The Identification of Enteric Fever-Specific Antigens for Population-Based Serosurveillance.

BACKGROUND: Enteric fever, caused by Salmonella enterica serovars Typhi and Paratyphi A, is a major public health problem in low- and middle-income countries. Moderate sensitivity and scalability of current methods likely underestimate enteric fever burden. Determining the serological responses to organism-specific antigens may improve incidence measures. METHODS: Plasma samples were collected from blood culture-confirmed enteric fever patients, blood culture-negative febrile patients over the course of 3 months, and afebrile community controls. A panel of 17 Salmonella Typhi and Paratyphi A antigens was purified and used to determine antigen-specific antibody responses by indirect ELISAs. RESULTS: The antigen-specific longitudinal antibody responses were comparable between enteric fever patients, patients with blood culture-negative febrile controls, and afebrile community controls for most antigens. However, we found that IgG responses against STY1479 (YncE), STY1886 (CdtB), STY1498 (HlyE), and the serovar-specific O2 and O9 antigens were greatly elevated over a 3-month follow up period in S. Typhi/S. Paratyphi A patients compared to controls, suggesting seroconversion. CONCLUSIONS: We identified a set of antigens as good candidates to demonstrate enteric fever exposure. These targets can be used in combination to develop more sensitive and scalable approaches to enteric fever surveillance and generate invaluable epidemiological data for informing vaccine policies. CLINICAL TRIAL REGISTRATION: ISRCTN63006567.

Phase 3 Efficacy Analysis of a Typhoid Conjugate Vaccine Trial in Nepal.

BACKGROUND: Salmonella Typhi is a major cause of fever in children in low- and middle-income countries. A typhoid conjugate vaccine (TCV) that was recently prequalified by the World Health Organization was shown to be efficacious in a human challenge model, but data from efficacy trials in areas where typhoid is endemic are lacking. METHODS: In this phase 3, randomized, controlled trial in Lalitpur, Nepal, in which both the participants and observers were unaware of the trial-group assignments, we randomly assigned children who were between 9 months and 16 years of age, in a 1:1 ratio, to receive either a TCV or a capsular group A meningococcal conjugate vaccine (MenA) as a control. The primary outcome was typhoid fever confirmed by blood culture. We present the prespecified analysis of the primary and main secondary outcomes (including an immunogenicity subgroup); the 2-year trial follow-up is ongoing. RESULTS: A total of 10,005 participants received the TCV and 10,014 received the MenA vaccine. Blood culture-confirmed typhoid fever occurred in 7 participants who received TCV (79 cases per 100,000 person-years) and in 38 who received MenA vaccine (428 cases per 100,000 person-years) (vaccine efficacy, 81.6%; 95% confidence interval, 58.8 to 91.8; P<0.001). A total of 132 serious adverse events (61 in the TCV group and 71 in the MenA vaccine group) occurred in the first 6 months, and 1 event (pyrexia) was identified as being vaccine-related; the participant remained unaware of the trial-group assignment. Similar rates of adverse events were noted in the two trial groups; fever developed in 5.0% of participants in the TCV group and 5.4% in the MenA vaccine group in the first week after vaccination. In the immunogenicity subgroup, seroconversion (a Vi IgG level that at least quadrupled 28 days after vaccination) was 99% in the TCV group (677 of 683 participants) and 2% in the MenA vaccine group (8 of 380 participants). CONCLUSIONS: A single dose of TCV was immunogenic and effective in reducing S. Typhi bacteremia in children 9 months to 16 years of age. (Funded by the Bill and Melinda Gates Foundation; Current Controlled Trials number, ISRCTN43385161.).

Gallbladder carriage generates genetic variation and genome degradation in Salmonella Typhi.

