Explore the vast range of titles available.

Relatively little is known of leprosy in Medieval Ireland; as an island located at the far west of Europe it has the potential to provide interesting insights in relation to the historical epidemiology of the disease. To this end the study focuses on five cases of probable leprosy identified in human skeletal remains excavated from inhumation burials. Three of the individuals derived from the cemetery of St Michael Le Pole, Golden Lane, Dublin, while single examples were also identified from Ardreigh, Co. Kildare, and St Patrick's Church, Armoy, Co. Antrim. The individuals were radiocarbon dated and examined biomolecularly for evidence of either of the causative pathogens, M. leprae or M. lepromatosis. Oxygen and strontium isotopes were measured in tooth enamel and rib samples to determine where the individuals had spent their formative years and to ascertain if they had undertaken any recent migrations. We detected M. leprae DNA in the three Golden Lane cases but not in the probable cases from either Ardreigh Co. Kildare or Armoy, Co. Antrim. M. lepromatosis was not detected in any of the burals. DNA preservation was sufficiently robust to allow genotyping of M. leprae strains in two of the Golden Lane burials, SkCXCV (12-13th century) and SkCCXXX (11-13th century). These strains were found to belong on different lineages of the M. leprae phylogenetic tree, namely branches 3 and 2 respectively. Whole genome sequencing was also attempted on these two isolates with a view to gaining further information but poor genome coverage precluded phylogenetic analysis. Data from the biomolecular study was combined with osteological, isotopic and radiocarbon dating to provide a comprehensive and multidisciplinary study of the Irish cases. Strontium and oxygen isotopic analysis indicate that two of the individuals from Golden Lane (SkCXLVIII (10-11th century) and SkCXCV) were of Scandinavian origin, while SkCCXXX may have spent his childhood in the north of Ireland or central Britain. We propose that the Vikings were responsible for introducing leprosy to Ireland. This work adds to our knowledge of the likely origins of leprosy in Medieval Ireland and will hopefully stimulate further research into the history and spread of this ancient disease across the world.

Mycobacterium avium subspecies paratuberculosis (Map) was monitored by quantitative PCR over a range of temporal and spatial scales in the River Tywi catchment. This study shows the persistence of Map over a 10-year period with little change, which correlates with the recognised levels of Johne’s disease in British herds over that period (aim 1). Map was quantified within the river at up to 108 cell equivalents L−1 and was shown to be consistently present when monitored over finer timescales (aim 4). Small wastewater treatment plants where the ingress of human-associated Map might be expected had no significant effect (aim 2). Map was found for the first time to be located in natural river foams providing another route for spread via aerosols (aim 5). This study provides evidence for the environmental continuum of Map from the grazing infected animal via rain driven runoff through field drains and streams into main rivers; with detection at a high frequency throughout the year. Should Map need to be monitored in the future, we recommend that weekly or monthly sampling from a fixed location on a river will capture an adequate representation of the flow dynamics of Map in a catchment (aim 3). The human exposure to Map during this process and its impact on human health remains unquantified.

The gold standard for clinical diagnosis of bacterial lower respiratory infections (LRIs) is culture, which has poor sensitivity and is too slow to guide early, targeted antimicrobial therapy. Metagenomic sequencing could identify LRI pathogens much faster than culture, but methods are needed to remove the large amount of human DNA present in these samples for this approach to be feasible. We developed a metagenomics method for bacterial LRI diagnosis that features efficient saponin-based host DNA depletion and nanopore sequencing. Our pilot method was tested on 40 samples, then optimized and tested on a further 41 samples. Our optimized method (6 h from sample to result) was 96.6% sensitive and 41.7% specific for pathogen detection compared with culture and we could accurately detect antibiotic resistance genes. After confirmatory quantitative PCR and pathobiont-specific gene analyses, specificity and sensitivity increased to 100%. Nanopore metagenomics can rapidly and accurately characterize bacterial LRIs and might contribute to a reduction in broad-spectrum antibiotic use.

A human genome is sequenced and assembled de novo using a pocket-sized nanopore device. We report the sequencing and assembly of a reference genome for the human GM12878 Utah/Ceph cell line using the MinION (Oxford Nanopore Technologies) nanopore sequencer. 91.2 Gb of sequence data, representing ∼30× theoretical coverage, were produced. Reference-based alignment enabled detection of large structural variants and epigenetic modifications. De novo assembly of nanopore reads alone yielded a contiguous assembly (NG50 ∼3 Mb). We developed a protocol to generate ultra-long reads (N50 > 100 kb, read lengths up to 882 kb). Incorporating an additional 5× coverage of these ultra-long reads more than doubled the assembly contiguity (NG50 ∼6.4 Mb). The final assembled genome was 2,867 million bases in size, covering 85.8% of the reference. Assembly accuracy, after incorporating complementary short-read sequencing data, exceeded 99.8%. Ultra-long reads enabled assembly and phasing of the 4-Mb major histocompatibility complex (MHC) locus in its entirety, measurement of telomere repeat length, and closure of gaps in the reference human genome assembly GRCh38.

Mycobacterium avium subspecies paratuberculosis (Map) causes Johne’s disease in animals and is significantly associated with Crohn’s disease (CD) in humans. Our previous studies have shown Map to be present in U.K. rivers due to land deposition from chronic livestock infection and runoff driven by rainfall. The epidemiology of CD in Cardiff showed a significant association with the River Taff, in which Map can be detected on a regular basis. We have previously hypothesized that aerosols from the river might influence the epidemiology of CD. In this preliminary study, we detected Map by quantitative PCR in one of five aerosol samples collected above the River Taff. In addition, we examined domestic showers from different regions in the U.K. and detected Map in three out of 30 independent samples. In detecting Map in river aerosols and those from domestic showers, this is the first study to provide evidence that aerosols are an exposure route for Map to humans and may play a role in the epidemiology of CD.

