Blue stain in a light microscope. The mosquito larvae were collected from different breeding habitats. A preliminary bioassay was performed by inoculating three loopful of 48 hours culture of spherical crystal protein producing Bacillus thuringiensis in a plastic cup containing 25 larvae and 100 ml of sterile distilled water. The cup was incubated at room temperature for 24 hours to observe the mortality of larvae. Further selective bioassay was performed with the isolate which showed 100% mortality, as described above in four replicates along with the negative and positive control. RESULTS: Out of 1385 Bacillus thuringiensis obtained from 454 soil samples, 766 (55.30%) were spherical crystal protein producers, among them, a single strain (14P2A) showed 100% mortality against mosquito larvae. The lethal concentration doses required to kill 50% and 90% of the larval population were 32.35 and 46.77 Parts per million respectively. CONCLUSIONS: The native Bacillus thuringiensis produces the crystal protein effective in killing mosquito larvae. The native Bacillus thuringiensis should be included as a tool to control mosquito-borne diseases in Nepal.

Antibiotic Susceptibility of Staphylococcus Aureus with VanA and MecA Genes.

BACKGROUND: Staphylococcus aureus (S.aureus) is an emerging antibiotic resistant bacterium responsible for various infections in human. Resistance to methicillin and vancomycin are of prime concern in S. aureus. The study aims to determine the minimum inhibitory concentration (MIC) of Vancomycin and evaluate the existence of mecA and vanA genes, associated with antibiotic resistance. METHODS: Clinical specimens from three Kathmandu hospitals were processed and S. aureus was identified using conventional microbiological procedures. MRSA was phenotypically identified with cefoxitin (30µg) disc diffusion, while vancomycin susceptibility was assessed using the Ezy MICTM stripes. The mecA and vanA genes were detected by polymerase chain reaction (PCR). RESULTS: Out of 266 S. aureus samples from various clinical specimen subjected for analysis, 77 (28.9%) were found methicillin-resistant (MRSA) and 10 (3.8%) were observed vancomycin-resistant (VRSA). Vancomycin resistant isolates showed a significant correlation between resistance to ampicillin, chloramphenicol, and cefoxitin. The mecA gene was found in 39 of the MRSA isolates, having 50.64% of MRSA cases, while the vanA gene was detected in 4 of the VRSA cases, constituting 40% of VRSA occurrences. CONCLUSIONS: The strains with higher vancomycin minimum inhibitory concentration values (≥ 1.5 µg/ml) displayed increased resistance rates to various antibiotics compared to strains with lower minimum inhibitory concentration values (< 1.5 µg/ml). The presence of vanA genes was strongly associated (100%) with vancomycin resistance, while the 10.3% mecA gene was identified from MRSA having resistance towards vancomycin also.

Extended Spectrum Beta Lactamase Escherichia Coli in Bagmati River, Kathmandu Valley.

BACKGROUND: Antimicrobial resistance organisms in the peripheral communities of an environment can be predicted by the presence of extended-spectrum beta-lactamase Escherichia coli in that environment. The close connectivity between humans and water sources can facilitate the entry of antimicrobial resistant organisms into the human ecosystem. The aim of this study was to assess beta lactamase producing Escherichia coli from Bagmati river within Kathmandu valley. METHODS: In the year 2020, a cross-sectional study was conducted on water samples collected from 66 locations along the Bagmati River. Coliforms were isolated by five tubes dilution method and identified by cultural and biochemical tests. Further Escherichia coli was isolated in eosin methylene blue agar at 44.5 °C. Antibiotic susceptibility test was performed by Kirby Bauer disk diffusion methods. Beta lactamase gene types were detected by using conventional multiplex polymerase chain reaction. RESULTS: A total of 615 bacterial isolates were identified among which 39 % (n=241) were Escherichia coli. Extended spectrum beta lactamase producing Escherichia coli was confirmed in 16.6 % (40/241) of total Escherichia coli isolates. Among 66 sites this isolate was detected in 26 (40 %) sampling sites excluding upstream regions. All the Escherichia coli isolates were multidrug resistance showing higher percentage (>99 %) of resistant for penicillin, tetracycline and erythromycin antibiotics. There were significant differences in resistance rate for cefotaxime and ceftazidime by extended spectrum beta lactamase producing and non-producing Escherichia coli (p<0.05). CONCLUSIONS: Presence of multidrug resistance extended spectrum beta lactamase producing Escherichia coli in river streams suggests the chances of circulating within river system and hence transmitting in human community. KEY WORDS: Bagmati river; drug resistance; escherichia coli; human.