Despite recent advances in typhoid fever control, asymptomatic carriage of Salmonella Typhi in the gallbladder remains poorly understood. Aiming to understand if S. Typhi becomes genetically adapted for long-term colonisation in the gallbladder, we performed whole genome sequencing on a collection of S. Typhi isolated from the gallbladders of typhoid carriers. These sequences were compared to contemporaneously sampled sequences from organisms isolated from the blood of acute patients within the same population. We found that S. Typhi carriage was not restricted to any particular genotype or conformation of antimicrobial resistance genes, but was largely reflective of S. Typhi circulating in the general population. However, gallbladder isolates showed a higher genetic variability than acute isolates, with median pairwise SNP distances of 21 and 13 SNPs (p = 2.8x10-9), respectively. Within gallbladder isolates of the predominant H58 genotype, variation was associated with a higher prevalence of nonsense mutations. Notably, gallbladder isolates displayed a higher frequency of non-synonymous mutations in genes encoding hypothetical proteins, membrane lipoproteins, transport/binding proteins, surface antigens, and carbohydrate degradation. Specifically, we identified several gallbladder-specific non-synonymous mutations involved in LPS synthesis and modification, with some isolates lacking the Vi capsular polysaccharide vaccine target due to the 134Kb deletion of SPI-7. S. Typhi is under strong selective pressure in the human gallbladder, which may be reflected phylogenetically by long terminal branches that may distinguish organisms from chronic and acute infections. Our work shows that selective pressures asserted by the hostile environment of the human gallbladder generate new antigenic variants and raises questions regarding the role of carriage in the epidemiology of typhoid fever.

Very long O-antigen chains of Salmonella Paratyphi A inhibit inflammasome activation and pyroptotic cell death.

Salmonella Paratyphi A (SPtA) remains one of the leading causes of enteric (typhoid) fever. Yet, despite the recent increased rate of isolation from patients in Asia, our understanding of its pathogenesis is incomplete. Here we investigated inflammasome activation in human macrophages infected with SPtA. We found that SPtA induces GSDMD-mediated pyroptosis via activation of caspase-1, caspase-4 and caspase-8. Although we observed no cell death in the absence of a functional Salmonella pathogenicity island-1 (SPI-1) injectisome, HilA-mediated overexpression of the SPI-1 regulon enhances pyroptosis. SPtA expresses FepE, an LPS O-antigen length regulator, which induces the production of very long O-antigen chains. Using a ΔfepE mutant we established that the very long O-antigen chains interfere with bacterial interactions with epithelial cells and impair inflammasome-mediated macrophage cell death. Salmonella Typhimurium (STm) serovar has a lower FepE expression than SPtA, and triggers higher pyroptosis, conversely, increasing FepE expression in STm reduced pyroptosis. These results suggest that differential expression of FepE results in serovar-specific inflammasome modulation, which mirrors the pro- and anti-inflammatory strategies employed by STm and SPtA, respectively. Our studies point towards distinct mechanisms of virulence of SPtA, whereby it attenuates inflammasome-mediated detection through the elaboration of very long LPS O-polysaccharides.

Tebipenem as an oral alternative for the treatment of typhoid caused by XDR Salmonella Typhi.

BACKGROUND: Antimicrobial therapy is essential for the treatment of enteric fever, the infection caused by Salmonella serovars Typhi and Paratyphi A. However, an increase in resistance to key antimicrobials and the emergence of MDR and XDR in Salmonella Typhi poses a major threat for efficacious outpatient treatments. OBJECTIVES: We recently identified tebipenem, an oral carbapenem licensed for use for respiratory tract infections in Japan, as a potential alternative treatment for MDR/XDR Shigella spp. Here, we aimed to test the in vitro antibacterial efficacy of this drug against MDR and XDR typhoidal Salmonella. METHODS: We determined the in vitro activity of tebipenem in time-kill assays against a collection of non-XDR and XDR Salmonella Typhi and Salmonella Paratyphi A (non-XDR) isolated in Nepal and Bangladesh. We also tested the efficacy of tebipenem in combination with other antimicrobials. RESULTS: We found that both XDR and non-XDR Salmonella Typhi and Salmonella Paratyphi A are susceptible to tebipenem, exhibiting low MICs, and were killed within 8-24 h at 2-4×MIC. Additionally, tebipenem demonstrated synergy with two other antimicrobials and could efficiently induce bacterial killing. CONCLUSIONS: Salmonella Paratyphi A and XDR Salmonella Typhi display in vitro susceptibility to the oral carbapenem tebipenem, while synergistic activity with other antimicrobials may limit the emergence of resistance. The broad-spectrum activity of this drug against MDR/XDR organisms renders tebipenem a good candidate for clinical trials.

