Drug-resistant Mycobacterium tuberculosis among Nepalese patients at a tuberculosis referral center.

BACKGROUND: Multidrug-resistant tuberculosis (MDR-TB), characterized by isoniazid and rifampicin resistance, is caused by chromosomal mutations that restrict treatment options and complicate tuberculosis management. This study sought to investigate the prevalence of pre-extensively drug-resistant (pre-XDR) and extensively drug-resistant (XDR) tuberculosis, as well as mutation pattern, in Nepalese patients with MDR/rifampicin-resistant (RR)-TB strains. METHODS: A cross-sectional study was conducted on MDR/RR-TB patients at the German Nepal Tuberculosis Project from June 2017 to June 2018. The MTBDRsl line probe assay identified pre-XDR-TB and XDR-TB. Pre-XDR-TB included MDR/RR-TB with resistance to any fluoroquinolone (FLQ), while XDR-TB included MDR/RR-TB with resistance to any FLQ and at least one additional group A drug. Mutation status was determined by comparing bands on reaction zones [gyrA and gyrB for FLQ resistance, rrs for SILD resistance, and eis for low-level kanamycin resistance, according to the GenoType MTBDRsl VER 2.0, Hain Lifescience GmbH, Nehren, Germany definition of pre-XDR and XDR] to the evaluation sheet. SPSS version 17.0 was used for data analysis. RESULTS: Out of a total of 171 patients with MDR/RR-TB, 160 had (93.57%) had MTBC, of whom 57 (35.63%) had pre-XDR-TB and 10 (6.25%) had XDR-TB. Among the pre-XDR-TB strains, 56 (98.25%) were FLQ resistant, while 1 (1.75%) was SLID resistant. The most frequent mutations were found at codons MUT3C (57.14%, 32/56) and MUT1 (23.21%, 13/56) of the gyrA gene. One patient had SLID resistant genotype at the MUT1 codon of the rrs gene (100%, 1/1). XDR-TB mutation bands were mostly detected on MUT1 (30%, 3/10) of the gyrA and rrs, MUT3C (30%, 3/10) of the gyrA, and MUT1 (30%, 3/10) of the rrs. CONCLUSIONS: Pre-XDR-TB had a significantly higher likelihood than XDR-TB, with different specific mutation bands present in gyrA and rrs genes.

Safety and immunogenicity of conjugate vaccine for typhoid (Vi-DT): Finding from an observer-blind, active-controlled, randomized, non-inferiority, phase III clinical trial among healthy volunteers.

Typhoid fever is a significant public health concern with most of the sufferers between 15 and 25 y of age in Nepal. We undertook this study to demonstrate Vi polysaccharide conjugated with diphtheria toxoid (Vi-DT) conjugate vaccine which is non-inferior to Typbar typhoid conjugate vaccine, a Vi polysaccharide vaccine conjugated with tetanus toxoid (Vi-TT) with a focus on the adult population from Dhulikhel Hospital which was one of the total four sites in Nepal. In this study, we assigned the eligible participants in 1:1:1:1 ratio by block randomization, and stratified into three age groups (6 months to less than 2 y, 2 y to less than 18 y, and 18 y to 45 y), allotted to Group A, B, C, and D. Group A, B, and C received 25 µg (0.5 mL) of Vi-DT study vaccine and participants in Group D received 25 µg (0.5 mL) Vi-TT vaccine. We descriptively analyzed safety in all the participants receiving one dose of the investigational vaccine. The anti-Vi-IgG seroconversion rate in Vi-DT recipients was 99.71% (97.5% CI 98.04-99.96; 344 of 345 participants) and 99.13% (94.27-99.87; 114 of 115) in Vi-TT recipients which indicates that Vi-DT vaccine is non-inferior to Vi-TT vaccine. In safety aspect, 16.81% of total subject had at least one solicited adverse reaction and 22.61% of the Vi-TT participants experienced at least one solicited adverse reaction with most of them being local adverse reactions. None of the enrolled participants reported serious adverse events. Our study shows that a single dose of the Vi-DT vaccine is immunogenic, safe to administer and non-inferior to the Vi-TT vaccine four weeks after vaccination.

Persistence of immunity following a single dose of inactivated poliovirus vaccine: a phase 4, open label, non-randomised clinical trial.

