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Classical Microbiological Diagnostics of Bacteremia: Are the Negative Results Really Negative? What is the Laboratory Result Telling Us About the "Gold Standard"?

ABSTRACT: Standard blood cultures require at least 24-120 h to be reported as preliminary positive. The objective of this study was to compare the reliability of Gram staining and fluorescent in-situ hybridization (FISH) for detecting bacteria in otherwise negative blood culture bottles. Ninety-six sets were taken from patients with a diagnosis of sepsis. Six incomplete blood culture sets and eight blood cultures sets demonstrating positive growth were excluded. We performed Gram stain and FISH on 82 sets taken from post-operative septic patients: 82 negative aerobic blood cultures, 82 anaerobic blood cultures, and 82 blood samples, as well as 57 blood samples taken from healthy volunteers. From the eighty-two blood sets analyzed from the septic patients, Gram stain visualized bacteria in 62.2% of blood samples, 35.4% of the negative aerobic bottles, and in 31.7% of the negative anaerobic bottles. Utilizing FISH, we detected bacteria in 75.6%, 56.1%, and 64.6% respectively. Among the blood samples from healthy volunteers, FISH detected bacteria in 64.9%, while Gram stain detected bacteria in only 38.6%. The time needed to obtain the study results using Gram stain was 1 h, for FISH 4 h, and for the culture method, considering the duration of growth, 5 days. Gram stain and FISH allow quick detection of bacteria in the blood taken directly from a patient. Finding phagocytosed bacteria, which were also detected among healthy individuals, confirms the hypothesis that blood microbiome exists.

SUBMITTER: Zrodlowski T

PROVIDER: S-EPMC7143506 | biostudies-literature | 2020 Feb

REPOSITORIES: biostudies-literature

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Classical Microbiological Diagnostics of Bacteremia: Are the Negative Results Really Negative? What is the Laboratory Result Telling Us About the "Gold Standard"?

Źródłowski Tomasz T Sobońska Joanna J Salamon Dominika D McFarlane Isabel M IM Ziętkiewicz Mirosław M Gosiewski Tomasz T

Microorganisms 20200229 3

Standard blood cultures require at least 24-120 h to be reported as preliminary positive. The objective of this study was to compare the reliability of Gram staining and fluorescent in-situ hybridization (FISH) for detecting bacteria in otherwise negative blood culture bottles. Ninety-six sets were taken from patients with a diagnosis of sepsis. Six incomplete blood culture sets and eight blood cultures sets demonstrating positive growth were excluded. We performed Gram stain and FISH on 82 sets ...[more]

PMID: 32121353

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Project description:OBJECTIVES:Culture-based diagnostics represent the standard of care in septic patients, but are highly insensitive and in many cases unspecific. We recently demonstrated the general feasibility of next-generation sequencing-based diagnostics using free circulating nucleic acids (cell-free DNA) in plasma samples of septic patients. Within the presented investigation, higher performance of next-generation sequencing-based diagnostics was validated by comparison to matched blood cultures. DESIGN:A secondary analysis of a prospective, observational, single-center study. SETTING:Surgical ICU of a university hospital and research laboratory. PATIENTS:Fifty patients with septic shock, 20 uninfected patients with elective surgery as control cohort. INTERVENTIONS:None. MEASUREMENTS AND MAIN RESULTS:From 256 plasma samples of 48 septic patients at up to seven consecutive time points within the 28-day observation period, cell-free DNA was isolated and analyzed by next-generation sequencing and relevance scoring. In parallel, results from culture-based diagnostics (e.g., blood culture) were obtained. Plausibility of blood culture and next-generation sequencing results as well as adequacy of antibiotic therapy was evaluated by an independent expert panel. In contrast to blood culture with a positivity rate of 33% at sepsis onset, the positivity rate for next-generation sequencing-based pathogen identification was 72%. Over the whole study period, blood culture positivity was 11%, and next-generation sequencing positivity was 71%. Ninety-six percent of positive next-generation sequencing results for acute sepsis time points were plausible and would have led to a change to a more adequate therapy in 53% of cases as assessed by the expert evaluation. CONCLUSIONS:Our results show that next-generation sequencing-based analyses of bloodstream infections provide a valuable diagnostic platform for the identification of clinically relevant pathogens with higher sensitivity and specificity than blood culture, indicating that patients might benefit from a more appropriate therapy based on next-generation sequencing-based diagnosis.

