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      Evaluation of Nucleic Acid Isothermal Amplification Methods for Human Clinical Microbial Infection Detection

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          Abstract

          Battling infection is a major healthcare objective. Untreated infections can rapidly evolve toward the condition of sepsis in which the body begins to fail and resuscitation becomes critical and tenuous. Identification of infection followed by rapid antimicrobial treatment are primary goals of medical care, but precise identification of offending organisms by current methods is slow and broad spectrum empirical therapy is employed to cover most potential pathogens. Current methods for identification of bacterial pathogens in a clinical setting typically require days of time, or a 4- to 8-h growth phase followed by DNA extraction, purification and PCR-based amplification. We demonstrate rapid (70–120 min) genetic diagnostics methods utilizing loop-mediated isothermal amplification (LAMP) to test for 15 common infection pathogen targets, called the Infection Diagnosis Panel (In-Dx). The method utilizes filtration to rapidly concentrate bacteria in sample matrices with lower bacterial loads and direct LAMP amplification without DNA purification from clinical blood, urine, wound, sputum and stool samples. The In-Dx panel was tested using two methods of detection: (1) real-time thermocycler fluorescent detection of LAMP amplification and (2) visual discrimination of color change in the presence of Eriochrome Black T (EBT) dye following amplification. In total, 239 duplicate samples were collected (31 blood, 122 urine, 73 mucocutaneous wound/swab, 11 sputum and two stool) from 229 prospectively enrolled hospital patients with suspected clinical infection and analyzed both at the hospital and by In-Dx. Sensitivity (Se) of the In-Dx panel targets pathogens from urine samples by In-Dx was 91.1% and specificity (Sp) was 97.3%, with a positive predictive value (PPV) of 53.7% and a negative predictive value (NPV) of 99.7% as compared to clinical microbial detection methods. Sensitivity of detection of the In-Dx panel from mucocutaneous swab samples was 65.5% with a Sp of 99.3%, and a PPV of 84% and NPV of 98% as compared to clinical microbial detection methods. Results indicate the LAMP-based In-Dx panel allows rapid and precise diagnosis of clinical infections by targeted pathogens across multiple culture types for point-of-care utilization.

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          Most cited references20

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          Coagulase-negative staphylococci.

          The definition of the heterogeneous group of coagulase-negative staphylococci (CoNS) is still based on diagnostic procedures that fulfill the clinical need to differentiate between Staphylococcus aureus and those staphylococci classified historically as being less or nonpathogenic. Due to patient- and procedure-related changes, CoNS now represent one of the major nosocomial pathogens, with S. epidermidis and S. haemolyticus being the most significant species. They account substantially for foreign body-related infections and infections in preterm newborns. While S. saprophyticus has been associated with acute urethritis, S. lugdunensis has a unique status, in some aspects resembling S. aureus in causing infectious endocarditis. In addition to CoNS found as food-associated saprophytes, many other CoNS species colonize the skin and mucous membranes of humans and animals and are less frequently involved in clinically manifested infections. This blurred gradation in terms of pathogenicity is reflected by species- and strain-specific virulence factors and the development of different host-defending strategies. Clearly, CoNS possess fewer virulence properties than S. aureus, with a respectively different disease spectrum. In this regard, host susceptibility is much more important. Therapeutically, CoNS are challenging due to the large proportion of methicillin-resistant strains and increasing numbers of isolates with less susceptibility to glycopeptides.
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            BreathDx – molecular analysis of exhaled breath as a diagnostic test for ventilator–associated pneumonia: protocol for a European multicentre observational study

            Background The diagnosis of ventilator-associated pneumonia (VAP) remains time-consuming and costly, the clinical tools lack specificity and a bedside test to exclude infection in suspected patients is unavailable. Breath contains hundreds to thousands of volatile organic compounds (VOCs) that result from host and microbial metabolism as well as the environment. The present study aims to use breath VOC analysis to develop a model that can discriminate between patients who have positive cultures and who have negative cultures with a high sensitivity. Methods/design The Molecular Analysis of Exhaled Breath as Diagnostic Test for Ventilator-Associated Pneumonia (BreathDx) study is a multicentre observational study. Breath and bronchial lavage samples will be collected from 100 and 53 intubated and ventilated patients suspected of VAP. Breath will be analysed using Thermal Desorption – Gas Chromatography – Mass Spectrometry (TD-GC-MS). The primary endpoint is the accuracy of cross-validated prediction for positive respiratory cultures in patients that are suspected of VAP, with a sensitivity of at least 99% (high negative predictive value). Discussion To our knowledge, BreathDx is the first study powered to investigate whether molecular analysis of breath can be used to classify suspected VAP patients with and without positive microbiological cultures with 99% sensitivity. Trial registration UKCRN ID number 19086, registered May 2015; as well as registration at www.trialregister.nl under the acronym ‘BreathDx’ with trial ID number NTR 6114 (retrospectively registered on 28 October 2016).
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              Centrifugal loop-mediated isothermal amplification microdevice for rapid, multiplex and colorimetric foodborne pathogen detection.