BACKGROUND: The polio eradication endgame required the withdrawal of Sabin type 2 from the oral poliovirus vaccine and introduction of one or more dose of inactivated poliovirus vaccine (IPV) into routine immunisation schedules. However, the duration of single-dose IPV immunity is unknown. We aimed to address this deficiency. METHODS: In this phase 4, open-label, non-randomised clinical trial, we assessed single-dose IPV immunity. Two groups of infants or children were screened: the first group had previously received IPV at 14 weeks of age or older (previous IPV group; age >2 years); the second had not previously received IPV (no previous IPV group; age 7-12 months). At enrolment, all participants received an IPV dose. Children in the no previous IPV group received a second IPV dose at day 30. Blood was collected three times in each group: on days 0, 7, and 30 in the previous IPV group and on days 0, 30, and 37 in the no previous IPV group. Poliovirus antibody was measured by microneutralisation assay. Immunity was defined as the presence of a detectable antibody or a rapid anamnestic response (ie, priming). We used the χ2 to compare proportions and the Mann-Whitney U test to assess continuous variables. To assess safety, vaccinees were observed for 30 min, caregivers for each participating child reported adverse events after each follow-up visit and were questioned during each follow-up visit regarding any adverse events during the intervening period. Adverse events were recorded and graded according to the severity of clinical symptoms. The study is registered with ClinicalTrials.gov, NCT03723837. FINDINGS: From Nov 18, 2018, to July 31, 2019, 502 participants enrolled in the study, 458 (255 [65%] boys and 203 [44%] girls) were included in the per protocol analysis: 234 (93%) in the previous IPV group and 224 (90%) in the no previous IPV group. In the previous IPV group, 28 months after one IPV dose 233 (>99%) of 234 children had persistence of poliovirus type 2 immunity (100 [43%] of 234 children were seropositive; 133 [99%] of 134 were seronegative and primed). In the no previous IPV group, 30 days after one IPV dose all 224 (100%) children who were type 2 poliovirus naive had seroconverted (223 [>99%] children) or were primed (one [<1%]). No adverse events were deemed attributable to study interventions. INTERPRETATION: A single IPV dose administered at 14 weeks of age or older is highly immunogenic and induces nearly universal type 2 immunity (seroconversion and priming), with immunity persisting for at least 28 months. The polio eradication initiative should prioritise first IPV dose administration to mitigate the paralytic burden caused by poliovirus type 2. FUNDING: WHO and Rotary International.

Comparison of Volunteer Non-Remunerated Donors and Replacement Donors.

BACKGROUND: There are mainly two types of blood donor in Nepal i.e., Voluntary Non-Remunerated Donor and Replacement Donor. The main aim of this study is to compare between Voluntary Non-Remunerated Donor and Replacement Donor. METHODS: This is retrospective and cross-sectional study conducted in the blood transfusion Service, Tribhuvan University Teaching Hospital. The study was conducted on both Voluntary Non-Remunerated Donor and Replacement Donor who came to blood transfusion Service and voluntary blood donation program. All the blood donors donating in blood transfusion Service were considered as the study population. RESULTS: Out of total 25951 donors, there were 15565(60.02%) Voluntary Non-Remunerated Donor and 10386(39.97%) Replacement Donor. On the whole, there were 21938(84.59%) male donors and 3995(15.40%) female donors. The male Replacement Donor was 14273(93.12%) followed by a male Voluntary Non-Remunerated Donor 7582(73.71%). The female Voluntary Non-Remunerated Donor was 2703(67.65 %) followed by female Replacement Donor 1292(32.34 %). CONCLUSIONS: This study concludes that the Voluntary Non-Remunerated Donor is quite low, in comparison to Replacement Donor. 100 % Voluntary Non-Remunerated Donor could not be achieved due to lack of awareness about health and voluntary type of blood donation.

Immune non-interference and safety study of Vi-DT typhoid conjugate vaccine with a measles, mumps and rubella containing vaccine in 9-15 months old Nepalese infants.

BACKGROUND: Typhoid fever is a common disease in developing countries especially in the Indian subcontinent and Africa. The available typhoid conjugate vaccines (TCV) have been found to be highly immunogenic in infants and children less than 2 years of age. Many countries are planning to adopt TCV in their routine EPI programs around 9 months of age when measles containing vaccines are given. Therefore, Vi-DT TCV was tested in 9-15 months aged healthy infants in Nepal to demonstrate non-interference with a measles containing vaccine. METHODS: This was a randomized, open label, phase III study to assess the immune non-interference, safety, and reactogenicity of Vi-DT typhoid conjugate vaccine when given concomitantly with measles, mumps and rubella (MMR) vaccine. A total of 360 participants aged 9-15 months were enrolled and randomized equally into Vi-DT + MMR (180 participants) or MMR alone (180 participants) group and were evaluated for immunogenicity and safety 28 days post vaccination. RESULTS: Using the immunogenicity set, difference between proportions (95% CI) of the Vi-DT + MMR group vs MMR alone group were -2.73% (-8.85, 3.38), -3.19% (-11.25, 4.88) and 2.91% (-3.36, 9.18) for sero-positivity rate of anti-measles, anti-mumps and anti- rubella, respectively. Only the lower bound of the range in difference of the proportions for sero-positivity rate of anti-mumps did not satisfy the non-inferiority criteria as it was above the -10% limit, which may not be of clinical significance. These results were confirmed in the per protocol set. There were no safety concerns reported from the study and both Vi-DT + MMR and MMR alone groups were comparable in terms of solicited and unsolicited adverse events . CONCLUSIONS: Results indicated that there is non-interference of MMR vaccine with Vi-DT and Vi-DT conjugate vaccine could be considered as an addition to the EPI schedule among children at risk of contracting typhoid.

