The rise of antibiotic-resistant bacteria has led to an urgent need for rapid detection of drug resistance in clinical samples, and improvements in global surveillance. Here we show how de Bruijn ...graph representation of bacterial diversity can be used to identify species and resistance profiles of clinical isolates. We implement this method for Staphylococcus aureus and Mycobacterium tuberculosis in a software package ('Mykrobe predictor') that takes raw sequence data as input, and generates a clinician-friendly report within 3 minutes on a laptop. For S. aureus, the error rates of our method are comparable to gold-standard phenotypic methods, with sensitivity/specificity of 99.1%/99.6% across 12 antibiotics (using an independent validation set, n=470). For M. tuberculosis, our method predicts resistance with sensitivity/specificity of 82.6%/98.5% (independent validation set, n=1,609); sensitivity is lower here, probably because of limited understanding of the underlying genetic mechanisms. We give evidence that minor alleles improve detection of extremely drug-resistant strains, and demonstrate feasibility of the use of emerging single-molecule nanopore sequencing techniques for these purposes.
Two billion people are infected with
, leading to 10 million new cases of active tuberculosis and 1.5 million deaths annually. Universal access to drug susceptibility testing (DST) has become a World ...Health Organization priority. We previously developed a software tool,
, which provided offline species identification and drug resistance predictions for
from whole genome sequencing (WGS) data. Performance was insufficient to support the use of WGS as an alternative to conventional phenotype-based DST, due to mutation catalogue limitations. Here we present a new tool,
, which provides the same functionality based on a new software implementation. Improvements include i) an updated mutation catalogue giving greater sensitivity to detect pyrazinamide resistance, ii) support for user-defined resistance catalogues, iii) improved identification of non-tuberculous mycobacterial species, and iv) an updated statistical model for Oxford Nanopore Technologies sequencing data.
is released under MIT license at https://github.com/mykrobe-tools/mykrobe. We incorporate mutation catalogues from the CRyPTIC consortium et al. (2018) and from Walker et al. (2015), and make improvements based on performance on an initial set of 3206 and an independent set of 5845
Illumina sequences. To give estimates of error rates, we use a prospectively collected dataset of 4362
. Using culture based DST as the reference, we estimate
to be 100%, 95%, 82%, 99% sensitive and 99%, 100%, 99%, 99% specific for rifampicin, isoniazid, pyrazinamide and ethambutol resistance prediction respectively. We benchmark against four other tools on 10207 (=5845+4362) samples, and also show that
gives concordant results with nanopore data. We measure the ability of
-based DST to guide personalized therapeutic regimen design in the context of complex drug susceptibility profiles, showing 94% concordance of implied regimen with that driven by phenotypic DST, higher than all other benchmarked tools.
Nature Communications 6: Article number: 10063 (2015); Published: 21 December 2015; Updated: 20 April 2016 In Supplementary Data 3 of this Article, one of the Staphylococcus aureus accession codes is ...incorrect, as follows: SRR2101499 should be ERR1197981.
IR spectroscopy is an excellent method for biological analyses. It enables the nonperturbative, label-free extraction of biochemical information and images toward diagnosis and the assessment of cell ...functionality. Although not strictly microscopy in the conventional sense, it allows the construction of images of tissue or cell architecture by the passing of spectral data through a variety of computational algorithms. Because such images are constructed from fingerprint spectra, the notion is that they can be an objective reflection of the underlying health status of the analyzed sample. One of the major difficulties in the field has been determining a consensus on spectral pre-processing and data analysis. This manuscript brings together as coauthors some of the leaders in this field to allow the standardization of methods and procedures for adapting a multistage approach to a methodology that can be applied to a variety of cell biological questions or used within a clinical setting for disease screening or diagnosis. We describe a protocol for collecting IR spectra and images from biological samples (e.g., fixed cytology and tissue sections, live cells or biofluids) that assesses the instrumental options available, appropriate sample preparation, different sampling modes as well as important advances in spectral data acquisition. After acquisition, data processing consists of a sequence of steps including quality control, spectral pre-processing, feature extraction and classification of the supervised or unsupervised type. A typical experiment can be completed and analyzed within hours. Example results are presented on the use of IR spectra combined with multivariate data processing.
Understanding molecular transport in the brain is critical to care and prevention of neurological disease and injury. A key question is whether transport occurs primarily by diffusion, or also by ...convection or dispersion. Dynamic contrast-enhanced (DCE-MRI) experiments have long reported solute transport in the brain that appears to be faster than diffusion alone, but this transport rate has not been quantified to a physically relevant value that can be compared to known diffusive rates of tracers.
In this work, DCE-MRI experimental data is analyzed using subject-specific finite-element models to quantify transport in different anatomical regions across the whole mouse brain. The set of regional effective diffusivities (Formula: see text), a transport parameter combining all mechanisms of transport, that best represent the experimental data are determined and compared to apparent diffusivity (Formula: see text), the known rate of diffusion through brain tissue, to draw conclusions about dominant transport mechanisms in each region.
In the perivascular regions of major arteries, Formula: see text for gadoteridol (550 Da) was over 10,000 times greater than Formula: see text. In the brain tissue, constituting interstitial space and the perivascular space of smaller blood vessels, Formula: see text was 10-25 times greater than Formula: see text.
The analysis concludes that convection is present throughout the brain. Convection is dominant in the perivascular space of major surface and branching arteries (Pe > 1000) and significant to large molecules (> 1 kDa) in the combined interstitial space and perivascular space of smaller vessels (not resolved by DCE-MRI). Importantly, this work supports perivascular convection along penetrating blood vessels.
The clinical and complete pathological response of a primary breast cancer to chemotherapy has been shown to be an important prognostic for survival. However, the majority of patients do not ...experience a complete pathological response to primary chemotherapy and the significance of lesser degrees of histological response is uncertain and the prognostic significance is unknown. The purpose of this study was to evaluate a new histological grading system to assess response of breast cancers to primary chemotherapy and to determine if such a system has prognostic value.
A consecutive series of 176 patients with large (≥4
cm) and locally advanced breast cancers were treated with multimodality therapy comprising primary chemotherapy, surgery, radiotherapy and tamoxifen. All underwent assessment of the primary breast tumour before and after completion of chemotherapy. Residual tumour was excised after completion of chemotherapy (mastectomy or wide local excision with axillary surgery). The removed tissue was assessed and response to chemotherapy graded using a five-point histological grading system based with the fundamental feature being a reduction in tumour cellularity; comparison being made with a pre-treatment core biopsy. All patients were followed up for 5 years or more. Pathological responses were compared to 5 year overall survival and disease-free survival using log rank tests.
The overall 5-year survival for all patients was 71%, and 5 year disease free interval was 60%. There was a significant correlation between pathological response using this new grading system and both overall survival (
P=0.02) and disease-free interval (
P=0.04). In a multivariate analysis of known prognostic factors, the Miller/Payne grading system was an independent predictor of overall patient survival.
This grading system, which assesses the histological response to primary chemotherapy, can predict overall survival and disease-free interval in patients with large and locally advanced breast cancers treated with such therapy. The relationship of degree of histological response to overall and disease-free survival has been shown in univariate and multivariate analyses and could potentially have an important role in the clinical management of patients with locally advanced breast cancer undergoing primary chemotherapy.
Aims
The Medicines and Healthcare products Regulatory Agency Yellow Card scheme (YCS) is the UK's system that collects spontaneous reports about suspected adverse drug reactions (ADRs). Reporting of ...suspected ADRs by young people (age <19 years) in the UK is extremely uncommon, driving efforts to improve awareness and reporting.
Methods
Quality improvement project, using an anonymous online survey about updated information for young people, distributed through school pupils (age 13–18 years) across the UK through the Alder Hey Research Ambassador programme.
Results
Research Ambassadors were recruited in 21 schools and colleges, generating 2933 responses (15 November 2022–08 April 2023); 6.3% of respondents had heard of the YCS, and 0.8% had previously reported a Yellow Card. There were 307 suspected drug–event combinations reported, 36 of which required attendance at hospital. The updated YCS reporting guide was understood by 92.8% of young people, and 90.8% reported knowing more about ADRs after reading the guide. The percentage of young people ‘Not comfortable’ reporting a suspected ADR decreased from 13.3% (before reading) to 4.1% after reading (P < .000001), and 84.5% of young people reported willingness to report a side effect in the future. The most common comments regarding further improvement of the information were content, or length of the text could be altered in some way (n = 543, 26.1%) and graphic design could be improved (n = 357, 17.2%).
Conclusions
The age‐appropriate information provided met many of their needs, increasing willingness to report. Integration into existing education curricula in the UK would facilitate knowledge transfer and improve reporting.
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