Quantification of Bacterial DNA in Sepsis

January 28, 2019 updated by: Region Örebro County

Quantification of Bacterial DNA in Septic Intensive Care Patients in Relation to Concentration of Antibiotics and Clinical Outcome: A Prospective Observation Study

The purpose of this study is to evaluate whether bacterial DNA clearance measured with droplet digital Polymerase Chain Reaction (ddPCR) can be used as a measure of bacterial load in septic intensive care patients. Furthermore, the aim is to examine a possible relation between clearance of bacterial DNA and clinical outcome in the septic patient, and the relationship between concentration of beta-lactam antibiotics and the clearance of bacterial DNA.

Study Overview

Status

Unknown

Intervention / Treatment

Detailed Description

Septic syndrome caused by bloodstream infections represents a major healthcare problem worldwide and is the leading cause of mortality in the non-cardiac intensive care unit (ICU). Despite ongoing research efforts in sepsis, the mortality remains as high as 12-30%.

In severe sepsis, the time to adequate antibiotic therapy has been clearly linked to survival. However, in the emergency room, it is challenging to identify the cause behind the clinical presentation and identify patients who would benefit the most from antibiotic treatment. An indiscriminate use of antibiotics inevitably leads to bacterial resistance and unacceptable adverse events. Thus, there is a great and unmet medical need to develop novel diagnostic instruments that could inform the clinician about who would need antibiotics and who would not benefit from it. Furthermore, early identification of the causal microorganism and the inflammatory status of the patients would improve decisions about treatments and reduce administration of antibiotics.

Today the gold standard for diagnosis of bloodstream infections (BSI) is blood culture followed by identification of species and resistance pattern by standard laboratory procedures. This procedure is associated with a high specificity but also with a low sensitivity. Probable reasons for a negative blood culture despite a strong clinical suspicion could be insufficient blood volume in the culture bottles or antibiotic treatment prior to blood sampling with a subsequent failure of the bacteria to grow in the bottles.

In recent years Polymerase Chain reaction (PCR) has been proposed as a method to determine bacterial DNA based on amplification of specific subunits (16S and 18S) of bacterial ribosomal RNA. PCR require very small amount of DNA. Another advantage is the possibility to determine bacterial species from non-viable bacteria by multiplex PCRs. However most of those multiplex PCRs have shown a limited sensitivity even though they also found clinical relevant pathogens that were not detected with blood cultures. Another limitation is that multiplex-PCR only detects at maximum 95% of all sepsis causing pathogens. In a recent published study the author concluded that a tested multiplex PCR "on whole blood specimens" in adjunct to current culture-based methods provided a clinical add-on value. The future role of PCR and other molecular techniques in the clinical setting needs further evaluation.

A new method that could improve the diagnostics of sepsis and also have the possibility to follow the bacterial DNA load during antibiotic treatment is Droplet Digital PCR (ddPCR). This technique enables an absolute quantification of the sepsis causing pathogen targeting either a species specific gene or the 16S ribosomal DNA (rDNA) gene, present in several copies per bacteria. In ddPCR, the sample is divided into about 20.000 water-in-oil droplets, and in all of them the PCR-reaction occurs. Following PCR, each droplet is analyzed to determine the fraction of PCR-positive droplets giving the amount of bacterial DNA in the original sample. The absolute target DNA template concentration can then be calculated, since the size of the droplets is known, and by using Poisson distribution statistics for the correction of more than one positive reaction in the same droplet.

To identify the sepsis causing pathogen sequencing of the 16S rDNA gene or a shotgun metagenomics approach could be used. In the latter, all DNA in a sample is sequenced using Next Generation Sequencing (NGS) and the sepsis causing pathogen can be both detected and aims to be quantified through bioinformatics data analysis.

