Search

IntroductionSalmonellaTyphi andSalmonellaParatyphi, fecal-oral transmitted bacterium have temporally and geographically heterogeneous pathways of transmission. Previous work in Kathmandu Nepal implicated stone waterspouts as a dominant transmission pathway after 77% of samples tested positive forS. Typhi and 70% forS. Paratyphi. Due to a falling water table, these spouts no longer provide drinking water, but typhoid fever persists, and the question of the disease’s dominant pathway of transmission remains unanswered.MethodsWe used environmental surveillance to detectS. Typhi and Paratyphi DNA from potential sources of transmission. We collected 1L drinking water samples from a population-based random sample of households between February and October 2019 Between November 2019 and July 2021, we collected monthly 50 mL river water samples from 19 sites along the rivers leading through the Kathmandu and Kavre Districts of Nepal. We processed drinking water samples using a single qpcr and processed river water samples using differential centrifugation and qPCR at time 0 and after 16 hours of culture enrichment. A 3-cycle threshold (Ct) decrease ofS. Typhi orS. Paratyphi, pre- and post-enrichment, was used as evidence of growth. We also performed structured observations of human-environment interactions to understand pathways of potential exposure.ResultsAmong 370 drinking water samples,S. Typhi was detected in 7 samples (1.8%) andS. Paratyphi A was detected in 4 (1.0%) samples. Among 381 river water samples,S. Typhi was detected in 171 (45%) andS. Paratyphi A was detected in 152 (42%) samples. Samples located upstream of the Kathmandu city center were positive forS. Typhi 12% of the time while samples from locations in and downstream the city had bacterial DNA detected 58% and 67% of the time respectively. Individuals were observed bathing in the rivers, washing clothes, and washing vegetables for sale in Kathmandu markets.ImplicationsThese results suggest that drinking water was not the dominant pathway of transmission ofS. Typhi andS. Paratyphi A in the Kathmandu Valley in 2019. The high degree of river water contamination and its use for washing vegetables raises the possibility that river systems srepresent an important source of typhoid exposure in Kathmandu.Author SummaryUnderstanding the dominant route of transmission of a pathogen is important for designing and implementing effective control strategies. Salmonella Typhi and Paratyphi which cause typhoid and paratyphoid fever infect an estimated 10 million people and kill more than 100,000 annually. In Kathmandu prior work suggested that stone spouts where people collected drinking water were contaminated and driving transmission of the diseases. However, many of these spouts no longer function, and people are still getting sick. We tested drinking water from households in this area as well as local river water and found that 13 drinking water samples were positive for S.Typhi and 15 were positive for S. Paratyphi and many river samples tested positive for these bacterium. River water samples were not often positive upstream of Kathmandu city center (12% positive for S.Typhi) but were often positive within the city center (58% positive for S.Typhi) and in rural areas up to 10 km downstream of the city (67% positive for S.Typhi). During sample collection, individuals were observed interacting with rivers by walking in them, washing clothes and washing vegetables for sale in markets. This study shows that drinking water may not be primary driver of typhoid transmission in the Kathmandu valley, but that sewage contaminated river water may be a foci of transmission into the wider population.

