Prognostic Significance of Bacterial Translocation Markers as Predictors of Infectious and Inflammatory Complications in Acute Mechanical Bowel Obstruction
Conditions: Intestinal Obstruction, Colorectal Cancer, Post-Op Complication, Systemic Inflammatory Response Syndrome, Colon Tumor
Sponsor: Karaganda Medical University



I. The aim of the study and introduction  
 
The aim of the study: 
Determine the diagnostic and prognostic significance of bacterial translocation as a predictor of 
the complications development in patients with malignant and benign acute bowel obstruction by 
assessing the relationship of biomarkers in the systemic circulation (LBP, sCD14-ST) with the 
detection of microorganism genes (16s rRNA) in mesenteric lymph nodes. 
 
Research objectives: 
1. 
Compare the level and dynamics of changes in the biomarkers of bacterial translocation 
(LBP, sCD14-ST) in the blood serum of operated colorectal cancer patients with and without acute 
bowel obstruction, as well as those who operated on for benign acute bowel obstruction.  
2. 
Determine the effect of clinical and morphological characteristics of a colon tumor on the 
frequency of bacterial translocation development.  
3. 
Evaluate the clinical and laboratory parameters of operated colorectal cancer patients with 
and without acute bowel obstruction, as well as those who operated on for benign acute bowel 
obstruction, depending on the presence of bacterial translocation and systemic inflammatory 
response syndrome.   
4. 
Identify the correlation between 16s rRNA in mesenteric lymph nodes and LBP, sCD14-
ST in blood serum.   
 
Introduction  
Despite modern approaches to the diagnosis and treatment of acute bowel obstruction (ABO), 
postoperative mortality ranges from 5 to 32%, and complications occur up 23% of cases [1, 2, 3]. 
One of the formidable infectious and inflammatory complications of ABO is sepsis. In ABO 
caused by colorectal cancer, sepsis occurs from 1.7 to 10.5% of cases, and in benign ABO from 3 
to 10.29% [4, 5, 6, 7]. The main component of the development of sepsis in ABO is bacterial 
translocation (BT) [8].   
Bacterial translocation is the migration of intestinal bacteria or their products through the intestinal 
mucosa into the mesenteric lymph nodes and further into normally sterile tissues and organs. For 
the first time, Durwanding [9] described the alleged migration of bacteria from the intestine in 
1881. R.D. Berg and A.W. Garlington first introduced the term “bacterial translocation” in 1979. 
The immune system of a healthy body quickly responds to the invasion of pathogenic 
microorganisms, thereby preventing their migration from the intestine into the systemic circulation 
[10]. Since severely ill patients are usually accompanied by systemic immunodeficiency or 
immunosuppression, the immune system is not able to eliminate pathogenic bacteria, and it leads 
to uncontrolled BT. In clinical practice, the study of the BT phenomenon is difficult, due to the 
inability to study the material of various tissues and organs for bacteriological research. Moreover, 
this method has low sensitivity, allows determining for the most part the aerobic flora and can give 
false negative results [11]. Therefore, other reliable BT markers are needed, the search for which 
can be carried out in this clinical study. 
Today there are several methods for detecting BT:  
1. direct method – the detection of 16s rRNA (ribosomal ribonucleic acid) in mesenteric lymph 
nodes (MLN); 
2. indirect method - the detection of serum lipopolysaccharide-binding protein (LBP) and 
presepsin (Soluble CD14 subtype or sCD14-ST). 
Mesenteric lymph nodes and vessels are probably the most important pathway for bacteria to 
spread from the intestines to the blood and other organs. Since MLN is usually sterile, the presence 
of viable bacteria in them is a marker of increased permeability of the intestinal barrier and 
bacterial translocation [12, 13]. 
LBP plays an important role in recognizing the main component of the bacteria outer cell wall - 
lipopolysaccharide (LPS). LBP sensitizes macrophage, monocyte and neutrophil receptors to LPS 

of bacteria, thereby activating the inflammatory signaling pathway [14, 15]. A number of studies 
have shown that LBP is a reliable biomarker of microbial translocation and sepsis [16, 17]. 
sCD14-ST has a high affinity for the lipopolysaccharides of the cell walls of gram-negative 
bacteria and peptidoglycans of gram-positive bacteria [18,19]. Presepsin has been identified as a 
biomarker of the early phase of sepsis and its level is a significant prognostic factor in outcomes 
in patients with sepsis [20, 21, 22].    
When analyzing the studies in the publication databases, it was found that more than  60% of the 
publications about BT is experimental, and most of the clinical studies were conducted in patients 
with HIV infection and cirrhosis.  BT in ABO is still not well understood, as is the role of microbial 
translocation in the development of SIRS and infectious and inflammatory complications. The 
questions of the diagnostic value of the proposed biomarkers, their study in dynamics, as well as 
the relationship between direct and indirect BT markers, remain unresolved. These unresolved 
problems require further in-depth study. Therefore, for the early diagnosis of infectious and 
inflammatory complications, it is necessary to study LBP, sCD-14 and 16sRNA as BT markers in 
patients with malignant and benign ABO, as well as in patients after planned surgical intervention 
for colon tumors. Based on changes in BT biomarkers in the blood serum, it’s suggested that 
patients with researched pathology can be stratified according to the risk level of developing 
infectious and inflammatory complications. It will be possible to take preventive measures to 
reduce the frequency and severity of these complications and mortality. 
 