BackgroundCulture-based microbiological investigation of hospital-acquired or ventilator-associated pneumonia (HAP or VAP) is insensitive, with aetiological agents often unidentified. This can lead to excess antimicrobial treatment of patients with susceptible pathogens, while those with resistant bacteria are treated inadequately for prolonged periods. Using PCR to seek pathogens and their resistance genes directly from clinical samples may improve therapy and stewardship.MethodsSurplus routine lower respiratory tract samples were collected from intensive care unit patients about to receive new or changed antibiotics for hospital-onset lower respiratory tract infections at 15 UK hospitals. Testing was performed using the BioFire FilmArray Pneumonia Panel (bioMérieux) and Unyvero Pneumonia Panel (Curetis). Concordance analysis compared machine and routine microbiology results, while Bayesian latent class (BLC) analysis estimated the sensitivity and specificity of each test, incorporating information from both PCR panels and routine microbiology.FindingsIn 652 eligible samples; PCR identified pathogens in considerably more samples compared with routine microbiology: 60.4% and 74.2% for Unyvero and FilmArray respectively vs 44.2% by routine microbiology. PCR tests also detected more pathogens per sample than routine microbiology. For common HAP/VAP pathogens, FilmArray had sensitivity of 91.7%–100.0% and specificity of 87.5%–99.5%; Unyvero had sensitivity of 50.0%–100.0%%, and specificity of 89.4%–99.0%. BLC analysis indicated that, compared with PCR, routine microbiology had low sensitivity, ranging from 27.0% to 69.4%.InterpretationConventional and BLC analysis demonstrated that both platforms performed similarly and were considerably more sensitive than routine microbiology, detecting potential pathogens in patient samples reported as culture negative. The increased sensitivity of detection realised by PCR offers potential for improved antimicrobial prescribing.

Mycobacterium avium subspecies paratuberculosis (Map) is the known cause of Johne’s disease (JD), a chronic inflammation of the intestine observed in livestock and wildlife. Although Map is significantly associated with Crohn’s disease (CD), its status as the causative agent remains unresolved (Chiodini et al., 2012).Pickup and co-workers (2006) developed a model of sources and sinks of Map in the environment in terms of human exposure (Pickup et al., 2006). The aims of this thesis were to refine and extend this model by investigating the presence of Map in the River Tywi catchment at various locations and intervals, in aerosols collected from the River Taff catchment, in domestic shower biofilms and in River Eden catchment field beck water. Allied to this, we studied how Map persists in the aquatic environment in microcosm experiments comprising of sterile lake water under two temperature regimes. A patient questionnaire was distributed at the Gastrointestinal Clinic at Royal Lancaster Infirmary which focused on the possible influence of geographic location on CD status.The Map status of the River Tywi catchment was confirmed as both IS900 and F57 were detected in river water collected from various locations in the catchment at various intervals. For the first time, Map was detected in aerosol samples collected in a non-farming environment, domestic shower biofilms and field beck water. The persistence of Map under environmental conditions was shown to be long term using a novel MAP2720c stress gene assay. In a preliminary study, CD status was shown to be significantly associated with the environment. Overall, this research reveals an extensive presence of Map in the environment and identifies novel sources that could result in exposure of humans to Map.

Lower respiratory infections (LRIs) accounted for three million deaths worldwide in 2016, the leading infectious cause of mortality. The 'gold standard' for investigation of bacterial LRIs is culture, which has poor sensitivity and is too slow to guide early antibiotic therapy. Metagenomic sequencing potentially could replace culture, providing rapid, sensitive and comprehensive results. We developed a metagenomics pipeline for the investigation of bacterial LRIs using saponin-based host DNA depletion combined with rapid nanopore sequencing. The first iteration of the pipeline was tested on respiratory samples from 40 patients. It was then refined to reduce turnaround and increase sensitivity, before testing a further 41 samples. The refined method was 96.6% concordant with culture for detection of pathogens and could accurately detect resistance genes with a turnaround time of six hours. This study demonstrates that nanopore metagenomics can rapidly and accurately characterise bacterial LRIs when combined with efficient human DNA depletion.

Nanopore sequencing is a promising technique for genome sequencing due to its portability, ability to sequence long reads from single molecules, and to simultaneously assay DNA methylation. However until recently nanopore sequencing has been mainly applied to small genomes, due to the limited output attainable. We present nanopore sequencing and assembly of the GM12878 Utah/Ceph human reference genome generated using the Oxford Nanopore MinION and R9.4 version chemistry. We generated 91.2 Gb of sequence data (~30x theoretical coverage) from 39 flowcells. De novo assembly yielded a highly complete and contiguous assembly (NG50 ~3Mb). We observed considerable variability in homopolymeric tract resolution between different basecallers. The data permitted sensitive detection of both large structural variants and epigenetic modifications. Further we developed a new approach exploiting the long-read capability of this system and found that adding an additional 5x-coverage of \"ultra-long\" reads (read N50 of 99.7kb) more than doubled the assembly contiguity. Modelling the repeat structure of the human genome predicts extraordinarily contiguous assemblies may be possible using nanopore reads alone. Portable de novo sequencing of human genomes may be important for rapid point-of-care diagnosis of rare genetic diseases and cancer, and monitoring of cancer progression. The complete dataset including raw signal is available as an Amazon Web Services Open Dataset at: https://github.com/nanopore-wgs-consortium/NA12878.