Decadal Analysis of ESBL-Escherichia coli Antibiotic Resistance Patterns in Urine Samples from Nepal: A Systematic Review and Meta-Analysis.

BACKGROUND: This systematic review aimed to determine the antimicrobial resistance pattern of the extended-spectrum ß-lactamases producing Escherichia coli (ESBL-EC) in urine samples in Nepal. METHODS: Systematic literature review was conducted to locate all articles reporting ESBL-EC in urine samples published between January 2012 to December 2022. The Egger's weighted regression analysis was done to assess the publication bias. A random-effects model was used to calculate the pooled prevalence and corresponding 95% confidence interval due to significant between-study heterogeneity. The strength of correlation between multidrug resistance and ESBL production in E.coli strains was determined using Pearson's correlation coefficient. The data were analyzed using R-language 4.2.2. software. RESULTS: The combined prevalence of E.coli in urine samples was found to be 14 % (95% CI, 11-18), while the overall pooled prevalence of ESBL E.coli and MDR E.coli were 30% (95% CI, 20-42) and 70% (95% CI, 38-90) respectively. A strong positive correlation of 0.99 (95% CI, 0.89-1.0) was found between ESBL production and MDR among E.coli isolates. Imipenem was the drug of choice against ESBL-E.coli in urine specimens. CONCLUSIONS: Our analyses showed the overall ESBL-EC and MDR-EC burden in Nepal is considerably high. Likewise, the study also infers an increasing trend of antibiotic resistance pattern of ESBL-EC in urine samples.

Application of Pediatric Risk of Mortality (PRISM) III Score in Predicting Mortality Outcomes.

BACKGROUND: Children admitted in a pediatric intensive care unit have a high risk of mortality. Pediatric risk of mortality III score in first 24 hours of admission has increasingly been used to predict mortality. The objective of this study was to evaluate the validity of Pediatric risk of mortality score in prediction of mortality among the patient admitted in pediatric intensive care unit. METHODS: This prospective observational study was conducted at pediatric intensive care unit of a government pediatric hospital from January to June 2021. Patients between 1 month to 14 years of age and meeting the inclusion criteria were enrolled. Pediatric risk of mortality III score was calculated within 24 hours of admission. Patients were followed up for outcome measure as survivors and non survivors. Chi square test and logistic regression analysis were used to find the association of predictors and the score. RESULTS: The mean Pediatric risk of mortality III score was lower in survivors than in non-survivors (4.67 ± 3.8 versus 14.10 ± 6.07; p<0.001). Those requiring inotropic and ventilator support have significantly higher mortality [49.4 versus 0.6 (p<0.001) and 81.8 versus 1.5 (p<0.001) respectively]. Minimum systolic blood pressure, abnormal pupillary reflex, increased blood urea nitrogen and decreased platelet were the significant (p<0.001) risk factors. The area under the Receiver Operating Characteristic curve was 0.916±0.024 (p<0.001) and goodness-of-fit test showed no significant difference between observed and expected mortalities (p=0.186). CONCLUSIONS: The Pediatric risk of mortality score constitutes a useful prognostic tool in predicting the mortality. KEY WORDS: Mortality; pediatrics; pediatric intensive care unit; risk score.

Source Tracing of Leishmania donovani in Emerging Foci of Visceral Leishmaniasis, Western Nepal.

We sequenced Leishmania donovani genomes in blood samples collected in emerging foci of visceral leishmaniasis in western Nepal. We detected lineages very different from the preelimination main parasite population, including a new lineage and a rare one previously reported in eastern Nepal. Our findings underscore the need for genomic surveillance.

Field assessment of the operating procedures of a semi-quantitative G6PD Biosensor to improve repeatability of routine testing.