Risk factors for the development of neonatal sepsis in a neonatal intensive care unit of a tertiary care hospital of Nepal.

BACKGROUND: Sepsis is an overwhelming and life-threatening response to bacteria in bloodstream and a major cause of neonatal morbidity and mortality. Understanding the etiology and potential risk factors for neonatal sepsis is urgently required, particularly in low-income countries where burden of infection is high and its epidemiology is poorly understood. METHODS: A prospective observational cohort study was conducted between April 2016 and October 2017 in a level three NICU at a tertiary care hospital in Nepal to determine the bacterial etiology and potential risk factors for neonatal sepsis. RESULTS: Among 142 NICU admitted neonates, 15% (21/142) and 32% (46/142) developed blood culture-positive and -negative neonatal sepsis respectively. Klebsiella pneumoniae (34%, 15/44) and Enterobacter spp. (25%, 11/44) were the most common isolates. The antimicrobial resistance of isolates to ampicillin (100%, 43/43), cefotaxime (74%, 31/42) and ampicillin-sulbactam (55%, 21/38) were the highest. BlaTEM (53%, 18/34) and blaKPC (46%, 13/28) were the commonest ESBL and carbapenemase genes respectively. In univariate logistic regression, the odds of sepsis increased with each additional day of use of invasive procedures such as mechanical ventilation (OR 1.086, 95% CI 1.008-1.170), umbilical artery catheter (OR 1.375, 95% CI 1.049-1.803), intravenous cannula (OR 1.140, 95% CI 1.062-1.225); blood transfusion events (OR 3.084, 95% CI 1.407-6.760); NICU stay (OR 1.109, 95% CI 1.040-1.182) and failure to breast feed (OR 1.130, 95% CI 1.060-1.205). Sepsis odds also increased with leukopenia (OR 1.790, 95% CI 1.04-3.082), increase in C-reactive protein (OR 1.028, 95% CI 1.016-1.040) and decrease in platelets count (OR 0.992, 95% CI 0.989-0.994). In multivariate analysis, increase in IV cannula insertion days (OR 1.147, 95% CI 1.039-1.267) and CRP level (OR 1.028, 95% CI 1.008-1.049) increased the odds of sepsis. CONCLUSIONS: Our study indicated various nosocomial risk factors and underscored the need to improve local infection control measures so as to reduce the existing burden of sepsis. We have highlighted certain sepsis associated laboratory parameters along with identification of antimicrobial resistance genes, which can guide for early and better therapeutic management of sepsis. These findings could be extrapolated to other low-income settings within the region.

A high prevalence of multi-drug resistant Gram-negative bacilli in a Nepali tertiary care hospital and associated widespread distribution of Extended-Spectrum Beta-Lactamase (ESBL) and carbapenemase-encoding genes.