Phenotypic detection of methicillin resistance, biofilm production, and inducible clindamycin resistance in Staphylococcus aureus clinical isolates in Kathmandu, Nepal.

INTRODUCTION: Methicillin resistance, inducible clindamycin resistance (ICR), biofilm production, and increased minimum inhibitory concentration (MIC) of vancomycin in Staphylococcus aureus are major causes of antibiotic treatment failure and increased morbidity and mortality. The surveillance of such isolates and the study of their antimicrobial pattern are essential in managing the infections caused by these isolates. This study aimed to determine methicillin resistance, biofilm production, and ICR in S. aureus isolates from a tertiary care hospital in Kathmandu, Nepal. MATERIALS AND METHODS: A total of 217 S. aureus isolated from different samples were processed following standard laboratory procedures. Antibiotic susceptibility testing was performed by the Kirby-Bauer disk diffusion technique. Methicillin-resistant S. aureus (MRSA) were identified by the cefoxitin disk diffusion test, and biofilm producers were examined using the microtiter plate technique. D-test and E-test were performed to determine inducible clindamycin resistance and minimum inhibitory concentration of vancomycin, respectively. RESULTS: Among the 217 S. aureus isolates, 78.3% were multidrug-resistant (MDR), 47.0% were MRSA, 62.2% were biofilm producers, and 50.7% showed ICR. All MRSA isolates exhibited MIC levels of vancomycin within the susceptible range. Biofilm producers and MRSA isolates showed elevated antimicrobial resistance. MRSA was significantly associated with MDR. Biofilm-producing and multidrug-resistant MRSA isolates showed significantly higher MIC levels of vancomycin (p = 0.0013 and < 0.0001, respectively), while ICR was significantly higher in MDR (p = 0.0001) isolates. CONCLUSION: High multidrug resistance, MRSA, and ICR in this study call for routine evaluation of antibiotic susceptibility patterns of S. aureus. Vancomycin can be used to treat serious staphylococcal infections. Clindamycin should be prescribed only after performing the D-test. Drugs like teicoplanin, chloramphenicol, doxycycline, amikacin, and levofloxacin can treat MRSA infections.

Diagnostic performance of GeneXpert MTB/RIF in detecting MTB in smear-negative presumptive TB patients.

BACKGROUND: Tuberculosis (TB) is a leading cause of morbidity and mortality worldwide. Control of TB is lingering by the lack of diagnostic tests that are simple, rapid, yet accurate. Thus, smear-negative pulmonary TB often misses the diagnosis. The study evaluated the performance of GeneXpert MTB/RIF assay for the detection of Mycobacterium tuberculosis (MTB). METHODS: The study was carried out from June to December 2016 in Nepal Tuberculosis Center, Bhaktapur, Nepal. A total of 173 sputum samples were collected and processed by microscopy [Auramine-O staining and Ziehl-Neelsen (ZN) staining], followed by GeneXpert MTB/RIF assay and culture in Lowenstein-Jensen (LJ) medium. RESULTS: Of 173 sputum samples, 162 (93.6%) were smear-negative. Of 162 smear-negative sputum samples, 35 (21.6%) were confirmed to have MTB by culture, and 31 (19.1%) by GeneXpert MTB/RIF assay. Of 31 GeneXpert-positive samples, 25 (80.6%) were susceptible, 4 (12.9%) were resistant, and 2 (6.45%) were intermediate to rifampicin. The sensitivity, specificity, positive predictive value, and negative predictive value of GeneXpert MTB/RIF assay for smear-negative sputum samples were 74.3%, 96.6%, 86.7%, and 92%, respectively. The GeneXpert MTB/RIF has a substantial diagnostic agreement of 90.91% with culture (Cohen's Kappa coefficient = 0.73). CONCLUSION: The diagnostic performance of GeneXpert MTB/RIF assay was almost on par with culture, and thus can be relied upon for MTB detection in smear-negative sputum samples.

Factors affecting willingness to participate in vaccine clinical trials in an underdeveloped country: perspective from Nepal.

Due to the inherent complex nature of clinical trials, individual's willingness to participate and hence, enrollment in a clinical trial maybe challenging. When it comes to vaccine clinical trial in children, informed consent needs to be secured from the parents or legally acceptable representatives (LARs). Some of the factors which contribute to hesitancy in taking part in clinical trials are based on the level of education, living standards, part of the world they live, associated burden of disease, fear of different procedures in clinical trial, side effects, limited understanding, limited time, and mistrust with Investigational product. This study included 201 parents/LARs, who approached Kanti Children Hospital site in Kathmandu with the interest to get their children enrolled in a vaccine clinical trial with objectives of describing the reasons for agreeing or disagreeing to participate in the vaccine clinical trial, factors affecting decision making, and finding the major concerns of parents/LARs. The acceptance for the study vaccine was 136 (67.7%) whereas denial was 65 (32.3%). This study showed that age, education level, family structure, advice from family and friends, and medical guidance play important roles in willingness of parents to get their child enrolled in the trial. If a proper counseling is done, fear of blood sampling is not a big factor which is contrary to the belief among clinical researchers. Safety of vaccine, frequency of injections, and cost of vaccine were the main concerns of the parents, which need to be addressed extensively while planning for any clinical trial in children.

