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      Evaluation of Real-Time PCR Coupled With Multiplex Probe Melting Curve Analysis for Pathogen Detection in Patients With Suspected Bloodstream Infections

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          Abstract

          Background: This study aimed to evaluate real-time polymerase chain reaction coupled with multiplex probe melting curve analysis (PCR-MCA) for pathogen detection in patients with suspected bloodstream infections (BSIs).

          Methods: A PCR-MCA assay was developed for simultaneous identification of 28 kinds of the most common pathogens and two resistance genes within a few hours. The diagnostic performance of the PCR-MCA assay was determined and compared to the results of blood culture.

          Results: A total of 2,844 consecutive new episodes of suspected BSIs in 2,763 patients were included in this study. There were 269 episodes of pathogens identified by blood culture. For all the pathogens tested, the PCR-MCA assay exhibited a sensitivity of 88.8% (239/269), specificity of 100% (2,575/2,575), and agreement of 98.9% (2,814/2,844). For the pathogens on the PCR-MCA list, the PCR-MCA results had a sensitivity of 99.2% (239/241), specificity of 100% (2,575/2,575), and agreement of 99.9% (2,814/2,816) compared with the results of blood culture. For seven samples with multiple pathogens identified simultaneously during one blood culture investigation, the PCR-MCA assay verified the results of the blood culture, with an agreement rate of 100% for each.

          Conclusion: The PCR-MCA assay could discover 88.8% of the pathogens in clinical practice, showing excellent diagnostic performance vs. that of blood culture for pathogen detection in patients with suspected BSIs, and would contribute to rapid diagnosis and correct antibiotic administration.

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          Most cited references 21

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          The Third International Consensus Definitions for Sepsis and Septic Shock (Sepsis-3).

          Definitions of sepsis and septic shock were last revised in 2001. Considerable advances have since been made into the pathobiology (changes in organ function, morphology, cell biology, biochemistry, immunology, and circulation), management, and epidemiology of sepsis, suggesting the need for reexamination.
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            Surviving Sepsis Campaign: international guidelines for management of severe sepsis and septic shock: 2008.