BACKGROUND: Multi-drug resistance (MDR) and extensive-drug resistance (XDR) associated with extended-spectrum beta-lactamases (ESBLs) and carbapenemases in Gram-negative bacteria are global public health concerns. Data on circulating antimicrobial resistance (AMR) genes in Gram-negative bacteria and their correlation with MDR and ESBL phenotypes from Nepal is scarce. METHODS: A retrospective study was performed investigating the distribution of ESBL and carbapenemase genes and their potential association with ESBL and MDR phenotypes in E. coli, Klebsiella spp., Enterobacter spp. and Acinetobacter spp. isolated in a major tertiary hospital in Kathmandu, Nepal, between 2012 and 2018. RESULTS: During this period, the hospital isolated 719 E. coli, 532 Klebsiella spp., 520 Enterobacter spp. and 382 Acinetobacter spp.; 1955/2153 (90.1%) of isolates were MDR and half (1080/2153) were ESBL producers. Upon PCR amplification, blaTEM (1281/1771; 72%), blaCTXM-1 (930/1771; 53%) and blaCTXM-8 (419/1771; 24%) were the most prevalent ESBL genes in the enteric bacilli. BlaOXA and blaOXA-51 were the most common blaOXA family genes in the enteric bacilli (918/1771; 25%) and Acinetobacter spp. (218/382; 57%) respectively. Sixteen percent (342/2153) of all isolates and 20% (357/1771) of enteric bacilli harboured blaNDM-1 and blaKPC carbapenemase genes respectively. Of enteric bacilli, Enterobacter spp. was the most frequently positive for blaKPC gene (201/337; 60%). The presence of each blaCTX-M and blaOXA were significantly associated with non-susceptibility to third generation cephalosporins (OR 14.7, p < 0.001 and OR 2.3, p < 0.05, respectively).The presence of each blaTEM, blaCTXM and blaOXA family genes were significantly associated with ESBL positivity (OR 2.96, p < 0.001; OR 14.2, p < 0.001 and OR 1.3, p < 0.05 respectively) and being MDR (OR 1.96, p < 0.001; OR 5.9, p < 0.001 and OR 2.3, p < 0.001 respectively). CONCLUSIONS: This study documents an alarming level of AMR with high prevalence of MDR ESBL- and carbapenemase-positive ESKAPE microorganisms in our clinical setting. These data suggest a scenario where the clinical management of infected patients is increasingly difficult and requires the use of last-resort antimicrobials, which in turn is likely to intensify the magnitude of global AMR crisis.

Assessing the Impact of a Vi-polysaccharide Conjugate Vaccine in Preventing Typhoid Infections Among Nepalese Children: A Protocol for a Phase III, Randomized Control Trial.

BACKGROUND: Enteric fever is estimated to affect 11-20 million people worldwide each year. Morbidity and mortality from enteric fever primarily occur in lower-income countries, with children under 5 years of age experiencing a significant portion of the burden. Over the last few decades, the control of enteric fever has focused primarily on improved water and sanitation, with the available vaccines unsuitable for children and primarily used by travelers. A new typhoid conjugate vaccine (Vi-TCV), prequalified by the World Health Organization (WHO) and highly immunogenic in children under 5, has the potential to reduce the typhoid burden in endemic countries. METHODS: This study is a double-blinded, randomized, controlled trial with a 2-year follow-up to assess the protective impact of the Vi-TCV vaccine, compared with a control vaccine, in children from 9 months to 16 years of age. The primary outcome of interest is the reduction in the number of culture-confirmed typhoid cases attributable to Vi-TCV. Approximately 20 000 children living in the Lalitpur district, within the Kathmandu valley, will be enrolled in the study and followed to measure both safety and efficacy data, which will include adverse events, hospitalizations, antibiotic use, and fever frequency. RESULTS: Both the intervention and control vaccines are WHO prequalified vaccines, which provide a health benefit to all participants. Children have been chosen to participate because they bear a substantial burden of both typhoid morbidity and mortality in this population. The results of this study will be disseminated through a series of published articles. The findings will also be made available to the participants and the broader community, as well as local stakeholders, within Nepal. CONCLUSIONS: This is the first large-scale, individually randomized, controlled trial of Vi-TCV in children in an endemic setting, and will provide new data on Vi-TCV field efficacy. With Vi-TCV introduction being considered in high-burden countries, this study will support important policy decisions. CLINICAL TRIALS REGISTRATION: The trial is registered on the ISRCTN registry (for details, see https://doi.org/10.1186/ISRCTN43385161; registry number: ISRCTN 43385161).

Logistics of Implementing a Large-scale Typhoid Vaccine Trial in Kathmandu, Nepal.