Project description:IntroductionSepsis and septic shock are the most severe forms of infection affecting predominantly elderly people, preterm and term neonates, and young infants. Even in high-income countries sepsis causes about 8% of admissions to pediatric intensive care units (PICUs). Early diagnosis, rapid anti-infective treatment, and prompt hemodynamic stabilization are crucial for patient survival. In this context, it is essential to identify the causative pathogen as soon as possible to optimize antimicrobial treatment. To date, culture-based diagnostic procedures (e.g., blood cultures) represent the standard of care. However, they have 2 major problems: on the one hand, in the case of very small sample volumes (and thus usually in children), they are not sufficiently sensitive. On the other hand, with a time-to-result of 2 to 5 days, blood cultures need a relatively long time for the anti-infective therapy to be calculated. To overcome these problems, culture-independent molecular diagnostic procedures such as unbiased sequence analysis of circulating cell-free DNA (cfDNA) from plasma samples of septic patients by next-generation sequencing (NGS) have been tested successfully in adult septic patients. However, these results still need to be transferred to the pediatric setting.MethodsThe Next GeneSiPS-Trial is a prospective, observational, non-interventional, multicenter study used to assess the diagnostic performance of an NGS-based approach for the identification of causative pathogens in (preterm and term) neonates (d1-d28, n = 50), infants (d29 to <1 yr, n = 50), and toddlers (1 yr to <5 yr, n = 50) with suspected or proven severe sepsis or septic shock (according to the pediatric sepsis definition) by the use of the quantitative sepsis indicating quantifier (SIQ) score in comparison to standard of care (culture-based) microbiological diagnostics. Potential changes in anti-infective treatment regimens based on these NGS results will be estimated retrospectively by a panel of 3 independent clinical specialists.DiscussionNeonates, infants, and young children are significantly affected by sepsis. Fast and more sensitive diagnostic approaches are urgently needed. This prospective, observational, non-interventional, multicenter study seeks to evaluate an NGS-based approach in critically ill children suffering from sepsis.Trial registrationDRKS-ID: DRKS00015705 (registered October 24, 2018). https://www.drks.de/drks_web/navigate.do?navigationId=trial.HTML&TRIAL_ID=DRKS00015705.

Project description:Blood cultures are central to the management of patients with sepsis and bloodstream infection. Clinical decisions depend on the timely availability of laboratory information, which, in turn, depends on the optimal laboratory processing of specimens. Discrete event simulation (DES) offers insights into where optimization efforts can be targeted. Here, we generate a detailed process map of blood culture processing within a laboratory and use it to build a simulator. Direct observation of laboratory staff processing blood cultures was used to generate a flowchart of the blood culture laboratory pathway. Retrospective routinely collected data were combined with direct observations to generate probability distributions over the time taken for each event. These data were used to inform the DES model. A sensitivity analysis explored the impact of staff availability on turnaround times. A flowchart of the blood culture pathway was constructed, spanning labeling, incubation, organism identification, and antimicrobial susceptibility testing. Thirteen processes in earlier stages of the pathway, not otherwise captured by routinely collected data, were timed using direct observations. Observations revealed that specimen processing is predominantly batched. Another eight processes were timed using retrospective data. A simulator was built using DES. Sensitivity analysis revealed that specimen progression through the simulation was especially sensitive to laboratory technician availability. Gram stain reporting time was also sensitive to laboratory scientist availability. Our laboratory simulation model has wide-ranging applications for the optimization of laboratory processes and effective implementation of the changes required for faster and more accurate results.ImportanceOptimization of laboratory pathways and resource availability has a direct impact on the clinical management of patients with bloodstream infection. This research offers an insight into the laboratory processing of blood cultures at a system level and allows clinical microbiology laboratories to explore the impact of changes to processes and resources.

Dataset Information

Classical Microbiological Diagnostics of Bacteremia: Are the Negative Results Really Negative? What is the Laboratory Result Telling Us About the "Gold Standard"?

Publications

Classical Microbiological Diagnostics of Bacteremia: Are the Negative Results Really Negative? What is the Laboratory Result Telling Us About the "Gold Standard"?

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