Safety and immunogenicity of the Vi-DT typhoid conjugate vaccine in healthy volunteers in Nepal: an observer-blind, active-controlled, randomised, non-inferiority, phase 3 trial.

BACKGROUND: Typhoid fever is an endemic disease in many low-income and middle-income countries. The 2018 WHO position paper recommends that countries should consider typhoid vaccination in high-risk groups and for outbreak control. To address the typhoid vaccine supply and demand gap, a typhoid Vi polysaccharide-diphtheria toxoid (Vi-DT) conjugate vaccine development effort was undertaken to achieve WHO prequalification and contribute to the global supply of typhoid conjugate vaccine. The main aim of this study was to show immune non-inferiority of the Vi-DT vaccine compared with the WHO prequalified Vi polysaccharide-tetanus toxoid (Vi-TT) conjugate vaccine (Typbar TCV; Bharat Biotech India, Hyderabad, India) in participants of various ages from an endemic country. METHODS: We did an observer-blind, active-controlled, randomised, non-inferiority, phase 3 trial at four hospitals in Kathmandu, Dhulikhel, Dharan, and Nepalgunj in Nepal. Eligible participants were healthy individuals aged 6 months to 45 years for whom informed consent was obtained, were willing to follow the study procedures and were available for the duration of the study. Patients with an acute or chronic illness that could interfere with interpretation of the study endpoints, or who were involved in any other clinical trial were excluded. Participants were randomly assigned (1:1:1:1) by block randomisation (block size of four and eight), stratified by age (6 months to <2 years, 2 years to <18 years, and 18 years to 45 years), into one of four groups (A-D). Participants in groups A-C received a single dose (25 µg; 0·5 mL) of Vi-DT test vaccine via intramuscular injection from one of three good manufacturing practice lots (group A received lot 1, group B received lot 2, and group C received lot 3), and those in group D received a single dose (25 µg; 0·5 mL) of the Vi-TT vaccine via intramuscular injection. All participants, site staff (except for those who administered the study vaccines), and those assessing the outcomes were masked to group assignment. The co-primary endpoints were: (1) non-inferiority of immunogenicity of the Vi-DT vaccine (pooled groups A-C) versus the Vi-TT vaccine (group D), measured by the anti-Vi IgG seroconversion rate at 4 weeks after vaccination; and (2) the lot-to-lot consistency of the Vi-DT vaccine, measured by immune equivalence of the anti-Vi IgG geometric mean titre (GMT) at 4 weeks after receipt of the three Vi-DT vaccine lots (lot 1 vs lot 2, lot 1 vs lot 3, and lot 2 vs lot 3). Non-inferiority of the Vi-DT vaccine compared with the Vi-TT vaccine was shown if the lower limit of the 97·5% CI for the difference between the seroconversion rates in Vi-DT vaccine groups A-C combined versus Vi-TT vaccine group D was above the predefined non-inferiority margin of -10%. Lot-to-lot immune equivalence was shown if the upper and lower bounds of the two-sided 99·17% CI around the GMT ratio for each pairwise lot-to-lot comparison was between 0·67 and 1·50, which is the predefined equivalence margin recommended by WHO. The co-primary immunogenicity endpoints were assessed in all randomised participants who had received their assigned vaccine and had completed at least one post-baseline immunogenicity assessment. Safety was descriptively summarised by group and age strata, and was assessed in all participants who had received one dose of the investigational vaccine. The trial is registered with ClinicalTrials.gov, NCT03933098. FINDINGS: Between Nov 20, 2019, and March 10, 2020, 1854 individuals were screened, of whom 1800 were enrolled and randomly assigned to groups A-D (450 participants in each group). 1786 (99·2%; 443 in group A, 450 in group B, 447 in group C, and 446 in group D) were included in the immunogenicity assessments at 4 weeks post vaccination, and all 1800 participants were included in the safety analysis. In the immunogenicity analysis, the anti-Vi-IgG seroconversion rate in all age strata was 99·33% (97·5% CI 98·61 to 99·68; 1331 of 1340 participants) in Vi-DT vaccine groups A-C and 98·88% (97·10 to 99·57; 441 of 446) in Vi-TT vaccine group D. The difference in seroconversion rates between Vi-DT vaccine groups A-C combined versus Vi-TT group D was 0·47% (97·5% CI -0·68 to 1·61), indicating non-inferiority of the Vi-DT vaccine. Anti-Vi-IgG GMT ratios at 4 weeks post-vaccination were 1·02 (99·17% CI 0·85 to 1·22) for lot 1 versus lot 2, 1·02 (0·85 to 1·23) for lot 1 versus lot 3, and 1·01 (0·84 to 1·21) for lot 2 versus lot 3, indicating lot-to-lot equivalence according to the predefined, WHO-recommended equivalence margin. The proportion of participants reporting adverse events was similar between Vi-DT vaccine groups A-C and Vi-TT vaccine group D; 260 (19·3%) of 1350 participants in Vi-DT vaccine groups A-C and 115 (25·6%) of 450 in Vi-TT vaccine group D reported solicited adverse events within 7 days after vaccination, and 208 (15·4%) in Vi-DT vaccine groups A-C and 76 (16·9%) in Vi-TT vaccine group D reported unsolicited adverse events within 4 weeks after vaccination. Seven serious adverse events (four [0·3%] participants in Vi-DT vaccine groups A-C and three [0·7%] in Vi-TT vaccine group D), including one death in the Vi-TT vaccine group, were reported during the 24-week follow-up period, none of which were considered related to the investigational product. INTERPRETATION: When administered as a single dose, the Vi-DT test vaccine was safe, immunogenic, and non-inferior to the Vi-TT vaccine at 4 weeks post vaccination. Equivalent immunogenicity of the three lots of Vi-DT vaccine was also shown, supporting the manufacturing process of this vaccine. Once prequalified by WHO, this vaccine could be an option for purchase by UN agencies. FUNDING: The Bill & Melinda Gates Foundation. TRANSLATION: For the Nepali translation of the abstract see Supplementary Materials section.