Typhoid fever is estimated to affect over 20 million people per year worldwide, with infants, children, and adolescents in south-central and southeast Asia experiencing the greatest burden of disease. The Typhoid Vaccine Acceleration Consortium (TyVAC) aims to support the introduction of typhoid conjugate vaccines into Gavi-eligible countries in an effort to reduce morbidity and mortality from typhoid. TyVAC-Nepal is a large-scale, participant- and observer-blind, individually randomized, controlled trial evaluating the efficacy of a newly developed typhoid conjugate vaccine in an urban setting in Nepal. In order to effectively deliver the trial, a number of key elements required meticulous planning. Public engagement strategies were considered early, and involved the implementation of a tiered approach. Approximately 300 staff were employed and trained in order to achieve the mass vaccination of 20 000 children aged 9 months to ≤16 years old over a 4-month period. There were 19 vaccination clinics established across the Lalitpur metropolitan city in the Kathmandu valley. Participants will be followed for 2 years post-vaccination to measure the rate reduction of blood culture-confirmed typhoid fever in the vaccination arm as compared to the control arm. The experience of conducting this large-scale vaccine trial suggests that comprehensive planning, continuous monitoring, and an ability to adapt plans in response to feedback are key.

Treatment Response in Enteric Fever in an Era of Increasing Antimicrobial Resistance: An Individual Patient Data Analysis of 2092 Participants Enrolled into 4 Randomized, Controlled Trials in Nepal.

BACKGROUND.: Enteric fever, caused by Salmonella Typhi and Salmonella Paratyphi A, is the leading cause of bacterial febrile disease in South Asia. METHODS.: Individual data from 2092 patients with enteric fever randomized into 4 trials in Kathmandu, Nepal, were pooled. All trials compared gatifloxacin with 1 of the following comparator drugs: cefixime, chloramphenicol, ofloxacin, or ceftriaxone. Treatment outcomes were evaluated according to antimicrobial if S. Typhi/Paratyphi were isolated from blood. We additionally investigated the impact of changing bacterial antimicrobial susceptibility on outcome. RESULTS.: Overall, 855 (41%) patients had either S. Typhi (n = 581, 28%) or S. Paratyphi A (n = 274, 13%) cultured from blood. There were 139 (6.6%) treatment failures with 1 death. Except for the last trial with ceftriaxone, the fluoroquinolone gatifloxacin was associated with equivalent or better fever clearance times and lower treatment failure rates in comparison to all other antimicrobials. However, we additionally found that the minimum inhibitory concentrations (MICs) against fluoroquinolones have risen significantly since 2005 and were associated with increasing fever clearance times. Notably, all organisms were susceptible to ceftriaxone throughout the study period (2005-2014), and the MICs against azithromycin declined, confirming the utility of these alternative drugs for enteric fever treatment. CONCLUSION.: The World Health Organization and local government health ministries in South Asia still recommend fluoroquinolones for enteric fever. This policy should change based on the evidence provided here. Rapid diagnostics are urgently required given the large numbers of suspected enteric fever patients with a negative culture.

Clinically and microbiologically derived azithromycin susceptibility breakpoints for Salmonella enterica serovars Typhi and Paratyphi A.

Azithromycin is an effective treatment for uncomplicated infections with Salmonella enterica serovar Typhi and serovar Paratyphi A (enteric fever), but there are no clinically validated MIC and disk zone size interpretative guidelines. We studied individual patient data from three randomized controlled trials (RCTs) of antimicrobial treatment in enteric fever in Vietnam, with azithromycin used in one treatment arm, to determine the relationship between azithromycin treatment response and the azithromycin MIC of the infecting isolate. We additionally compared the azithromycin MIC and the disk susceptibility zone sizes of 1,640 S. Typhi and S. Paratyphi A clinical isolates collected from seven Asian countries. In the RCTs, 214 patients who were treated with azithromycin at a dose of 10 to 20 mg/ml for 5 to 7 days were analyzed. Treatment was successful in 195 of 214 (91%) patients, with no significant difference in response (cure rate, fever clearance time) with MICs ranging from 4 to 16 µg/ml. The proportion of Asian enteric fever isolates with an MIC of ≤ 16 µg/ml was 1,452/1,460 (99.5%; 95% confidence interval [CI], 98.9 to 99.7) for S. Typhi and 207/240 (86.3%; 95% CI, 81.2 to 90.3) (P < 0.001) for S. Paratyphi A. A zone size of ≥ 13 mm to a 5-µg azithromycin disk identified S. Typhi isolates with an MIC of ≤ 16 µg/ml with a sensitivity of 99.7%. An azithromycin MIC of ≤ 16 µg/ml or disk inhibition zone size of ≥ 13 mm enabled the detection of susceptible S. Typhi isolates that respond to azithromycin treatment. Further work is needed to define the response to treatment in S. Typhi isolates with an azithromycin MIC of >16 µg/ml and to determine MIC and disk breakpoints for S. Paratyphi A.