During the septic course the patient go from vasodilation to vascular leakage and finally a hypotension. This together with added intravenous fluid treatment will increase the distribution volume with an altered pharmacokinetic state for added antibiotics. Furthermore, decreased kidney function is common in ICU patients and augmented creatinine clearance during the acute phase of sepsis is described with increasing frequency. Therefore, it is difficult to anticipate the optimal antibiotic dose for the septic patient. Indeed, it is shown that in a considerable portion of ICU patients an adequate antibiotic exposure is not achieved and this is associated with negative clinical outcome.

A dysregulated host response is a cornerstone of the current understanding of sepsis pathophysiology. Initially during the sepsis course a proinflammatory response is induced by early activation genes, including cytokines associated with inflammation: tumor necrosis factor (TNF), Interleukin-1(IL-1), IL-12 and IL-18. Simultaneously an immune suppression is induced by anti-inflammatory cytokines i.e IL-10 and transforming growth factor. A disrupted homeostasis of the pro- and anti-inflammatory response is suggested to be part of the dysregulated host response in sepsis although clinically available biomarkers for its identification are lacking. Furthermore, the regulatory mediators involved in this process and the association to bacterial clearance during sepsis needs further investigation.

The gastrointestinal tract may be a source of bacteria and a worsening of the septic inflammation in ICU patients, if the epithelial barrier is disrupted. Thus intact gastrointestinal function is critical in severely diseased patients. There are no clinically available biomarkers of gastrointestinal function; however the recently described acute gastrointestinal injury (AGI) score has been shown to correlate with adverse outcome in ICU-patients.

The aim of the study is to investigate whether bacterial DNA in whole blood measured with ddPCR can be used for assessment of bacterial clearance in septic intensive care patients. To do so the investigators will search for a potential association between bacterial DNA clearance and clearance of bacteremia detected by blood cultures.

Further, the aim is to examine a possible relation between clearance of bacterial DNA, biomarkers of dysregulated host response, and clinical outcome in the septic patient. Outcome is measured as: mortality on ICU, mortality <28 days, days in the ICU, change in Sequential Organ Failure Assessment score(SOFA), time in assisted ventilation, lactate clearance, need of vasopressors, and severity of gastrointestinal injury. Further, the aim is to study the relationship between concentration of beta-lactam antibiotics i.e. the minimal inhibitory concentration for the bacteria (T>MIC) detected and the clearance of bacterial DNA. The material collected will also contribute to reveal the relationship between ddPCR and shot gun sequencing in detecting the sepsis causing bacteria.

Study Type

Observational

Enrollment (Anticipated)

60

Contacts and Locations

This section provides the contact details for those conducting the study, and information on where this study is being conducted.

Study Contact

Study Contact Backup

Study Locations

Participation Criteria

Researchers look for people who fit a certain description, called eligibility criteria. Some examples of these criteria are a person's general health condition or prior treatments.

Eligibility Criteria

Ages Eligible for Study

18 years and older (Adult, Older Adult)

Accepts Healthy Volunteers

No

Genders Eligible for Study

All

Sampling Method

Probability Sample

Study Population

Patients at the emergency department

Description

Inclusion Criteria:

  1. Adult patient at the emergency department with verified or suspected sepsis and who is in need of intensive care.
  2. Informed Consent by patient or legal representative.
  3. Indication for beta-lactam antibiotics

Exclusion Criteria:

  1. < 18 years
  2. Patients and/or relatives unable to understand study information.
  3. Anaphylactic allergy to beta-lactam antibiotics
  4. Patients already treated with antibiotics

Study Plan

This section provides details of the study plan, including how the study is designed and what the study is measuring.

How is the study designed?

Design Details

  • Observational Models: Cohort
  • Time Perspectives: Prospective

Cohorts and Interventions

Group / Cohort
Intervention / Treatment
intensive care patients with sepsis
Adult patients with verified or suspected sepsis admitted to the intensive care department from the emergency department
Blood samples taken every third hour during the first 48 hours of intensive care

What is the study measuring?