            To provide an update to the original Surviving Sepsis Campaign clinical management guidelines, "Surviving Sepsis Campaign Guidelines for Management of Severe Sepsis and Septic Shock," published in 2004. Modified Delphi method with a consensus conference of 55 international experts, several subsequent meetings of subgroups and key individuals, teleconferences, and electronic-based discussion among subgroups and among the entire committee. This process was conducted independently of any industry funding. We used the Grades of Recommendation, Assessment, Development and Evaluation (GRADE) system to guide assessment of quality of evidence from high (A) to very low (D) and to determine the strength of recommendations. A strong recommendation (1) indicates that an intervention's desirable effects clearly outweigh its undesirable effects (risk, burden, cost) or clearly do not. Weak recommendations (2) indicate that the tradeoff between desirable and undesirable effects is less clear. The grade of strong or weak is considered of greater clinical importance than a difference in letter level of quality of evidence. In areas without complete agreement, a formal process of resolution was developed and applied. Recommendations are grouped into those directly targeting severe sepsis, recommendations targeting general care of the critically ill patient that are considered high priority in severe sepsis, and pediatric considerations. Key recommendations, listed by category, include early goal-directed resuscitation of the septic patient during the first 6 hrs after recognition (1C); blood cultures before antibiotic therapy (1C); imaging studies performed promptly to confirm potential source of infection (1C); administration of broad-spectrum antibiotic therapy within 1 hr of diagnosis of septic shock (1B) and severe sepsis without septic shock (1D); reassessment of antibiotic therapy with microbiology and clinical data to narrow coverage, when appropriate (1C); a usual 7-10 days of antibiotic therapy guided by clinical response (1D); source control with attention to the balance of risks and benefits of the chosen method (1C); administration of either crystalloid or colloid fluid resuscitation (1B); fluid challenge to restore mean circulating filling pressure (1C); reduction in rate of fluid administration with rising filing pressures and no improvement in tissue perfusion (1D); vasopressor preference for norepinephrine or dopamine to maintain an initial target of mean arterial pressure > or = 65 mm Hg (1C); dobutamine inotropic therapy when cardiac output remains low despite fluid resuscitation and combined inotropic/vasopressor therapy (1C); stress-dose steroid therapy given only in septic shock after blood pressure is identified to be poorly responsive to fluid and vasopressor therapy (2C); recombinant activated protein C in patients with severe sepsis and clinical assessment of high risk for death (2B except 2C for postoperative patients). In the absence of tissue hypoperfusion, coronary artery disease, or acute hemorrhage, target a hemoglobin of 7-9 g/dL (1B); a low tidal volume (1B) and limitation of inspiratory plateau pressure strategy (1C) for acute lung injury (ALI)/acute respiratory distress syndrome (ARDS); application of at least a minimal amount of positive end-expiratory pressure in acute lung injury (1C); head of bed elevation in mechanically ventilated patients unless contraindicated (1B); avoiding routine use of pulmonary artery catheters in ALI/ARDS (1A); to decrease days of mechanical ventilation and ICU length of stay, a conservative fluid strategy for patients with established ALI/ARDS who are not in shock (1C); protocols for weaning and sedation/analgesia (1B); using either intermittent bolus sedation or continuous infusion sedation with daily interruptions or lightening (1B); avoidance of neuromuscular blockers, if at all possible (1B); institution of glycemic control (1B), targeting a blood glucose < 150 mg/dL after initial stabilization (2C); equivalency of continuous veno-veno hemofiltration or intermittent hemodialysis (2B); prophylaxis for deep vein thrombosis (1A); use of stress ulcer prophylaxis to prevent upper gastrointestinal bleeding using H2 blockers (1A) or proton pump inhibitors (1B); and consideration of limitation of support where appropriate (1D). Recommendations specific to pediatric severe sepsis include greater use of physical examination therapeutic end points (2C); dopamine as the first drug of choice for hypotension (2C); steroids only in children with suspected or proven adrenal insufficiency (2C); and a recommendation against the use of recombinant activated protein C in children (1B). There was strong agreement among a large cohort of international experts regarding many level 1 recommendations for the best current care of patients with severe sepsis. Evidenced-based recommendations regarding the acute management of sepsis and septic shock are the first step toward improved outcomes for this important group of critically ill patients.
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              New developments in the diagnosis of bloodstream infections.

              New techniques have emerged for the detection of bacteria in blood, because the blood culture as gold standard is slow and insufficiently sensitive when the patient has previously received antibiotics or in the presence of fastidious organisms. DNA-based techniques, hybridisation probes, and PCR-based detection or protein-based detection by mass spectroscopy are aimed at rapid identification of bacteria and provide results within 2 h after the first signal of growth in conventional blood cultures. Also, detection of microorganisms directly in blood by pathogen-specific or broad-range PCR assays (eubacterial or panfungal) shows promising results. Interpretation is complex, however, because of detection of DNA rather than living pathogens, the risk of interfering contamination, the presence of background DNA in blood, and the lack of a gold standard. As these techniques are emerging, clinical value and cost-effectiveness have to be assessed. Nevertheless, molecular assays are expected eventually to replace the current conventional microbiological techniques for detection of bloodstream infections.
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                Author and article information

                Contributors
                Journal
                Front Cell Infect Microbiol
                Front Cell Infect Microbiol
                Front. Cell. Infect. Microbiol.
                Frontiers in Cellular and Infection Microbiology
                Frontiers Media S.A.
                2235-2988
                22 October 2019
                2019
                : 9
                10.3389/fcimb.2019.00361
                6817478
                Copyright © 2019 Xiao, Shen, Zhao, Yao, Yang and Niu.

                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) and the copyright owner(s) 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.

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                Figures: 0, Tables: 5, Equations: 0, References: 21, Pages: 11, Words: 7589
                Categories
                Cellular and Infection Microbiology
                Original Research

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