Phenotypic variation in the lipopolysaccharide O-antigen of Salmonella Paratyphi A and implications for vaccine development.

Enteric fever remains a major public health problem in South and Southeast Asia. The recent roll-out of the typhoid conjugate vaccine protecting against S. Typhi exhibits great promise for disease reduction in high burden areas. However, some endemic regions remain vulnerable to S. Paratyphi A due to a lack of licensed vaccines and inadequate WASH. Several developmental S. Paratyphi A vaccines exploit O-antigen as the target antigen. It has been hypothesised that O-antigen is under selective and environmental pressure, with mutations in O-antigen biosynthesis genes being reported, but their phenotypic effects are unknown. Here, we aimed to evaluate O-antigen variation in S. Paratyphi A originating from Nepal, and the potential effect of this variation on antibody binding. O-antigen variation was determined by measuring LPS laddering shift following electrophoresis; this analysis was complemented with genomic characterisation of the O-antigen region. We found structural O-antigen variation in <10 % of S. Paratyphi A organisms, but a direct underlying genetic cause could not be identified. High-content imaging was performed to determine antibody binding by commercial O2 monoclonal (mAb) and polyclonal antibodies, as well as polyclonal sera from convalescent patients naturally infected with S. Paratyphi A. Commercial mAbs detected only a fraction of an apparently "clonal" bacterial population, suggesting phase variation and nonuniform O-antigen composition. Notably, and despite visible subpopulation clusters, O-antigen structural changes did not appear to affect the binding ability of polyclonal human antibody considerably, which led to no obvious differences in the functionality of antibodies targeting organisms with different O-antigen conformations. Although these results need to be confirmed in organisms from alternative endemic areas, they are encouraging the use of O-antigen as the target antigen in S. Paratyphi A vaccines.

A retrospective investigation of the population structure and geospatial distribution of Salmonella Paratyphi A in Kathmandu, Nepal.

Salmonella Paratyphi A, one of the major etiologic agents of enteric fever, has increased in prevalence in recent decades in certain endemic regions in comparison to S. Typhi, the most prevalent cause of enteric fever. Despite this increase, data on the prevalence and molecular epidemiology of S. Paratyphi A remain generally scarce. Here, we analysed the whole genome sequences of 216 S. Paratyphi A isolates originating from Kathmandu, Nepal between 2005 and 2014, of which 200 were from patients with acute enteric fever and 16 from the gallbladder of people with suspected chronic carriage. By exploiting the recently developed genotyping framework for S. Paratyphi A (Paratype), we identified several genotypes circulating in Kathmandu. Notably, we observed an unusual clonal expansion of genotype 2.4.3 over a four-year period that spread geographically and systematically replaced other genotypes. This rapid genotype replacement is hypothesised to have been driven by both reduced susceptibility to fluoroquinolones and genetic changes to virulence factors, such as functional and structural genes encoding the type 3 secretion systems. Finally, we show that person-to-person is likely the most common mode of transmission and chronic carriers seem to play a limited role in maintaining disease circulation.

Prevalence and genetic analysis of phenotypically Vi- negative Salmonella typhi isolates in children from Kathmandu, Nepal.