Prevalence of multidrug-resistance and bla VIM and bla IMP genes among gram-negative clinical isolates in tertiary care hospital, Kathmandu, Nepal.

BACKGROUND AND OBJECTIVES: Carbapenems have been the choice of antibiotics for the treatment of infections caused by multidrug-resistant bacteria. The main objective of this study was to determine the prevalence of carbapenemase (bla VIM and bla IMP ) producing isolates among Enterobacteriaceae, Pseudomonas aeruginosa, and Acinetobacter baumannii. MATERIALS AND METHODS: A total of 1,151 clinical samples were collected from the patients visiting Annapurna Neurological Institute and Allied Science and Annapurna Research Centre, Kathmandu, between June 2017 and January 2018. Antibiotic susceptibility testing (AST) was performed on the Enterobacteriaceae, P. aeruginosa and A. baumannii isolates using the Kirby-Bauer disk diffusion method. The modified Hodge test (MHT) was performed on the carbapenem-resistant isolates to confirm carbapenemase production. DNA was extracted and then screened for bla VIM and bla IMP genes by multiplex PCR. RESULTS: Of the total 1,151 clinical samples, 253 (22.0%) showed positive growth. Of them, 226 (89.3%) were identified as Enterobacteriaceae, P. aeruginosa, and A. baumannii. Among the 226 isolates, 106 (46.9%) were multidrug-resistant. Out of the 106, 97 (91.5%) isolates showed resistance to at least one of the carbapenem used. Among the 97 carbapenem-resistant isolates, 67 (69.1%) showed the modified Hodge test (MHT) positive results. bla VIM and bla IMP were detected in 40 and 38 isolates respectively using multiplex PCR assay. CONCLUSION: This study determined a high prevalence of MDR and carbapenem resistance among Enterobacteriaceae, P. aeruginosa, and A. baumannii as detected by the presence of bla VIM and bla IMP genes. This study recommends the use of rapid and advanced diagnostic tools along with conventional phenotypic detection methods in the clinical settings for early detection and management of drug-resistant pathogens to improve treatment strategies.

Prevalence of carbapenemase-producing Klebsiella pneumoniae at a tertiary care hospital in Kathmandu, Nepal.