Insertion sequence contributes to the emergence of carbapenem resistance by dissemination of carbapenemase genes and providing promoter for their overexpression. This study aims to ascertain the occurrence of ISAba1-linked OXA carbapenemase genes and its relevance to carbapenem resistance level inAcinetobacter baumannii. This hospital based descriptive study was conducted at Shahid Gangalal National Heart Center, Kathmandu, Nepal. An overall of 1,291 clinical specimens received for routine culture and antibiotic susceptibility testing throughout the study period were included in this study. Identification ofAcinetobacter baumanniiwas validated through detection of intrinsicblaOXA-51-likegene by polymerase chain reaction (PCR). Antibiotic susceptibility was tested by Kirby-Bauer disc diffusion approach and minimum inhibitory concentration (MIC) of meropenem was assessed through agar dilution method. Uniplex PCR assays were performed to detect genes encoding oxacillinases and ISAba1. Upstream association of insertion element, ISAba1to oxacillinase genes was assessed through PCR mapping strategy using ISAba1F and OXA-51R/OXA-23R primers. Out of the 340 bacteria isolated, only 40 (11.8%) wereAcinetobacter baumannii. All isolates were resistant against meropenem with MIC value ranging from 16-256 μg/ml.blaOXA-23-likegenes was present in every isolate butblaOXA-58in just two isolates (5%). All isolates had ISAba1either aboveblaOXA-23-likeorblaOXA-51-likegene. Higher MIC90value of meropenem (243.20 μg/ml) was found inA.baumanniicluster with ISAba1-linked upstream to bothblaOXA-23-likeandblaOXA-51-likegenes, thus depicting their eminent role to enhanced carbapenem resistance.Acinetobacter baumanniiisolates with ISAba1-linked oxacillinase genes are rapidly emerging in clinical settings of Nepal. Thus, medical communities need to be prepared and enable targeted approaches for managing burgeoning problem of carbapenem resistance.

Environmental surveillance, using detection ofSalmonellaTyphi DNA, has emerged as a potentially useful tool to identify typhoid-endemic settings; however, it is relatively costly and requires molecular diagnostic capacity. We sought to determine whetherS. Typhi bacteriophages are abundant in water sources in a typhoid-endemic setting, using low-cost assays. We collected drinking and surface water samples from urban, peri-urban and rural areas in 4 regions of Nepal. We performed a double agar overlay withS. Typhi to assess the presence of bacteriophages. We isolated and tested phages against multiple strains to assess their host range. We performed whole genome sequencing of isolated phages, and generated phylogenies using conserved genes.S. Typhi-specific bacteriophages were detected in 54.9% (198/361) of river water samples and 6.3% (1/16) drinking water samples from the Kathmandu Valley and Kavrepalanchok. Water samples collected within or downstream of population-dense areas were more likely to be positive (72.6%, 193/266) than those collected upstream from population centers (5.3%, 5/95) (p=0.005). In urban Biratnagar and rural Dolakha, where typhoid incidence is low, only 6.7% (1/15, Biratnagar) and 0% (0/16, Dolakha) samples contained phages. AllS. Typhi phages were unable to infect otherSalmonellaand non-Salmonellastrains, nor a Vi-knockoutS. Typhi strain. Representative strains fromS. Typhi lineages were variably susceptible to the isolated phages. Phylogenetic analysis showed thatS. Typhi phages belonged to two different viral families (AutographiviridaeandSiphoviridae) and clustered in three distinct groups.S. Typhi bacteriophages were highly abundant in surface waters of typhoid-endemic communities but rarely detected in low typhoid burden communities. Bacteriophages recovered were specific forS. Typhi and required Vi polysaccharide for infection. Screening small volumes of water with simple, low-cost plaque assays enables detection ofS. Typhi phages and should be further evaluated as a scalable tool for typhoid environmental surveillance.HighlightsTyphoid phages are detectable in surface water using simple assays, in communities with high typhoid burden.Bacteriophages are highly specific forS. Typhi and required Vi polysaccharide for infection.S. Typhi phages have a broad lytic activity against theS. Typhi strains circulating in Nepal.Phage plaque assay can be used as a low-cost tool to identify communities where typhoid is endemic.The high abundance of phages in river water suggest that this could be an alternative to molecular methods for environmental surveillance for typhoid.