The Vi capsular polysaccharide (ViPS) protects Salmonella enterica subspecies enterica serotype Typhi (S.Typhi) in vivo by multiple mechanisms. Recent microbiological reports from typhoid endemic countries suggest that acapsulate S.Typhi may occur in nature and contribute to clinical typhoid fever that is indistinguishable from disease caused by capsulate strains. The prevalence and genetic basis of ViPS-negative S.Typhi isolates in children from Kathmandu, Nepal, were tested in 68 isolates. Although 5.9% of isolates tested negative for capsular expression by slide agglutination tests, a novel multiplex PCR assay and individual PCR analyses demonstrated the presence of all 14 genes responsible for the synthesis, transportation and regulation of the ViPS. These data suggest that phenotypically acapsulate S.Typhi may not have a genetic basis for the same.

Perspectives of pharmacy employees on an inappropriate use of antimicrobials in Kathmandu, Nepal.

BACKGROUND: Unregulated antimicrobial use is common in both hospital and community settings of low- and middle-income countries (LMICs). However, discrete data regarding the use/misuse of antimicrobials at pharmacies in LMICs are limited. This study was conducted to understand knowledge, attitude, and practice of pharmacy employees on antimicrobial dispensing in Nepal. METHODS: We conducted a cross-sectional survey using a structured questionnaire on 801 pharmacy employees working in community and hospital pharmacies located in Lalitpur metropolitan city (LMC) of Kathmandu, Nepal between April 2017 and March 2019. RESULTS: A majority (92%) of respondents agreed that demand for non-prescription antimicrobials was common. Asking for prescription before dispensing was ranked as the first preference by majority (69%) of participants. Suspected respiratory tract infection was the most common reason demanding for non-prescription antimicrobials with the highest mean rank of 1.5. Azithromycin was the most commonly prescribed and sold antimicrobial, as reported by 46% and 48% of participants respectively. A majority (87%) of respondents agreed on antimicrobial resistance (AMR) to be a global public health threat; and misuse/overuse of antimicrobials was perceived as the most common cause of AMR with a mean rank of 1.93. CONCLUSION: Our study revealed that unfounded dispensing and use of antimicrobials is prevalent among pharmacies in Kathmandu, Nepal. This over reliance on antimicrobials, notably azithromycin, may escalate burden of AMR. We identified several drivers of inappropriate antimicrobial dispensing practice in pharmacies, which will aid public health authorities in addressing these issues. Further studies considering role of other stakeholders, such as doctors, veterinarians, general public, and policy makers are required to obtain a more holistic perspectives on practices of antimicrobial use so to curb the extant AMR crisis.

Direct inference and control of genetic population structure from RNA sequencing data.

RNAseq data can be used to infer genetic variants, yet its use for estimating genetic population structure remains underexplored. Here, we construct a freely available computational tool (RGStraP) to estimate RNAseq-based genetic principal components (RG-PCs) and assess whether RG-PCs can be used to control for population structure in gene expression analyses. Using whole blood samples from understudied Nepalese populations and the Geuvadis study, we show that RG-PCs had comparable results to paired array-based genotypes, with high genotype concordance and high correlations of genetic principal components, capturing subpopulations within the dataset. In differential gene expression analysis, we found that inclusion of RG-PCs as covariates reduced test statistic inflation. Our paper demonstrates that genetic population structure can be directly inferred and controlled for using RNAseq data, thus facilitating improved retrospective and future analyses of transcriptomic data.

Highly resistant Salmonella enterica serovar Typhi with a novel gyrA mutation raises questions about the long-term efficacy of older fluoroquinolones for treating typhoid fever.

As a consequence of multidrug resistance, clinicians are highly dependent on fluoroquinolones for treating the serious systemic infection typhoid fever. While reduced susceptibility to fluoroquinolones, which lessens clinical efficacy, is becoming ubiquitous, comprehensive resistance is exceptional. Here we report ofloxacin treatment failure in typhoidal patient infected with a novel, highly fluoroquinolone-resistant isolate of Salmonella enterica serovar Typhi. The isolation of this organism has serious implications for the long-term efficacy of ciprofloxacin and ofloxacin for typhoid treatment.