              We present a centrifugal microfluidic device which enables multiplex foodborne pathogen identification by loop-mediated isothermal amplification (LAMP) and colorimetric detection using Eriochrome Black T (EBT). Five identical structures were designed in the centrifugal microfluidic system to perform the genetic analysis of 25 pathogen samples in a high-throughput manner. The sequential loading and aliquoting of the LAMP cocktail, the primer mixtures, and the DNA sample solutions were accomplished by the optimized zigzag-shaped microchannels and RPM control. We targeted three kinds of pathogenic bacteria (Escherichia coli O157:H7, Salmonella typhimurium and Vibrio parahaemolyticus) and detected the amplicons of the LAMP reaction by the EBT-mediated colorimetric method. For the limit-of-detection (LOD) test, we carried out the LAMP reaction on a chip with serially diluted DNA templates of E. coli O157:H7, and could observe the color change with 380 copies. The used primer sets in the LAMP reaction were specific only to the genomic DNA of E. coli O157:H7, enabling the on-chip selective, sensitive, and high-throughput pathogen identification with the naked eyes. The entire process was completed in 60min. Since the proposed microsystem does not require any bulky and expensive instrumentation for end-point detection, our microdevice would be adequate for point-of-care (POC) testing with high simplicity and high speed, providing an advanced genetic analysis microsystem for foodborne pathogen detection.
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                Author and article information

                Contributors
                Journal
                Front Microbiol
                Front Microbiol
                Front. Microbiol.
                Frontiers in Microbiology
                Frontiers Media S.A.
                1664-302X
                12 December 2017
                2017
                : 8
                : 2211
                Affiliations
                [1] 1Department of Osteopathic Medical Specialties, Section of Emergency Medicine, College of Osteopathic Medicine, Michigan State University , East Lansing, MI, United States
                [2] 2Civil and Environmental Engineering, Michigan State University , East Lansing, MI, United States
                [3] 3The Center for Microbial Ecology, Michigan State University , East Lansing, MI, United States
                [4] 4Bioresources Unit, Austrian Institute of Technology GmbH , Tulln, Austria
                [5] 5Department of Microbiology, Sparrow Laboratories, Sparrow Health System , Lansing, MI, United States
                [6] 6Department of Microbiology and Molecular Genetics, Michigan State University , East Lansing, MI, United States
                Author notes

                Edited by: John W. A. Rossen, University Medical Center Groningen, Netherlands

                Reviewed by: Roel Nijhuis, Leiden University Medical Center, Netherlands; Andrew F. Gardner, New England Biolabs, United States; Guido van Marle, University of Calgary, Canada

                *Correspondence: Brett E. Etchebarne, madcow@ 123456msu.edu

                Present address: Timothy A. Johnson, Department of Animal Sciences, Purdue University, West Lafayette, IN, United States

                This article was submitted to Infectious Diseases, a section of the journal Frontiers in Microbiology

                Article
                10.3389/fmicb.2017.02211
                5732957
                29b7c8bb-0843-42f5-af18-cde155a4a648
                Copyright © 2017 Etchebarne, Li, Stedtfeld, Nicholas, Williams, Johnson, Stedtfeld, Kostic, Khalife, Tiedje, Hashsham and Hughes.

                This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) or licensor are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.

                History
                : 06 June 2017
                : 26 October 2017
                Page count
                Figures: 2, Tables: 4, Equations: 0, References: 26, Pages: 13, Words: 0
                Funding
                Funded by: Michigan State University 10.13039/100007709
                Award ID: Clinical Translational Sciences Institute Seed Grant #224
                Award ID: College of Osteopathic Medical Specialties Research Award
                Categories
                Microbiology
                Methods

                Microbiology & Virology
                clinical pathogen infection,rapid detection,direct amplification,sepsis

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