Background: \ud The emergence of increasingly antimicrobial-resistant Salmonella enterica serovar Typhi (S Typhi) threatens to undermine effective treatment and control. Understanding where antimicrobial resistance in S Typhi is emerging and spreading is crucial towards formulating effective control strategies.\ud \ud Methods: \ud In this genomic epidemiology study, we sequenced the genomes of 3489 S Typhi strains isolated from prospective enteric fever surveillance studies in Nepal, Bangladesh, Pakistan, and India (between 2014 and 2019), and combined these with a global collection of 4169 S Typhi genome sequences isolated between 1905 and 2018 to investigate the temporal and geographical patterns of emergence and spread of antimicrobial-resistant S Typhi. We performed non-parametric phylodynamic analyses to characterise changes in the effective population size of fluoroquinolone-resistant, extensively drug-resistant (XDR), and azithromycin-resistant S Typhi over time. We inferred timed phylogenies for the major S Typhi sublineages and used ancestral state reconstruction methods to estimate the frequency and timing of international and intercontinental transfers.\ud \ud Findings: \ud Our analysis revealed a declining trend of multidrug resistant typhoid in south Asia, except for Pakistan, where XDR S Typhi emerged in 2016 and rapidly replaced less-resistant strains. Mutations in the quinolone-resistance determining region (QRDR) of S Typhi have independently arisen and propagated on at least 94 occasions, nearly all occurring in south Asia. Strains with multiple QRDR mutations, including triple mutants with high-level fluoroquinolone resistance, have been increasing in frequency and displacing strains with fewer mutations. Strains containing acrB mutations, conferring azithromycin resistance, emerged in Bangladesh around 2013 and effective population size of these strains has been steadily increasing. We found evidence of frequent international (n=138) and intercontinental transfers (n=59) of antimicrobial-resistant S Typhi, followed by local expansion and replacement of drug-susceptible clades.\ud \ud Interpretation: \ud Independent acquisition of plasmids and homoplastic mutations conferring antimicrobial resistance have occurred repeatedly in multiple lineages of S Typhi, predominantly arising in south Asia before spreading to other regions.\ud \ud Funding: \ud Bill & Melinda Gates Foundation.

The incidence of enteric fever, an invasive bacterial infection caused by typhoidal Salmonellae (Salmonella enterica serovars Typhi and Paratyphi), is largely unknown in regions without blood culture surveillance. The aim of this study was to evaluate whether new diagnostic serological markers for typhoidal Salmonella can reliably estimate population-level incidence.We collected longitudinal blood samples from patients with blood culture-confirmed enteric fever enrolled from surveillance studies in Bangladesh, Nepal, Pakistan, and Ghana between 2016 and 2021 and conducted cross-sectional serosurveys in the catchment areas of each surveillance site. We used ELISAs to measure quantitative IgA and IgG antibody responses to hemolysin E and S Typhi lipopolysaccharide. We used Bayesian hierarchical models to fit two-phase power-function decay models to the longitudinal antibody responses among enteric fever cases and used the joint distributions of the peak antibody titres and decay rate to estimate population-level incidence rates from cross-sectional serosurveys.The longitudinal antibody kinetics for all antigen-isotypes were similar across countries and did not vary by clinical severity. The seroincidence of typhoidal Salmonella infection among children younger than 5 years ranged between 58·5 per 100 person-years (95% CI 42·1-81·4) in Dhaka, Bangladesh, to 6·6 per 100 person-years (4·3-9·9) in Kavrepalanchok, Nepal, and followed the same rank order as clinical incidence estimates.The approach described here has the potential to expand the geographical scope of typhoidal Salmonella surveillance and generate incidence estimates that are comparable across geographical regions and time.BillMelinda Gates Foundation.For the Nepali, Bengali and Urdu translations of the abstract see Supplementary Materials section.