Genomic epidemiology, antimicrobial resistance and virulence factors of Enterobacter cloacae complex causing potential community-onset bloodstream infections in a tertiary care hospital of Nepal.

Objectives: Community-onset bloodstream infections (BSIs) caused by carbapenemase-producing Enterobacter cloacae complex (ECC) species are increasing internationally. This observation suggests that ECC are emerging pathogens, requiring for detailed understanding on their genomic epidemiology including transmission dynamics and antimicrobial resistance profiles. Patients and methods: We performed WGS on 79 Enterobacter spp. isolated from the patients with clinically significant BSIs and admitted to emergency department of a major tertiary hospital in Nepal between April 2016 and October 2017. Results: We identified 5 species and 13 STs of ECC. Enterobacter xiangfangensis ST171, one of the globally emerging carbapenem resistant ECC clones with epidemic potential, was the most prevalent (42%). Phylogenetic analysis showed a large (>19 400 SNPs) core genome SNP distance across major STs, which was minimal (<30 SNPs) among the isolates of each prevalent ST, suggesting the relatively recent importation of major STs followed by local clonal expansions. Genomic evidence for resistance to all major antimicrobial classes except for colistin and macrolides was detected. A limited number of isolates also carried bla NDM-1 (n = 2) and bla OXA-48 (n = 1) carbapenemase genes. Virulence factors encoding siderophores (24%), T6SSD (25%) and fimbriae (54%) were detected. Conclusions: Our study highlighted that MDR ECC clones are important pathogens of BSIs in community. Though of low prevalence, carbapenem resistance observed in our ECC isolates raised concern about further community dissemination, underscoring the need for community surveillance to identify MDR ECC clones with epidemic potential.

Epidemiology, etiology, and diagnosis of health care acquired pneumonia including ventilator-associated pneumonia in Nepal.

Epidemiologic data regarding health care acquired pneumonia (HAP) and ventilator-associated pneumonia (VAP) from Nepal are negligible. We conducted a prospective observational cohort study in the intensive care unit (ICU) of a major tertiary hospital in Nepal between April 2016 and March 2018, to calculate the incidence of VAP, and to describe clinical variables, microbiological etiology, and outcomes. Four hundred and thirty-eight patients were enrolled in the study. Demographic data, medical history, antimicrobial administration record, chest X-ray, biochemical, microbiological and haematological results, acute physiology and chronic health evaluation II score and the sequential organ failure assessment scores were recorded. Categorical variables were expressed as count and percentage and analyzed using the Fisher's exact test. Continuous variables were expressed as median and interquartile range and analyzed using Kruskal-Wallis rank sum test and the pairwise Wilcoxon rank-sum test. 46.8% (205/438) of the patients required intubation. Pneumonia was common in both intubated (94.14%; 193/205) and non-intubated (52.36%; 122/233) patients. Pneumonia developed among intubated patients in the ICU had longer days of stay in the ICU (median of 10, IQR 5-15, P< 0.001) when compared to non-intubated patients with pneumonia (median of 4, IQR 3-6, P< 0.001). The incidence rate of VAP was 20% (41/205) and incidence density was 16.45 cases per 1,000ventilator days. Mortality was significantly higher in patients with pneumonia requiring intubation (44.6%, 86/193) than patients with pneumonia not requiring intubation (10.7%, 13/122, p<0.001, Fisher's exact test). Gram negative bacteria such as Klebsiella and Acinetobacter species were the dominant organisms from both VAP and non-VAP categories. Multi-drug resistance was highly prevalent in bacterial isolates associated with VAP (90%; 99/110) and non-VAP categories (81.5%; 106/130). HAP including VAP remains to be the most prevalent hospital-acquired infections (HAIs) at Patan hospital. A local study of etiological agents and outcomes of HAP and VAP are required for setting more appropriate guidelines for management of such diseases.