AIM: Although carbapenem is the last-resort drug for treating drug-resistant Gram-negative bacterial infections, prevalence of carbapenem-resistant bacteria has substantially increased worldwide owing to irrational use of antibiotics particularly in developing countries like Nepal.  Therefore, this study was aimed to determine the prevalence of carbapenemase-producing K. pneumoniae and to detect the carbapenemase genes (blaNDM-2 and blaOXA-48) in at a tertiary care hospital in Nepal. MATERIALS AND METHODS: A hospital-based cross-sectional study was carried out from June 2018 to January 2019 at the Microbiology Laboratory of Annapurna Neurological Institute and Allied Sciences, Kathmandu, Nepal. Different clinical samples were collected and cultured in appropriate growth media. Biochemical tests were performed for the identification of K. pneumoniae. Antibiotic susceptibility testing (AST) was performed by the Kirby-Bauer disc diffusion method. The modified Hodge test (MHT) was performed to detect carbapenemase producers. The plasmid was extracted by the modified alkaline hydrolysis method. Carbapenemase-producing K. pneumoniae were further confirmed by detecting blaNDM-2 and blaOXA-48 genes by PCR using specific forward and reverse primers followed by gel electrophoresis. RESULTS: Out of the total 720 samples, 38.9% (280/720) were culture positive. K. pneumoniae was the most predominant isolate 31.4% (88/280). Of 88 K. pneumoniae isolates, 56.8% (50/88) were multi-drug resistant (MDR), and 51.1% (45/88) were MHT positive. Colistin showed the highest sensitivity (100%; 88/88), followed by tigecycline (86.4%; 76/88). blaNDM-2 and blaOXA-48 genes were detected in 24.4% (11/45) and 15.5% (7/45) of carbapenemase-producing K. pneumoniae isolates, respectively. CONCLUSION: The rate of MDR and carbapenemase production was high in the K. pneumoniae isolates. Colistin and tigecycline could be the drug of choice for the empirical treatments of MDR and carbapenemase-producing K. pneumoniae. Our study provides a better understanding of antibiotic resistance threat and enables physicians to select the most appropriate antibiotics.

Prevalence of multidrug-resistant and extended-spectrum beta-lactamase producing Gram-negative isolates from clinical samples in a tertiary care hospital of Nepal.

BACKGROUND: The existence of multidrug-resistant organisms, including extended-spectrum beta-lactamases (ESBLs), is on rise across the globe and is becoming a severe problem. Knowledge of the prevalence and antibiogram profile of such isolates is essential to develop an appropriate treatment methodology. This study aimed to study the prevalence of Gram-negative isolates exhibiting ESBL at a tertiary care hospital and study their antibiogram profile. METHODS: A cross-sectional study was conducted at Shahid Gangalal National Heart Centre, Kathmandu, Nepal, from June 2018 to November 2018. A total of 770 clinical samples were collected and identified using the conventional biochemical tests following the Clinical and Laboratory Standard Institute (CLSI) guidelines. Antimicrobial susceptibility testing (AST) was performed using the standardized Kirby-Bauer disk diffusion method. The screening test for ESBL producers was performed as recommended by the CLSI and the confirmatory test was performed phenotypically using the E-test. RESULTS: Out of the 92 isolates, 84 (91.3%) were multidrug-resistant, and 47 (51.1%) were found to be potential ESBL producers. Of these, 16 isolates were confirmed ESBL producers by the E-test. Escherichia coli and Klebsiella pneumoniae were the predominant isolates and were also the major ESBL producers. Besides polymyxin B (100% sensitive), meropenem and imipenem showed high efficacy against the ESBL producers. CONCLUSION: Multidrug resistance was very high; however, ESBL production was low. Polymyxin B and carbapenems are the choice of drugs against ESBL producers but should be used only as the last line drugs.

Challenges and opportunities in setting up a phase III vaccine clinical trial in resource limited settings: Experience from Nepal.

Clinical trials are complicated, time-consuming and costly. From the initial screening, informed consent and recruitment of the participants' to study completion, the sponsor must undertake a wide array of complex and closely monitored operations, complying with international standards for human subject research and local requirements. Conducting these studies in an underdeveloped country, with limited resources, infrastructure, and experience with regulated clinical trials adds to this complexity. The initial site selection, set up and preparatory activities for the clinical trial are crucial to minimizing the risks to both participants and to successful completion during the subsequent study execution.In this paper, we describe the experience and lessons learned of building clinical trial site capacity in terms of infrastructure and human resource development for a Phase III vaccine clinical trial. We believe that sharing the experience of setting up a clinical trial in a resource-limited country will enable other entities contemplating clinical research in these countries, to prepare and plan ahead, to minimize the impact of barriers, and to contribute to bringing more studies to the countries where people live with the burden of vaccine-preventable, poverty-associated diseases.

Prevalence of intestinal parasitosis and associated risk factors among school children of Saptari district, Nepal: a cross-sectional study.

BACKGROUND: Intestinal parasitosis, caused by both helminths and protozoans, are among the most prevalent infections, especially in developing countries. Enteric parasites continue to be a major cause of parasitic diseases which is the most common among street and school going children with poor sanitation. This cross-sectional study was carried out to determine the prevalence and potential risk factors of intestinal parasitosis among school going children of two schools of Saptari district of southern Nepal. Stool samples were collected in a clean, dry, screw-capped, and wide-mouthed plastic container, fixed with 10% formal-saline solution, and transported to the laboratory for further microscopic analysis by following concentration technique. RESULTS: Out of the 285 stool samples analysed, 94 (33%) were positive for the parasitosis. Presence of intestinal parasites was marginally more in rural school (44.6%) than in urban (30%) (P < 0.05). Giardia lamblia was highly prevalent (15.4%) followed by Entamoeba histolytica-like (7.7%), E. coli (7%), Ascaris lumbricoides (1.8%), and Hymenolepis nana (1.08%), respectively. Children of the age group 11-15 years were highly affected (44.2%) compared to younger age groups. The findings of intestinal parasitosis in the study population were statistically significant with family income, hand-washing habit, type of drinking water, and availability of a toilet facility at home (P < 0.05). Over 85% of infection was associated with parasitosis that indicated mainly waterborne infection rather than soil-borne helminths. CONCLUSIONS: Poor hygiene measures and farming occupation are identified as major risk factors of parasitic infections, so sanitation especially focusing on safe drinking water along with multi intervention strategies must be emphasized in the Saptari district of Nepal to reduce the burden of parasitic diseases in school children.