Primary Outcome Measures

Outcome Measure
Measure Description
Time Frame
Bacterial DNA measured by 16S ddPCR
Time Frame: 48 hours
copies/mL
48 hours

Secondary Outcome Measures

Outcome Measure
Measure Description
Time Frame
Concentration of beta-lactam antibiotics
Time Frame: 48 hours
mg/mL
48 hours
Lactate clearance
Time Frame: 48 hours
mmol/L, clearance %
48 hours
SOFA
Time Frame: ICU stay up to 30 days
Sequential Organ Failure Assessment score, from 0(normal) to 24(highest organ dysfunction)
ICU stay up to 30 days
Time in assisted ventilation
Time Frame: ICU stay up to 30 days
hours
ICU stay up to 30 days
Need of vasopressors
Time Frame: ICU stay up to 30 days
mcg/kg/hour
ICU stay up to 30 days
Days in the ICU
Time Frame: ICU stay up to 30 days
Days
ICU stay up to 30 days
AGI score
Time Frame: ICU stay up to 30 days
Acute Gastrointestinal Injury Score 0(normal) to 4(life-threatening GI complications)
ICU stay up to 30 days
ICU mortality
Time Frame: ICU stay up to 30 days
Occurence of death during ICU stay
ICU stay up to 30 days
28-day mortality
Time Frame: 28 days
Occurence of death at day 28
28 days
Gastrointestinal complications
Time Frame: 28 days
Occurence of gastrointestinal complications including GI-bleeding, GI-ischemia, pancreatitis and ileus
28 days
Intestinal fatty acid binding protein (I-FABP)
Time Frame: 48 hours
picogram/mL, biomarker of gastrointestinal injury
48 hours
Citrulline
Time Frame: 48 hours
nmol/mL, biomarker of gastrointestinal injury
48 hours
Nuclear DNA
Time Frame: 48 hours
copies/mL
48 hours
Cytokines (Th1 and Th2 signature panel)
Time Frame: 48 hours
pg/ml
48 hours
Blood cell populations
Time Frame: 48 hours
Mean fluorescence intensity/ Antibodies/cell. Monocytes, granulocytes, T-cells, and myeloid derived stem cells including surface markers such as PD-1(Programmed cell death protein 1),PD-L1 (Programmed death ligand 1), and HLA-DR (Human leukocyte Antigen - DR isotype)
48 hours
HLA-DR messenger RNA (mRNA)
Time Frame: 48 hours
ratio/reference gene
48 hours
Procalcitonin
Time Frame: 48 hours
mcg/L
48 hours
MicroRNA (miRNA)
Time Frame: 48 hours
Relative expression/ratio reference gene, transcriptomic and quantitative PCR based measurement
48 hours

Collaborators and Investigators

This is where you will find people and organizations involved with this study.

Investigators

  • Principal Investigator: Jan Källman, PhD, Region Örebro County
  • Principal Investigator: Hans Hjelmqvist, Phd, Region Örebro County

Study record dates

These dates track the progress of study record and summary results submissions to ClinicalTrials.gov. Study records and reported results are reviewed by the National Library of Medicine (NLM) to make sure they meet specific quality control standards before being posted on the public website.

Study Major Dates

Study Start (Actual)

January 7, 2019

Primary Completion (Anticipated)

December 31, 2021

Study Completion (Anticipated)

May 31, 2022

Study Registration Dates

First Submitted

December 4, 2018

First Submitted That Met QC Criteria

December 19, 2018

First Posted (Actual)

December 20, 2018

Study Record Updates

Last Update Posted (Actual)

January 29, 2019

Last Update Submitted That Met QC Criteria

January 28, 2019

Last Verified

December 1, 2018

More Information

Terms related to this study

Plan for Individual participant data (IPD)

Plan to Share Individual Participant Data (IPD)?

Undecided

Drug and device information, study documents

Studies a U.S. FDA-regulated drug product

No

Studies a U.S. FDA-regulated device product

No

This information was retrieved directly from the website clinicaltrials.gov without any changes. If you have any requests to change, remove or update your study details, please contact register@clinicaltrials.gov. As soon as a change is implemented on clinicaltrials.gov, this will be updated automatically on our website as well.

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