BackgroundThe incidence of enteric fever, an invasive bacterial infection caused by typhoidal Salmonellae, is largely unknown in regions lacking blood culture surveillance. New serologic markers have proven accurate in diagnosing enteric fever, but whether they could be used to reliably estimate population-level incidence is unknown.MethodsWe collected longitudinal blood samples from blood culture-confirmed enteric fever cases enrolled from surveillance studies in Bangladesh, Nepal, Pakistan, and Ghana and conducted cross-sectional serosurveys in the catchment areas of each surveillance site. We used ELISAs to measure quantitative IgA and IgG antibody responses to Hemolysin E (HlyE) and S. Typhi lipopolysaccharide (LPS). We used Bayesian hierarchical models to fit two-phase power-function decay models to the longitudinal antibody responses among enteric fever cases and used the joint distributions of the peak antibody titers and decay rate to estimate population-level incidence rates from cross-sectional serosurveys.FindingsThe longitudinal antibody kinetics for all antigen-isotypes were similar across countries and did not vary by clinical severity. The seroincidence of typhoidal Salmonella infection among children InterpretationThe approach described here has the potential to expand the geographic scope of typhoidal Salmonella surveillance and generate incidence estimates that are comparable across geographic regions and time.FundingThis work was supported by the Bill and Melinda Gates Foundation (INV-000572).Research in contextEvidence before this studyPrevious studies have identified serologic responses to two antigens (Hemolysin E [HlyE] and Salmonella lipopolysaccharide [LPS]) as promising diagnostic markers of acute typhoidal Salmonella infection. We reviewed the evidence for seroepidemiology tools for enteric fever available as of November 01, 2021, by searching the National Library of Medicine article database and medRxiv for preprint publications, published in English, using the terms “enteric fever”, “typhoid fever”, “Salmonella Typhi”, “Salmonella Paratyphi”, “typhoidal Salmonella”, “Hemolysin E”, “Salmonella lipopolysaccharide”, “seroconversion”, “serosurveillance”, “seroepidemiology”, “seroprevalence” and “seropositivity.” We found no studies using HlyE or LPS as markers to measure the incidence or prevalence of enteric fever in a population. Anti-Vi IgG responses were used as a marker of population seroprevalence in cross-sectional studies conducted in South Africa, Fiji, and Nepal, but were not used to calculate population-based incidence estimates.Added value of this studyWe developed and validated a method to estimate typhoidal Salmonella incidence in cross-sectional population samples using antibody responses measured from dried blood spots. First, using longitudinal dried blood spots collected from over 1400 blood culture-confirmed cases in four countries, we modeled the longitudinal dynamics of antibody responses for up to two years following infection, accounting for heterogeneity in antibody responses and age-dependence. We found that longitudinal antibody responses were highly consistent across four countries on two continents and did not differ by clinical severity. We then used these antibody kinetic parameters to estimate incidence in population-based samples in six communities across the four countries, where concomitant population-based incidence was measured using blood cultures. Seroincidence estimates were much higher than blood-culture-based case estimates across all six sites, suggestive of a high incidence of asymptomatic or unrecognized infections. Still, the rank order of seroincidence and culture-based incidence rates were the same, with the highest rates in Bangladesh and lowest in Ghana.Implications of all the available evidenceMany at-risk low- and middle-income countries lack data on typhoid incidence needed to inform and evaluate vaccine introduction. Even in countries where incidence estimates are available, data are typically geographically and temporally sparse due to the resources necessary to initiate and sustain blood culture surveillance. We found that typhoidal Salmonella infection incidence can be estimated from community-based serosurveys using dried blood spots, representing an efficient and scalable approach for generating the typhoid burden data needed to inform typhoid control programs in resource-constrained settings.

The emergence of increasingly antimicrobial-resistant (AMR) Salmonella enterica serovar Typhi (S. Typhi) threatens to undermine effective treatment and control. Here, aiming to investigate the temporal and geographic patterns of emergence and spread of AMR S. Typhi, we sequenced 3,489 S. Typhi isolated from prospective surveillance in South Asia and combined these with a global collection of 4,169 S. Typhi genomes. Our analysis revealed that independent acquisition of plasmids and homoplastic mutations conferring AMR have occurred repeatedly in multiple lineages of S. Typhi, predominantly arising in South Asia. We found evidence of frequent international and intercontinental transfers of AMR S. Typhi, followed by rapid expansion and replacement of antimicrobial-susceptible clades.