In remote communities, diagnosis of G6PD deficiency is challenging. We assessed the impact of modified test procedures and delayed testing for the point-of-care diagnostic STANDARD G6PD (SDBiosensor, RoK), and evaluated recommended cut-offs. We tested capillary blood from fingerpricks (Standard Method) and a microtainer (BD, USA; Method 1), venous blood from a vacutainer (BD, USA; Method 2), varied sample application methods (Methods 3), and used micropipettes rather than the test's single-use pipette (Method 4). Repeatability was assessed by comparing median differences between paired measurements. All methods were tested 20 times under laboratory conditions on three volunteers. The Standard Method and the method with best repeatability were tested in Indonesia and Nepal. In Indonesia 60 participants were tested in duplicate by both methods, in Nepal 120 participants were tested in duplicate by either method. The adjusted male median (AMM) of the Biosensor Standard Method readings was defined as 100% activity. In Indonesia, the difference between paired readings of the Standard and modified methods was compared to assess the impact of delayed testing. In the pilot study repeatability didn't differ significantly (p = 0.381); Method 3 showed lowest variability. One Nepalese participant had <30% activity, one Indonesian and 10 Nepalese participants had intermediate activity (≥30% to <70% activity). Repeatability didn't differ significantly in Indonesia (Standard: 0.2U/gHb [IQR: 0.1-0.4]; Method 3: 0.3U/gHb [IQR: 0.1-0.5]; p = 0.425) or Nepal (Standard: 0.4U/gHb [IQR: 0.2-0.6]; Method 3: 0.3U/gHb [IQR: 0.1-0.6]; p = 0.330). Median G6PD measurements by Method 3 were 0.4U/gHb (IQR: -0.2 to 0.7, p = 0.005) higher after a 5-hour delay compared to the Standard Method. The definition of 100% activity by the Standard Method matched the manufacturer-recommended cut-off for 70% activity. We couldn't improve repeatability. Delays of up to 5 hours didn't result in a clinically relevant difference in measured G6PD activity. The manufacturer's recommended cut-off for intermediate deficiency is conservative.

Epidemiological, Serological, and Entomological Investigation of New Visceral Leishmaniasis Foci in Nepal.

The aim of this study was to explore epidemiological, serological, entomological, and social aspects of visceral leishmaniasis (VL) in new foci in Nepal. The study was conducted in 11 villages of five districts that had been previously free of VL but that reported new cases between 2019 and 2021. We screened 1,288 inhabitants using rK39 tests and investigated the epidemiological and clinical characteristics of 12 recent VL cases. A total of 182 community members were interviewed about knowledge, attitude, and practices regarding VL. They then underwent an awareness training; 40 of them had a second interview at 6 months to assess the training impact. Vector surveys were conducted in six houses per village to assess sandfly density and infection rates. The prevalence of VL infection was 0.5% and 3.2% among screened populations in Dolpa and Kavre districts, respectively, while the other districts had no rK39-positive cases. No association between travel history and VL infection was found. Phlebotomus argentipes sandflies were collected in three districts at high altitudes (from 1,084 to 4,450 m). None of the sandflies captured had Leishmania donovani DNA. People in new foci were not aware of VL symptoms, vectors, or preventive measures. The training significantly improved their knowledge and practice in seeking medical care in case of illness. The epidemiological, serological, and entomological investigations suggest indigenous focal transmission of VL. An integrated package of strategic interventions should be implemented by the national VL elimination program in districts with new VL foci.

Assessment of the impact of implementation research on the Visceral Leishmaniasis (VL) elimination efforts in Nepal.

Nepal, Bangladesh, and India signed a Memorandum of Understanding (MoU) in 2005 to eliminate visceral leishmaniasis (VL) as a public health problem from the Indian subcontinent by 2015. By 2021, the number of reported VL cases in these countries had declined by over 95% compared to 2007. This dramatic success was achieved through an elimination programme that implemented early case detection and effective treatment, vector control, disease surveillance, community participation, and operational research that underpinned these strategies. The experience offered an opportunity to assess the contribution of implementation research (IR) to VL elimination in Nepal. Desk review and a stakeholder workshop was conducted to analyse the relationship between key research outputs, major strategic decisions in the national VL elimination programme, and annual number of reported new cases over time between 2005 and 2023. The results indicated that the key decisions across the strategic elements, throughout the course of the elimination programme (such as on the most appropriate tools for diganostics and treatment, and on best strategies for case finding and vector management), were IR informed. IR itself responded dynamically to changes that resulted from interventions, addressing new questions that emerged from the field. Close collaboration between researchers, programme managers, and implementers in priority setting, design, conduct, and review of studies facilitated uptake of evidence into policy and programmatic activities. VL case numbers in Nepal are now reduced by 90% compared to 2005. Although direct attribution of disease decline to research outputs is difficult to establish, the Nepal experience demonstrates that IR can be a critical enabler for disease elimination. The lessons can potentially inform IR strategies in other countries with diseases targeted for elimination.

