BackgroundChildren are substantially affected by enteric fever in most settings with a high burden of the disease, including Nepal. However pathogen population structure and transmission dynamics are poorly delineated in young children, the proposed target group for immunization programs. Here we present whole genome sequencing and antimicrobial susceptibility data on 198 S. Typhi and 66 S. Paratyphi A isolated from children aged 2 months to 15 years of age during blood culture surveillance at Patan Hospital, Nepal, 2008–2016.Principal findingsS. Typhi was the dominant agent and comprised several distinct genotypes, dominated by 4.3.1 (H58). The heterogeneity of genotypes in children under five was reduced compared to data from 2005–2006, attributable to ongoing clonal expansion of H58. Most isolates (86%) were non-susceptible to fluoroquinolones, associated mainly with S. Typhi H58 lineage II and S. Paratyphi A harbouring mutations in the quinolone resistance-determining region (QRDR); non-susceptible strains from these groups accounted for 50% and 25% of all isolates. Multi-drug resistance (MDR) was rare (3.5% of S. Typhi, 0 S. Paratyphi A) and restricted to chromosomal insertions of resistance genes in H58 lineage I strains. Temporal analyses revealed a shift in dominance from H58 Lineage I to H58 Lineage II, with the latter being significantly more common after 2010. Comparison to global data sets showed the local S. Typhi and S. Paratyphi A strains had close genetic relatives in other South Asian countries, indicating regional strain circulation. Multiple imports from India of ciprofloxacin-resistant H58 lineage II strains were identified, but these were rare and showed no evidence of clonal replacement of local S. Typhi.SignificanceThese data indicate that enteric fever in Nepal continues to be a major public health issue with ongoing inter- and intra-country transmission, and highlights the need for regional coordination of intervention strategies. The absence of a S. Paratyphi A vaccine is cause for concern, given its prevalence as a fluoroquinolone resistant enteric fever agent in this setting.

NDM-producing Klebsiella pneumoniae strains represent major clinical and infection control challenges, particularly in resource-limited settings with high rates of antimicrobial resistance. Determining whether transmission occurs at a gene, plasmid, or bacterial strain level and within hospital and/or the community has implications for monitoring and controlling spread. Whole-genome sequencing (WGS) is the highest-resolution typing method available for transmission epidemiology. We sequenced carbapenem-resistant K. pneumoniae isolates from 26 individuals involved in several infection case clusters in a Nepali neonatal unit and 68 other clinical Gram-negative isolates from a similar time frame, using Illumina and PacBio technologies. Within-outbreak chromosomal and closed-plasmid structures were generated and used as data set-specific references. Three temporally separated case clusters were caused by a single NDM K. pneumoniae strain with a conserved set of four plasmids, one being a 304,526-bp plasmid carrying blaNDM-1. The plasmids contained a large number of antimicrobial/heavy metal resistance and plasmid maintenance genes, which may have explained their persistence. No obvious environmental/human reservoir was found. There was no evidence of transmission of outbreak plasmids to other Gram-negative clinical isolates, although blaNDM variants were present in other isolates in different genetic contexts. WGS can effectively define complex antimicrobial resistance epidemiology. Wider sampling frames are required to contextualize outbreaks. Infection control may be effective in terminating outbreaks caused by particular strains, even in areas with widespread resistance, although this study could not demonstrate evidence supporting specific interventions. Larger, detailed studies are needed to characterize resistance genes, vectors, and host strains involved in disease, to enable effective intervention.

BackgroundSalmonella Typhi (S. Typhi) causes typhoid fever, which remains an important public health issue in many developing countries. Kathmandu, the capital of Nepal, is an area of high incidence and the pediatric population appears to be at high risk of exposure and infection.MethodsWe recently defined the population structure of S. Typhi, using new sequencing technologies to identify nearly 2,000 single nucleotide polymorphisms (SNPs) that can be used as unequivocal phylogenetic markers. Here we have used the GoldenGate (Illumina) platform to simultaneously type 1,500 of these SNPs in 62 S. Typhi isolates causing severe typhoid in children admitted to Patan Hospital in Kathmandu.ResultsEight distinct S. Typhi haplotypes were identified during the 20-month study period, with 68% of isolates belonging to a subclone of the previously defined H58 S. Typhi. This subclone was closely associated with resistance to nalidixic acid, with all isolates from this group demonstrating a resistant phenotype and harbouring the same resistance-associated SNP in GyrA (Phe83). A secondary clone, comprising 19% of isolates, was observed only during the second half of the study.ConclusionsOur data demonstrate the utility of SNP typing for monitoring bacterial populations over a defined period in a single endemic setting. We provide evidence for genotype introduction and define a nalidixic acid resistant subclone of S. Typhi, which appears to be the dominant cause of severe pediatric typhoid in Kathmandu during the study period.

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Typhoid fever is a major public health problem in developing countries, conservatively estimated to occur in 17 million cases and be responsible for 200,000 deaths annually. We investigated the acquisition of natural immunity to Salmonella enterica serovar Typhi in a region where typhoid is endemic by testing sera from an age-stratified sample of 210 healthy participants in Kathmandu, Nepal, for bactericidal activity toward S. Typhi and for anti-Vi capsular polysaccharide antibodies. Bactericidal titers in children were significantly lower than those in newborns and adults (P < 0.0001). Anti-S. Typhi bactericidal geometric mean titers were age dependent, increasing 10-fold during childhood. Anti-Vi polysaccharide antibody geometric mean concentrations were also lower in children than in adults. Data presented here indicate the possibility of a relationship between low levels of bactericidal activity toward S. Typhi in serum and susceptibility to disease, as observed for other polysaccharide-encapsulated bacteria.

The data on the burden of invasive bacterial infections and pneumonia from this study suggest that vaccines in development against Salmonella Typhi and the pneumococcus have the potential to significantly improve the health of children in Nepal.

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Since its first identification in Scotland, over 1000 cases of unexplained pediatric hepatitis in children have been reported worldwide, including 278 cases in the UK 1 . Here we report investigation of 38 cases, 66 age-matched immunocompetent controls and 21 immunocompromised comparator subjects, using a combination of genomic, transcriptomic, proteomic and immunohistochemical methods. We detected high levels of adeno-associated virus 2 (AAV2) DNA in liver, blood, plasma or stool from 27/28 cases. We found low levels of Adenovirus (HAdV) and Human Herpesvirus 6B (HHV-6B), in 23/31 and 16/23 respectively of the cases tested. In contrast, AAV2 was infrequently detected at low titre in blood or liver from control children with HAdV, even when profoundly immunosuppressed.AAV2, HAdV and HHV-6 phylogeny excluded emergence of novel strains in cases. Histological analyses of explanted livers showed enrichment for T-cells and B-lineage cells.Proteomic comparison of liver tissue from cases and healthy controls, identified increased expression of HLA class 2, immunoglobulin variable regions and complement proteins.HAdV and AAV2 proteins were not detected in the livers. Instead, we identified AAV2 DNA complexes reflecting both HAdV and HHV-6B-mediated replication. We hypothesize that high levels of abnormal AAV2 replication products aided by HAdV and in severe cases HHV-6B, may have triggered immune-mediated hepatic disease in genetically and immunologically predisposed children.