Distribution and pyrethroid resistance status of Aedes aegypti and Aedes albopictus populations and possible phylogenetic reasons for the recent invasion of Aedes aegypti in Nepal.

BACKGROUND: When the first systematic list of mosquitoes in Nepal was reported in 1990, there was no description of Aedes aegypti (L.), while Aedes albopictus (Skuse) has been included in the Stegomyia subgroup since the 1950s. The first record of Ae. aegypti in Nepal was reported in 2009, suggesting some coincidence between the invasion of this species and the first record of dengue fever in Nepal in 2006. RESULTS: We performed a field survey of the distribution and insecticide susceptibility of Ae. aegypti and Ae. albopictus in Nepal in 2017 and 2018. Mosquito larvae were collected from used tires located along the streets of Kathmandu, Bharatpur and Pokhara, and a simplified bioassay was used to assess the susceptibility of the larvae to pyrethroid insecticides using d-allethrin. The presence or absence of point mutations in the voltage-gated sodium channel was also detected by direct sequencing. V1016G was detected at a high frequency and a strong correlation was observed between the frequencies of V1016G and susceptibility indices in Ae. aegypti populations. F1534C was also detected at a relatively low frequency. In Ae. albopictus populations, susceptibilities to d-allethrin were high and no point mutations were detected. Analysis of the cytochrome c oxidase subunit 1 (cox1) gene was performed for assessing genetic diversity and the existence of two strains were identified in Ae. aegypti populations. One consisted of 9 globally-distributed haplotypes while the other was derived from an African haplotype. CONCLUSIONS: The high pyrethroid resistance, high V1016G frequency, and relatively low quantity of insecticides used to control dengue vectors in Nepal may have resulted in only weak selection pressure favoring insecticide resistance and could support the hypothesis that this species has recently been introduced from neighboring Asian countries where pyrethroid resistance is relatively widespread.

Intestinal Parasitic Infections among Prison Inmates in Kathmandu Nepal.

BACKGROUND: Prison inmates are at high risk of intestinal parasitic infections. Thus, we studied intestinal parasitic infections among inmates of the Central Jail, Kathmandu, Nepal. METHODS: Morning stool samples from 400 inmates (M=282 and F=118), were collected in a clean, dry and wide-mouthed plastic container. The samples were transported to the research laboratory of Shi-Gan International College of Science and Technology, and were fixed using 10% formal saline. Then, samples were processed by formal ether sedimentation concentration technique and were observed microscopically by direct-smear technique. RESULTS: Six percent (24/400) samples were positive for intestinal parasites, with a gender ratio (M:F) 1.7:1. But, co-parasitism was not observed. Intestinal parasitic infections were higher among 21-40 years age-group, 3.5% (14/262). Similarly, intestinal parasitic infections were higher among 'Dalits' ethnic group, 21.1% (4/19). As compared to helminths, more protozoans, 62.5% (15/24), were observed. Giardia lamblia, 41.67% (10/24), was the most common protozoans while Trichuris trichiura, 25.0% (6/24), was the most common helminths. CONCLUSIONS: Intestinal parasitic infections were lower among the inmates of Central jail, but such conditions cannot be presumed in other peripheral settings. Pure and safe drinking water supply and the effective deworming campaign can further reduce this figure at this setting and peripheral jails across the country.

Epidemiology of urinary tract infection and antimicrobial resistance in a pediatric hospital in Nepal.

BACKGROUND: Urinary tract infection is an infection affecting infants and children. The aim of this study was to determine the etiology of urinary tract infection along with their antimicrobial resistance. METHODS: This cross-sectional study was conducted from June 2015 to January 2016 at Siddhi Memorial Hospital, Bhaktapur, Nepal. Urine samples were first cultured on cystine lactose electrolyte deficient agar and blood agar by semi-quantitative technique, and then incubated aerobically for 18-24 h at 37 °C. The identified bacterial isolates were tested for antimicrobial susceptibility by Kirby Bauer disc diffusion technique. RESULTS: Of 1599 urine samples, 12.3% samples showed significant bacterial growth. E. coli (58.7%) was the most common pathogen, followed by Klebsiella pneumoniae (22.5%). Most of the isolates were resistant to ampicillin and co-trimoxazole, while least were resistant to amikacin and nitrofurantoin. Higher multi-drug resistance (61.9%) was observed among isolates. CONCLUSIONS: E. coli and Klebsiella spp. were predominant cause of pediatric urinary tract infection in children. Higher susceptibility observed against aminoglycosides and nitrofurans make these drugs suitable in emergency.