Follow-up assessment of visceral leishmaniasis treated patients and the impact of COVID-19 on control services in Nepal.

BACKGROUND: Follow-up assessment of visceral leishmaniasis (VL) treated cases is important to monitor the long term effectiveness of treatment regimens. The main objective of this study was to identify the gaps and challenges in the follow-up of treated VL cases, to monitor treatment outcome and to assess the impact of COVID-19 on VL elimination services and activities. METHODS: Clinicians treating VL patients, district focal persons for VL, and patients treated for VL in seven high endemic districts in Nepal during 2019-2022 were interviewed to collect data on challenges in the follow-up of VL treated patients as per national strategy. RESULTS: Follow up status was poor in two districts with the largest number of reported cases. The majority of cases were children under 10 years of age (44.2%). Among 104 VL treated cases interviewed, 60.6% mentioned that clinicians had called them for follow-up but only 37.5% had complied. Among 112 VL treated cases followed up, 8 (7.14%) had relapse and 2 (1.8%) had PKDL. Among 66 cases who had VL during the COVID-19 lock down period, 32 (48.5%) were diagnosed within 1 week; however, 10 (15.1%) were diagnosed only after 4 weeks or more. During the COVID-19 pandemic, there was no active search for VL because of budget constraints and lack of diagnostic tests, and no insecticide spraying was done. CONCLUSION: Relapses and PKDL are challenges for VL elimination and a matter of concern. Successful implementation of the national strategy for follow up of treated VL cases requires addressing elements related to patients (awareness, transport, communication) clinicians (compliance) and organization of service delivery (local health worker training and deployment). COVID-19 did not have much impact on VL diagnosis and treatment; however, public health programmes including active case detection and insecticide spraying for vector control were severely reduced.

Epidemiological, serological, and entomological aspects of visceral leishmaniasis (VL) in suspected new VL foci in Bangladesh.

The study aimed to explore epidemiological, serological, and entomological aspects of visceral leishmaniasis (VL) in suspected new VL foci and assess the knowledge, attitude, and practices of the community living in the alleged new VL foci. The study investigated new visceral leishmaniasis (VL) cases reported between 2019 and 2020 in four sub-districts (Dharmapasha, Hakimpur, Islampur and Savar) where we tested 560 members using the rK39 rapid test and conducted vector collections in six neighbouring houses of the index cases to assess sandfly density and distribution, examined sandflies' infection, and determined the spatial relationship with VL infection. Furthermore, we highlighted the importance of early detection, and community awareness in controlling the spread of the disease. The study screened 1078 people from 231 households in the four sub-districts for fever, history of visceral leishmaniasis (VL), and PKDL-like skin lesions. Among sub-districts, positivity rate for rK39 rapid test was highest (3.5 %) in Savar. Sandflies were present across all areas except in Dharmapasha, but all 21 collected female P. argentipes sandflies were negative for Leishmania parasite DNA. We found one person from Islampur with a history of VL, and one from Islampur and another one from Savar had PKDL. After the awareness intervention, more people became familiar with VL infection (91.2 %), and their knowledge concerning sandflies being the vector of the disease and the risk of having VL increased significantly (30.1 %). The study found no active case in the suspected new foci, but some asymptomatic individuals were present. As sandfly vectors exist in these areas, the National Kala-azar Elimination Programme (NKEP) should consider these areas as kala-azar endemic and initiate control activities as per national guidelines.

Assessment of treatment outcomes of visceral leishmaniasis (VL) treated cases and impact of COVID-19 on VL management and control services in Bangladesh.

