Clinical trial NCT03782454: Quantification of Bacterial DNA in Sepsis
Official title: Quantification of Bacterial DNA in Septic Intensive Care Patients in Relation to Concentration of Antibiotics and Clinical Outcome: A Prospective Observation Study
Study type: OBSERVATIONAL
Overall status: UNKNOWN
Conditions: Septic Shock
Sepsis Bacterial
Sepsis
Keywords: Bacterial DNA clearance
Droplet digital PCR
Betalactam antibiotics
Acute gastrointestinal index
Cytokines
Lead sponsor: Region Örebro County
Sponsor class: OTHER
First posted: 2018-12-20
Enrollment: 60
Sex: ALL
Minimum age (years): 18
Standard ages: ADULT
OLDER_ADULT
Accepts healthy volunteers: no
Observational model: COHORT
Time perspective: PROSPECTIVE
Sampling method: PROBABILITY_SAMPLE
Study population: Patients at the emergency department

Brief summary: 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.

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.

Eligibility criteria: 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

Primary outcomes:
measure: Bacterial DNA measured by 16S ddPCR
timeFrame: 48 hours
Secondary outcomes:
measure: Concentration of beta-lactam antibiotics
timeFrame: 48 hours
measure: Lactate clearance
timeFrame: 48 hours
measure: SOFA
timeFrame: ICU stay up to 30 days
measure: Time in assisted ventilation
timeFrame: ICU stay up to 30 days
measure: Need of vasopressors
timeFrame: ICU stay up to 30 days
measure: Days in the ICU
timeFrame: ICU stay up to 30 days
measure: AGI score
timeFrame: ICU stay up to 30 days
measure: ICU mortality
timeFrame: ICU stay up to 30 days
measure: 28-day mortality
timeFrame: 28 days
measure: Gastrointestinal complications
timeFrame: 28 days
measure: Intestinal fatty acid binding protein (I-FABP)
timeFrame: 48 hours
measure: Citrulline
timeFrame: 48 hours
measure: Nuclear DNA
timeFrame: 48 hours
measure: Cytokines (Th1 and Th2 signature panel)
timeFrame: 48 hours
measure: Blood cell populations
timeFrame: 48 hours
measure: HLA-DR messenger RNA (mRNA)
timeFrame: 48 hours
measure: Procalcitonin
timeFrame: 48 hours
measure: MicroRNA (miRNA)
timeFrame: 48 hours
Other outcomes:
Locations:
city: Örebro
country: Sweden
facility: University Hospital in Örebro
Study officials:
affiliation: Region Örebro County
name: Jan Källman, PhD
role: PRINCIPAL_INVESTIGATOR
affiliation: Region Örebro County
name: Hans Hjelmqvist, Phd
role: PRINCIPAL_INVESTIGATOR
References: