Yersinia pestis, the aetiological agent of the plague, causes sporadic disease in endemic areas of the world and is classified as a National Institute of Allergy and Infectious Diseases Category A Priority Pathogen because of its potential to be used as a bioweapon. Health departments, hospitals and government agencies need the ability to rapidly identify and characterize cultured isolates of this bacterium. Assays have been developed to perform this function; however, they are limited in their ability to distinguish Y. pestis from Yersinia pseudotuberculosis. This report describes the creation of a real-time PCR assay using Taqman probes that exclusively identifies Y. pestis using a unique target sequence of the yihN gene on the chromosome. As with other Y. pestis PCR assays, three major genes located on each of the three virulence plasmids were included: lcrV on pCD1, caf1 on pMT1 and pla on pPCP1. The quadruplex assay was validated on a collection of 192 Y. pestis isolates and 52 near-neighbour isolates. It was discovered that only 72 % of natural plague isolates from the states of New Mexico and Utah harboured all three virulence plasmids. This quadruplex assay proved to be 100 % successful in differentiating Y. pestis from all near neighbours tested and was able to reveal which of the three virulence plasmids a particular isolate possessed.
BACKGROUND: Intraoperative hypotension is common and associated with organ injury and death, although randomized data showing a causal relationship remain sparse. A risk-adjusted measure of intraoperative hypotension may therefore contribute to quality improvement efforts. METHODS: The measure we developed defines hypotension as a mean arterial pressure <65 mm Hg sustained for at least 15 cumulative minutes. Comparisons are based on whether clinicians have more or fewer cases of hypotension than expected over 12 months, given their patient mix. The measure was developed and evaluated with data from 225,389 surgeries in 5 hospitals. We assessed discrimination and calibration of the risk adjustment model, then calculated the distribution of clinician-level measure scores, and finally estimated the signal-to-noise reliability and predictive validity of the measure. RESULTS: The risk adjustment model showed acceptable calibration and discrimination (area under the curve was 0.72 and 0.73 in different validation samples). Clinician-level, risk-adjusted scores varied widely, and 36% of clinicians had significantly more cases of intraoperative hypotension than predicted. Clinician-level score distributions differed across hospitals, indicating substantial hospital-level variation. The mean signal-to-noise reliability estimate was 0.87 among all clinicians and 0.94 among clinicians with >30 cases during the 12-month measurement period. Kidney injury and in-hospital mortality were most common in patients whose anesthesia providers had worse scores. However, a sensitivity analysis in 1 hospital showed that score distributions differed markedly between anesthesiology fellows and attending anesthesiologists or certified registered nurse anesthetists; score distributions also varied as a function of the fraction of cases that were inpatients. CONCLUSIONS: Intraoperative hypotension was common and was associated with acute kidney injury and in-hospital mortality. There were substantial variations in clinician-level scores, and the measure score distribution suggests that there may be opportunity to reduce hypotension which may improve patient safety and outcomes. However, sensitivity analyses suggest that some portion of the variation results from limitations of risk adjustment. Future versions of the measure should risk adjust for important patient and procedural factors including comorbidities and surgical complexity, although this will require more consistent structured data capture in anesthesia information management systems. Including structured data on additional risk factors may improve hypotension risk prediction which is integral to the measure’s validity.
Clostridium botulinum is the aetiological agent of botulism, a disease marked by flaccid paralysis that can progress to asphyxiation and death. This species is defined by the production of one of the botulinum neurotoxins (BoNTs), which are the most potent toxins known. Because of their potency, these toxins have the potential to be used as biological weapons, and therefore C. botulinum has been classified as a category A select agent. There are four related but antigenically distinct BoNT types that cause disease in humans, A, B, E and F. The mouse bioassay is the current gold standard by which BoNTs are confirmed. However, this method is expensive, slow and labour-intensive. Although PCR-based assays have been used extensively for the detection of BoNT-producing bacteria in food, animals and faecal samples, and recently to help diagnose disease in humans, no real-time quantitative PCR (qPCR) assay has yet been developed that can identify and differentiate all four BoNTs that cause disease in humans. This report describes the development of a qPCR single-tube assay that uniquely identifies these four BoNTs responsible for human disease. A total of 79 C. botulinum isolates with varying toxin types was evaluated in this study, as well as numerous near-neighbours and other bacterial species. The results showed that this quadruplex assay was capable of detecting any of the four toxin genes in a given sample at a sensitivity of about 130-840 fg genomic DNA and could detect the presence of up to all four BoNT genes simultaneously in a given sample. The assay was also functional in the presence of extraneous organic matter commonly found in various environmental samples. Abbreviations: BoNT, botulinum neurotoxin; qPCR, real-time quantitative PCR. Details of qPCR results for individual bacterial isolates are available as supplementary material with the online version of this paper.
A recent advance in biotechnology has been the understanding of gene silencing. The process, elucidated in Caenorhabditis elegans and known as RNA interference (RNAi), is a vital defence mechanism in plants, fungi and arthropods. Key aspects of RNAi are specificity for precise silencing of RNA sequences; ability to transfer silencing from cell to cell and host to non-host; and potential to function in many organisms. This work investigates whether a plant-produced RNAi signal can induce specific gene silencing in non-viral pathogens that have intimate contact with host cytoplasm. By transforming a plant pest with the gfp gene, and subsequently introducing RNAi constructs targeting the gfp sequence, it should be possible to observe a gfp silencing response. In concurrent experiments appropriate plant pathogens that have intimate connections with host cytoplasm, such as Sclerotinia sclerotiorum and Plasmodiophora brassicae, will be transformed with the gfp gene and used to challenge the RNAi expressing host plant. If the system is functional, then the silencing of pathogen GFP expression should be observed upon infection of the RNAi expressing host plant. Future work will identify genes essential to pathogenicity of target pathogens and use these to design RNAi constructs for plant transformation.
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