BACKGROUND: COVID-19 has largely impacted the management of Visceral leishmaniasis (VL), like several other Neglected Tropical Diseases. The impact was particularly evident in Lower and Middle-Income countries where the already inadequate healthcare resources were diverted to managing the COVID-19 pandemic. Bangladesh achieved the elimination target for VL in 2016. To sustain this success, early diagnosis and treatment, effective vector control, and periodic surveillance are paramount. However, the specific control measures for VL in Bangladesh that were hampered during COVID-19 and their extent are unknown. METHODS: This study aimed at identifying the gaps and challenges in the follow-up of treated VL patients by interviewing both the treated VL cases and their health service providers. We followed VL cases treated between 2019 and 2020 in five VL endemic subdistricts (upazilas) both retrospectively and prospectively to monitor clinical improvement, relapse, or other consequences. Moreover, interviews were conducted with the health service providers to assess the impact of COVID-19 on VL case detection, treatment, reporting, vector control operations, and logistic supply chain management. RESULTS: There was no added delay for VL diagnosis; however, VL treatment initiation and reporting time increased almost two-fold due to COVID-19. Indoor Residual Spraying activity was significantly hampered due to a shortage of insecticides. Out of 44 enrolled and treated VL patients, two relapsed (4.5 %), two developed Para Kala-Azar Dermal Leishmaniasis (4.5 %), and three (6.8 %) Post Kala-Azar Dermal Leishmaniasis (PKDL). The health service providers highlighted patients` unwillingness to visit the hospital, financial constraints, and distance from the hospitals as the main reasons for missed follow-up visits (20.5 %). Building good communication in the community, awareness schemes, and incentive-based approaches were suggested as possible solutions to mitigate these problems. CONCLUSION: Long-term follow-up is required for the early detection and management of VL relapse and PKDL cases. Effective vector control measures, capacity development, and identification of new VL hotspots are pivotal in the VL endemic regions to sustain the elimination goal.

Burden of human metapneumovirus infections among children with acute respiratory tract infections attending a Tertiary Care Hospital, Kathmandu.

BACKGROUND: Acute respiratory infections (ARIs) are one of the most common causes of mortality and morbidity worldwide. Every year millions of children suffer from viral respiratory tract infections (RTIs) ranging from mild to severe illnesses. Human Metapneumovirus (HMPV) is among the most frequent viruses responsible for RTIs. However, HMPV infections and their severity among children have not been explored yet in Nepal. PURPOSE: Therefore, the study was focused on HMPV infections and other potential viral etiologies or co-infections using multiplex PCR among children attending Kanti Children's Hospital and assessed the clinical characteristics of the infections as well as found the co-infections. A hospital-based cross-sectional study was designed and a convenience sampling method was used to enroll children of less than 15 years with flu-like symptoms from both outpatients and inpatients departments over three months of the study period. RESULTS: HMPV infection (13.3%) was the most predominant infection among the different viral infections in children with ARIs in Kanti Children's Hospital. The HMPV was more prevalent in the age group less than three years (21.8%). Cough and fever were the most common clinical features present in all children infected with HMPV followed by rhinorrhea, sore throat, and wheezing. HMPV-positive children were diagnosed with pneumonia (42.9%), bronchiolitis (28.5%), upper respiratory tract infections (14.3%), and asthma (14.3%). The prevalence of HMPV was high in late winter (14.3%) followed by early spring (13.5%). CONCLUSIONS: This study provides the baseline information on HMPV and associated co-infection with other respiratory viruses for the differential diagnosis based on molecular methods and also the comparison of clinical presentations among the different respiratory syndromes.

Genotypically Confirmed Vancomycin-Resistant Staphylococcus aureus With vanB Gene Among Clinical Isolates in Kathmandu.

Purpose: Methicillin-resistant Staphylococcus aureus, a common bacterial pathogen causes various infections. The acquisition of various antimicrobial-resistant genes in S. aureus has led to the transformation of this bacterium into a superbug. Vancomycin resistance among MRSA isolates is an emerging threat in empirical therapy of various infections. The study was hence aimed to find out the susceptibility status of S. aureus isolates toward vancomycin and detect mecA, vanA, and vanB genes among the isolates. Methods: A total of 1245 clinical samples from the participants attending a tertiary care hospital in Kathmandu were processed. S. aureus isolated from the samples were subjected to antibiotic susceptibility patterns using the modified Kirby-Bauer disk diffusion method. Agar dilution method was used to determine the minimum inhibitory concentration of vancomycin. The antibiotic-resistant genes such as mecA, vanA, and vanB among S. aureus isolates were screened by a conventional polymerase chain reaction. Results: Of 1245 samples, 80 S. aureus were identified. Out of which, 47.5% (38/80) were phenotypically confirmed MRSA isolates. mecA gene was detected in 84.2% (32/38) of MRSA isolates. 10.5% (4/38) were confirmed as vancomycin-intermediate S. aureus (VISA) by MIC determination. None of the isolates was positive for the vanA gene; however, 2 isolates were found to possess the vanB gene. The 2 isolates have vancomycin MIC breakpoints of 4 to 8 µg/mL. Conclusion: There might be a spreading of vancomycin resistance among S. aureus, creating serious public health problems. Therefore, measures to limit vancomycin resistance should be considered in healthcare facilities as immediately as possible.

Molecular detection of plasmid mediated blaTEM, blaCTX-M,and blaSHV genes in Extended Spectrum ß-Lactamase (ESBL) Escherichia coli from clinical samples.

BACKGROUND: Extended spectrum ß-lactamases (ESBLs) are a group of beta-lactamase enzymes that confer resistance to the oxyimino-cephalosporins and monobactams. The emergence of ESBL - producing genes possesses a serious threat for treating infections since it is associated with multi-drug resistance. This study was focused to identify the ESBLs producing genes from Escherichia coli isolates from clinical samples from a referral-level tertiary care hospital in Lalitpur. METHODS: This was a cross-sectional study conducted from September 2018 to April 2020 at the Microbiology Laboratory of Nepal Mediciti Hospital. Clinical samples were processed, and culture isolates were identified and characterized following standard microbiological techniques. An antibiotic susceptibility test was performed by a modified Kirby-Bauer disc diffusion method as recommended by Clinical and Laboratory Standard Institute guidelines.Extended -spectrum beta-lactamases were phenotypically confirmed by the combined disc method. The ESBL-producing genes blaTEM, blaCTX-M and blaSHV were confirmed by PCR. RESULTS: Of the 1449 total E. coli isolates, 22.29% (323/1449) isolates were multi-drug resistant (MDR). Among the total MDR E. coli isolates, 66.56% (215/323) were ESBL producers. The maximum number of ESBL E. coli was isolated from urine 90.23% (194) followed by sputum 5.58% (12), swab 2.32% (5), pus 0.93% (2), and blood 0.93% (2). The antibiotic susceptibility pattern of ESBL E. coli producers showed the highest sensitivity toward tigecycline (100%) followed by polymyxin b, colistin and meropenem. Out of 215 phenotypically confirmed ESBL E. coli, only 86.51% (186) isolates were found to be positive by PCR for either blaTEM or blaCTX-M genes. Among the ESBL genotypes, the most common were blaTEM 63.4% (118) followed by blaCTX-M 36.6% (68). CONCLUSION: The emergence of MDR and ESBL - producing E. coli isolates with high antibiotic - resistant rates to commonly used antibiotics and increased predominance of major gene types blaTEM is a serious concern to the clinicians and microbiologists. Periodic monitoring of antibiotic susceptibility and associated genes would help guide the rationale use of antibiotics for treating the predominant pathogen E. coli in the hospitals and healthcare facilities of the communities.

Detection of blaNDM-1,mcr-1 and MexB in multidrug resistant Pseudomonas aeruginosa isolated from clinical specimens in a tertiary care hospital of Nepal.