Enteric fever remains a major cause of morbidity in developing countries with poor sanitation conditions that enable fecal contamination of water distribution systems. Historical evidence has shown that contamination of water systems used for household consumption or agriculture are key transmission routes for Salmonella Typhi and Salmonella Paratyphi A. The World Health Organization now recommends that typhoid conjugate vaccines (TCV) be used in settings with high typhoid incidence; consequently, governments face a challenge regarding how to prioritize typhoid against other emerging diseases. A key issue is the lack of typhoid burden data in many low- and middle-income countries where TCV could be deployed. Here we present an argument for utilizing environmental sampling for the surveillance of enteric fever organisms to provide data on community-level typhoid risk. Such an approach could complement traditional blood culture-based surveillance or even replace it in settings where population-based clinical surveillance is not feasible. We review historical studies characterizing the transmission of enteric fever organisms through sewage and water, discuss recent advances in the molecular detection of typhoidal Salmonella in the environment, and outline challenges and knowledge gaps that need to be addressed to establish environmental sampling as a tool for generating actionable data that can inform public health responses to enteric fever.

Regulating immunity is a leading target for cancer therapy. Here, we show that the anti-tumor immune response can be modulated by the brain’s reward system, a key circuitry in emotional processes. Activation of the reward system in tumor-bearing mice (Lewis lung carcinoma (LLC) and B16 melanoma) using chemogenetics (DREADDs), resulted in reduced tumor weight. This effect was mediated via the sympathetic nervous system (SNS), manifested by an attenuated noradrenergic input to a major immunological site, the bone marrow. Myeloid derived suppressor cells (MDSCs), which develop in the bone marrow, became less immunosuppressive following reward system activation. By depleting or adoptively transferring the MDSCs, we demonstrated that these cells are both necessary and sufficient to mediate reward system effects on tumor growth. Given the central role of the reward system in positive emotions, these findings introduce a physiological mechanism whereby the patient’s psychological state can impact anti-tumor immunity and cancer progression., Neural activation can have wide ranging effects beyond central and peripheral nervous system. This work shows that chemogenetic activation of the brain’s reward system ventral tegmental area (VTA) can boost mice’s immune function, confer anti-tumor immunity, and reduce tumor mass in experimental rodent models of lung carcinoma and melanoma.

Cancer and autoimmune diseases are fundamentally different pathological conditions. In cancer, the immune response is suppressed and unable to eradicate the transformed self-cells, while in autoimmune diseases it is hyperactivated against a self-antigen, leading to tissue injury. Yet, mechanistically, similarities in the triggering of the immune responses can be observed. In this review, we highlight some parallel aspects of the microenvironment in cancer and autoimmune diseases, especially hypoxia, and the role of macrophages, neutrophils, and their interaction. Macrophages, owing to their plastic mode of activation, can generate a pro- or antitumoral microenvironment. Similarly, in autoimmune diseases, macrophages tip the Th1/Th2 balance via various effector cytokines. The contribution of neutrophils, an additional plastic innate immune cell population, to the microenvironment and disease progression is recently gaining more prominence in both cancer and autoimmune diseases, as they can secrete cytokines, chemokines, and reactive oxygen species (ROS), as well as acquire an enhanced ability to produce neutrophil extracellular traps (NETs) that are now considered important initiators of autoimmune diseases. Understanding the contribution of macrophages and neutrophils to the cancerous or autoimmune microenvironment, as well as the role their interaction and cooperation play, may help identify new targets and improve therapeutic strategies.