Intestinal parasitic infections among public and private schoolchildren of Kathmandu, Nepal: prevalence and associated risk factors.

OBJECTIVE: Intestinal parasitic infections (IPIs) are a major cause of morbidity among children in developing countries. Investigation about the etiological agents and socio-ecological pattern of the infection would help to design better preventive strategy. The previous studies reported high prevalence of IPIs among schoolchildren of Nepal. Though these data may be essential for the policymakers and researchers, in Kathmandu, the capital of Nepal it remains unexplored whether the types of school and socioeconomic status affect the IPIs or not. The present study is an extension of previous works to investigate causative agents and associated risk factors. We examined 508 stool samples of schoolchildren from two schools by formal-ether concentration technique and analyzed the data based on school types. RESULTS: The overall IPIs rate was 19.9% (n = 101) with the dominance of protozoans (78.4%) over helminths (21.6%). Giardia duodenalis (32.7%) and Ascaris lumbricoides (21.8%) were the most commonly detected protozoan and helminth species respectively. Prevalence of IPIs was higher among children from public school (26.1%) than private school (12.1%). Higher infection rates were found among farmer's children (29.0%) and Dalit children (36.2%). These findings reveal the different prevalence of IPIs among public and private schoolchildren and suggest the need of effective preventive measures.

Diarrheal disease outbreak in Gaidatar village of Rautahat District, Nepal.

OBJECTIVE: Diarrheal diseases, including cholera, remain a major public health concern in developing countries like Nepal. This study investigated a diarrheal outbreak that affected over 1500 people in Gaidatar village of Rautahat district in central Nepal and sought to identify the source and causation of the disease. Stool samples were collected from individuals with acute diarrheal illness (n = 16) and healthy non-diarrheal children (n = 39), along with samples from local drinking water sources (n = 8) and their sewage system (n = 10). None of the individuals were sampled multiple times. Diarrheic stool and sewage samples were analysed for the presence of Vibrio cholerae, while coliforms were tested in drinking water samples following standard microbiological protocols. Enteric parasites were tested in both diarrheic and non-diarrheic stool samples. RESULTS: Vibrio cholerae O1 Ogawa serotype was isolated in 18.7% of the diarrheic stool and 20.0% of the sewage. Coliforms were found in all drinking water samples, with 87.5% testing positive for fecal coliform. Additionally, 43.6% of the stool samples (n = 55) had at least one of the intestinal parasites tested, primarily Giardia lamblia (21.8%). However, almost all parasites were found in non-diarrheal stool. Taken together, our results provide evidence that the diarrheal outbreak was associated with V. cholerae O1 Ogawa serotype, possibly transmitted through the drinking water sources contaminated with fecal matters from their sewage (drainage) system. These findings warrant regular surveillance of drinking water sources to help prevent future outbreaks.

Thrombocytosis as a Predictor of Serious Bacterial Infection in Febrile Infants.

BACKGROUND: Most of the febrile infants <90 days old will have no more than a mild viral infection but there is a substantial minority that will be diagnosed as having serious bacterial infection at a reported prevalence of 10-14%. A simple, readily available, inexpensive diagnostic marker that yields results quickly and also accurately identifies bacterial infections in febrile infants would be of great value in management of these infants. This study aims to assess the role of thrombocytosis in predicting serious bacterial infection in young febrile infants beyond neonatal period. METHODS: A hospital based cross-sectional observational study was conducted from May 2016 to April 2017 on 76 febrile infants of age group 29-90 days in Kanti Children's Hospital. RESULTS: The incidence of serious bacterial infection was found 43 (56.6%). Thrombocytosis, elevated C-reactive protein and pyuria were significantly higher in serious bacterial infection cases (p value <0.05). Thrombocytosis alone had the sensitivity of only 53.5%, but had specificity of 90.9%. Elevated C-reactive protein had the best sensitivity (81.4%). Combination of leukocytosis, elevated C-reactive protein, pyuria and thrombocytosis had better sensitivity (93.0%) than these parameters alone. The overall ability of platelet count to identify infants with SBI was only moderate (AUC: 0.722). Elevated C-reactive protein was found to have better ability to identify infants with serious bacterial infection (AUC: 0.846). CONCLUSIONS: Thrombocytosis is a common finding in young infants diagnosed with serious bacterial infection. It has however, moderate ability in identifying infants with serious bacterial infection. Combining thrombocytosis with elevated C-reactive protein, leukocytosis and pyuria has better sensitivity in diagnosing serious bacterial infection than these individual parameters alone. Hence, combining these parameters may help in early prediction of febrile young infants at risk of serious bacterial infection.