INTRODUCTION: Pseudomonas aeruginosa is an opportunistic pathogen, which causes healthcare-associated infections in immunosuppressed patients. They exhibit resistance to multiple classes of antibiotics via various mechanisms such as the over-expression of efflux pumps, decreased production of the outer membrane protein (D2 porin), over-expression of the chromosomally encoded AmpC cephalosporinase, modification of drugs, and mutation(s) at the target site of the drug. The bacteria also develop antibiotic resistance through the acquisition of resistance genes carried on mobile genetic elements. Limited data on phenotypic as well as genotypic characterization of MDR P. aeruginosa in Nepal infers the needs for this study. This study was carried out to determine the prevalence rate of metallo-ß-lactamase (MBL-producer) as well as colistin resistant multidrug resistant (MDR) P. aeruginosa in Nepal and also to detect MBL, colistin resistance, and efflux pump encoding genes i.e. blaNDM-1, mcr-1 and MexB respectively in MDR P. aeruginosa isolated from clinical samples. METHODS/METHODOLOGY: A total of 36 clinical isolates of P. aeruginosa were collected. All bacterial isolates were phenotypically screened for antibiotic susceptibility using Kirby Bauer Disc Diffusion method. All the multidrug resistant P. aeruginosa were phenotypically screened for MBL producer by Imipenem-EDTA combined disc diffusion test (CDDT). Similarly, MIC value for colistin was also determined by broth microdilution method. Genes encoding carbapenemase (blaNDM-1), colistin resistant (mcr-1) and efflux pump activity (MexB) were assayed by PCR. RESULTS: Among 36 P. aeruginosa, 50% were found to be MDR among which 66.7% were found to be MBL producer and 11.2% were found to be colistin resistant. Among MDR P. aeruginosa, 16.7%, 11.2% and 94.4% were found to be harbouring blaNDM-1, mcr-1 and MexB genes respectively. CONCLUSION: In our study, carbapenemase production (encoded by blaNDM-1), colistin resistant enzyme production (encoded by mcr-1), and expression of efflux pump (encoded by MexB) are found to be one of the major causes of antibiotic resistance in P. aeruginosa. Therefore, periodic phenotypic as well as genotypic study in Nepal on P. aeruginosa would provide the scenario of resistance pattern or mechanisms in P. aeruginosa. Furthermore, new policies or rules can be implemented in order to control the P. aeruginosa infections.

Antibiotics Prescription, Dispensing Practices and Antibiotic Resistance Pattern in Common Pathogens in Nepal: A Narrative Review.

Antimicrobial resistance (AMR) is increasing and it is a serious public health problem worldwide. Nepal is considered as one of the contributors for rising AMR due to the most prevalent irrational use of antibiotics. In this review, we have assessed the practices of antibiotic prescription and dispensing, and antibiotic resistance of commonly encountered bacteria in Nepal. There is exponential increase of therapeutic consumption of antibiotics either without clinician's prescription or irrational prescription. Almost half of the population in Nepal was found to purchase antibiotics easily from the nearby pharmacies without clinician's prescription. Irrational prescription is exceeded in remote areas which could be due to lack of access with health posts and hospitals. The third generation cephalosporins, which are considered as the last resort antibiotics were found to be relatively prescribed and dispensed higher as compared to other classes of antibiotics. Despite the existing limited functional surveillance system, antibiotic resistance among bacteria is increasing in Nepal because of irrational prescription, dispensing and consumption of antibiotics without prescription.

Prevalence of Multidrug Resistant Pseudomonas aeruginosa Isolated from Clinical Specimens in Tertiary Care Hospital.

BACKGROUND: Pseudomonas aeruginosa is an opportunistic pathogen, which causes nosocomial infections in human. The rapid increase in drug resistance of this pathogen is a global concern. The aim of this study was to determine the clinical burden of P.aeruginosa, its antibiotic susceptibility pattern along with metallo-ß-lactamase (MBL) detection. METHODS: The descriptive cross-sectional study was conducted in Upendra Devkota Memorial National Institute of Neurological and Allied Sciences from January to August 2021. Isolation and identification of P. aeruginosa from clinical specimens was performed by using standard laboratory procedure. All bacterial isolates were phenotypically screened for multidrug resistance using Kirby Bauer disc diffusion method. All the multidrug resistant P.aeruginosa were phenotypically screened for MBL producer by Imipenem-EDTA combined disc diffusion test (CDDT). RESULTS: A total of 770 samples were processed of which 36 isolates of P. aeruginosa were obtained. P.aeruginosa was isolated mainly from tracheal aspirates, sputum, blood and urine. Among 36 isolates, 50% were found to be multidrug resistant (MDR). More percentage of P.aeruginosa isolates were found resistant to aztreonam, ofloxacin and levofloxacin (52.8%). Furthermore, this study reveals antibiotics like piperacillin/tazobactam and carbapenem were found to be good choice for the treatment of infection caused by this organism. Among MDR isolates 66.7% were found to be MBL producer. CONCLUSIONS: The data in this study highlights the prevalence of multidrug resistant, MBL producer, and colistin resistant P.aeruginosa in clinical specimens. In this study, carbapenems and piperacillin/tazobactam were found to be most effective antimicrobial drugs for empirical therapy in P.aeruginosa infections.