With fewer than 200 tigers (Panthera tigris tigris) left in Nepal, that are generally confined to five protected areas across the Terai Arc Landscape, genetic studies are needed to provide crucial information on diversity and connectivity for devising an effective country-wide tiger conservation strategy. As part of the Nepal Tiger Genome Project, we studied landscape change, genetic variation, population structure, and gene flow of tigers across the Terai Arc Landscape by conducting Nepal's first comprehensive and systematic scat-based, non-invasive genetic survey. Of the 770 scat samples collected opportunistically from five protected areas and six presumed corridors, 412 were tiger (57%). Out of ten microsatellite loci, we retain eight markers that were used in identifying 78 individual tigers. We used this data set to examine population structure, genetic variation, contemporary gene flow, and potential population bottlenecks of tigers in Nepal. We detected three genetic clusters consistent with three demographic sub-populations and found moderate levels of genetic variation (H-e = 0.61, A(R) = 3.51) and genetic differentiation (F-ST = 0.14) across the landscape. We detected 3-7 migrants, confirming the potential for dispersal-mediated gene flow across the landscape. We found evidence of a bottleneck signature likely caused by large-scale land-use change documented in the last two centuries in the Terai forest. Securing tiger habitat including functional forest corridors is essential to enhance gene flow across the landscape and ensure long-term tiger survival. This requires cooperation among multiple stakeholders and careful conservation planning to prevent detrimental effects of anthropogenic activities on tigers. USAID [FOG AID-367-G-11-00001]; Katheryn Fuller Fellowship (WWF US); Wildlife Conservation Society (WCS); Virginia Tech (VT) This work was supported by USAID FOG AID-367-G-11-00001. Netra Sharma and Bronwyn Llewellyn were involved with the project and helped in conceptualization. The Katheryn Fuller Fellowship (WWF US), Wildlife Conservation Society (WCS), and Virginia Tech (VT) supported Kanchan Thapa.

Previously, we have identified a new epitope in EMMPRIN, a multifunctional protein that mediates tumor cell–macrophage interactions and induces both MMP-9 and VEGF. Here, we synthesized this epitope as an octa-branched multiple antigenic peptide (MAP) to vaccinate mice implanted with subcutaneous syngeneic colon (CT26), prostate (TRAMP-C2) or renal (RENCA) cell line carcinomas. Vaccination inhibited, and sometimes regressed, tumor growth in a dose-dependent manner, reaching 94%, 71% and 72% inhibition, respectively, at a 50 μg dose (p < 0.01). Mice with regressed tumors demonstrated immune memory, preventing tumor recurrence upon re-implantation (p < 0.001). When tumor cells were administered through the tail vein to generate lung metastases, vaccination reduced the number of metastatic foci (by 15- and 23-folds, p < 0.001), and increased the median survival time by 25% and 53% in RENCA and CT26 metastases, respectively (p < 0.01) relative to scrambled-MAP controls. No significant adverse responses were observed in all experiments. We show that the tumor microenvironment was immune modulated, as vaccination induced production of EMMPRIN-specific antibodies, increased CD8+ T cells infiltration and cytotoxicity, alleviated immune suppression by decreasing TGFβ concentrations, reduced angiogenesis and cell proliferation, and enhanced apoptosis. Thus, our successful active peptide vaccination strategy differs from previous, unsuccessful attempts, both in the selected target (the EMMPRIN epitope) and in the use of a modified, MAP configuration, and demonstrates that this may be an efficient approach for the treatment and prevention of some types of cancer.

As part of a comprehensive health care programme for people who use drugs in Nepal, HIV and viral hepatitis B and C status--including risk factors, HCV-genotypes and co-infections--as well as two IL28B Single-nucleotide polymorphisms (SNPs) were assessed for a random sample of 401 people who inject drugs in three regions of Nepal: mid-western Terrai (Nepalgunj), the eastern region (Dharan, Biratnagar) and the central region (Kathmandu, Lalitpur and Chitwan). Individuals were included who showed at least a minimum of health care seeking behaviour. This latter criterion was defined by being registered with any organisation offering health services. The average age of the participants was 30.5 yrs, and the average length of intravenous drug use was 8.5 yrs. The prevalence rates of HBsAg, anti-HIV antibodies and HCV-RNA were 3.5%, 13.8% and 41.9%, respectively. Spontaneous HCV clearance was evident in 16% of all of those who tested positive for anti-HCV antibodies. Independent risk factors for HCV-RNA positivity were age, gender, geographical region, duration of injecting drug use, history of imprisonment and HIV co-infection. In the age group ≤24 yrs, the rate of spontaneous HCV clearance was 43.5%. Overall, 59.8% of HCV infections were caused by HCV genotype 3 and 40.2% by HCV genotype 1. No other HCV genotypes were identified in this study. The IL28B SNP rs12979860 and rs8099917 were identified in 122 patients, and 75.4% of all participants had both favourable genotypes rs12979860 C/C and rs8099917 T/T.