Prevalence of post kala-azar dermal leishmaniasis (PKDL) and treatment seeking behavior of PKDL patients in Nepal.

BACKGROUND: In Nepal, the burden of post kala-azar dermal leishmaniasis (PKDL) is not known since there is no active case detection of PKDL by the national programme. PKDL patients could pose a challenge to sustain visceral leishmaniasis (VL) elimination. The objective of this study was to determine the prevalence of PKDL and assess PKDL patients' knowledge on VL and PKDL, and stigma associated with PKDL. METHODOLOGY/PRINCIPAL FINDINGS: Household surveys were conducted in 98 VL endemic villages of five districts that reported the highest number of VL cases within 2018-2021. A total of 6,821 households with 40373 individuals were screened for PKDL. Cases with skin lesions were referred to hospitals and examined by dermatologists. Suspected PKDL cases were tested with rK39 and smear microscopy from skin lesions. An integrated diagnostic approach was implemented in two hospitals with a focus on management of leprosy cases where cases with non-leprosy skin lesions were tested for PKDL with rK39. Confirmed PKDL patients were interviewed to assess knowledge and stigma associated with PKDL, using explanatory model interview catalogue (EMIC) with maximum score of 36. Among 147 cases with skin lesions in the survey, 9 (6.12%) were confirmed as PKDL by dermatologists at the hospital. The prevalence of PKDL was 2.23 per 10,000 population. Among these 9 PKDL cases, 5 had a past history of VL and 4 did not. PKDL cases without a past history of VL were detected among the "new foci", Surkhet but none in Palpa. None of the cases negative for leprosy were positive for PKDL. There was very limited knowledge of PKDL and VL among PKDL cases. PKDL patients suffered to some degree from social and psychological stigma (mean ± s.d. score = 17.89 ± 12.84). CONCLUSIONS/SIGNIFICANCE: Strengthening the programme in PKDL case detection and management would probably contribute to sustenance of VL elimination. Awareness raising activities to promote knowledge and reduce social stigma should be conducted in VL endemic areas.

Multi-drug Resistance, ß-Lactamases Production, and Coexistence of bla NDM-1 and mcr-1 in Escherichia coli Clinical Isolates From a Referral Hospital in Kathmandu, Nepal.

The ability of pathogenic Escherichia coli to produce carbapenemase enzymes is a characteristic that allows them to resist various antibiotics, including last-resort antibiotics like colistin and carbapenem. Our objectives were to identify rapidly developing antibiotic resistance (AR), assess ß-lactamases production, and detect mcr-1 and bla NDM-1 genes in the isolates. A prospective cross-sectional study was carried out in a referral hospital located in Kathmandu from November 2019 to December 2020 using standard laboratory and molecular protocols. Among 77 total E. coli isolates, 64 (83.1%) of them were categorized as MDR. Phenotypically 13 (20.3%) colistin-resistant, 30 (46.9%) ESBL and 8 (12.5%) AmpC producers, and 5 (7.8%) ESBL/AmpC co-producers were distributed among MDR-E. coli. Minimum inhibitory concentrations (MIC) against the majority of MDR isolates were exhibited at 1 g/L. Of these 77 E. coli isolates, 24 (31.2%) were carbapenem-resistant. Among these carbapenem-resistant bacteria, 11 (45.9%) isolates were reported to be colistin-resistant, while 15 (62.5%) and 2 (8.3%) were MBL and KPC producers, respectively. Out of 15 MBL producers, 6 (40%) harbored bla NDM-1, and 8 (61.5%) out of 13 colistin-resistant pathogens possessed mcr-1. The resistance by colistin- and carbapenem were statistically associated (P < .001). However, only 2 (18.2%) of the co-resistant bacteria were found to have both genes. Our study revealed the highly prevalent MDR and the carbapenem-resistant E. coli and emphasized that the pathogens possess a wide range of capabilities to synthesize ß-lactamases. These findings could assist to expand the understanding of AR in terms of enzyme production.