II. Criteria for the selection of research participants 
1. Number of participants: 150 participants.  
2. Sex: all. 
3. Gender based: no. 
4. Age: from 18 years old. 
5. Nationality: the racial and ethnic distribution of the participants is unimportant, so people 
with different nationalities and ethnicities will be included in the study. 
6. Criteria for inclusion: - patients with malignant acute bowel obstruction,  
             - patients with benign acute bowel obstruction, 
             - colorectal cancer patients without acute bowel obstruction             
(planned operations). 
8. Criteria for exclusion: - age less than 18,  
    - pregnancy,  
    - patients with paralytic acute bowel obstruction,  
    - patients with HIV infection, liver cirrhosis,  
    - patient with an infectious process due to another pathology.  
 
III. Methods and procedures 
 
Methods 
The study materials are blood serum and (MLN).  
Venous blood sampling will be performed 1 hour before surgery, 24 and 72 hours after it. Venous 
blood will be collected in 5 ml vacutainers with a coagulation activator and a serum gel separator. 
It will be centrifuged for 20 minutes at 1000 x g, after which the gel completely separates the 
serum from the clot, forming a tight barrier. The obtained sample of freshly prepared serum will 
be stored at -200 to -800C for up to 2 mesenteric lymph nodes months, in order to avoid any loss 
of biological activity and contamination. No repeated freeze/thaw cycles is allowed. ELISA Kit 
for Lipopolysaccharide Binding Protein (LBP, Human) and for Presepsin (sCD14-ST, Human), 
from Cloud-Clone Corp. will be used to determine any presence of LBP and sCD14-ST. The 
analysis will be performed according to the manufacturer's instructions for an ELISA EVOLIS 
robotic system from BioRad.  
 

The operating surgeon will perform a MLN sampling in sterile conditions during surgery after 
resection of the intestine from the mesentery of the gross specimen. MLN will be placed in a sterile 
tube without any fillers. The DNA will be extracted by the GeneJET Genomic DNA Purification 
Kit manufactured by Thermo Fisher Scientific, USA, in accordance with the manufacturer’s 
instructions. The 16s rRNA bacteria in MLN will be detected by using real-time PCR and BIO-
RAD CFX96 amplifier with 16s rRNA direct and reverse primers (U16SRT-F 
FACTCCTACGGGAGGGAGGCAGGT and U16SRT-R TATTACCGCGGCTGCTGGGC). 
 
During the implementation, the resources of the Collective Use Laboratory of Research Center 
NJSC "Karaganda Medical University" will be used. 
 
Arms  
Assigned Interventions 
Group 1 
60 patients with malignant acute bowel 
obstruction 
Diagnostic Test: LBP  
     Determine any presence of LBP in blood 
serum by ELISA method 1 hour before 
surgery, 24 and 72 hours after it.  
Diagnostic Test: sCD14-ST 
    Determine any presence of sCD14-ST in 
blood serum by ELISA method 1 hour before 
surgery, 24 and 72 hours after it.  
Diagnostic Test: 16s rRNA 
    Determine any presence of 16s rRNA in 
mesenteric lymph nodes by PCR method. 
Group 2 
60 colorectal cancer patients without acute 
bowel obstruction (planned operations) 
Diagnostic Test: LBP  
    Determine any presence of LBP in blood 
serum by ELISA method 1 hour before 
surgery, 24 and 72 hours after it.  
Diagnostic Test: sCD14-ST 
    Determine any presence of sCD14-ST in 
blood serum by ELISA method 1 hour before 
surgery, 24 and 72 hours after it.  
Diagnostic Test: 16s rRNA 
    Determine any presence of 16s rRNA in 
mesenteric lymph nodes by PCR method. 
Group 3 
30 
patients 
with 
benign 
acute 
bowel 
obstruction  
Diagnostic Test: LBP  
    Determine any presence of LBP in blood 
serum by ELISA method 1 hour before 
surgery, 24 and 72 hours after it.  
Diagnostic Test: sCD14-ST 
    Determine any presence of sCD14-ST in 
blood serum by ELISA method 1 hour before 
surgery, 24 and 72 hours after it.  
Diagnostic Test: 16s rRNA 
    Determine any presence of 16s rRNA in 
mesenteric lymph nodes by PCR method. 
 
All groups will be stratified in subgroups by the presence or absence of postoperative infectious 
and inflammatory complications, systemic inflammatory response syndrome (SIRS) and organ 
dysfunctions according to the SOFA criteria (Sequential Organ Failure Assessment).  
 
 
 

 
 
N=150, 
age >18,  
men, women, 
60 patients with malignant 
acute bowel obstruction 
30 patients with benign 
acute bowel obstruction  
SIRS 
No-SIRS 
Complications 
No Complications 
Organ dysfunction 
No Organ dysfunction 
 
SIRS 
No-SIRS 
 
 
Complications 
No Complications 
 
Organ dysfunction 
No Organ dysfunction 
Complications 
No Complications 
 
SIRS 
No-SIRS 
 
Exclusion Criteria: 
- age<18 
-pregnancy 
-paralytic ABO 
-HIV infection 
-liver cirrhosis 
-infectious process due to 
another pathology 
Organ dysfunction 
No Organ dysfunction 
60 colorectal cancer 
patients without acute 
bowel obstruction (planned 
operations) 
Determination of LBP and 
sCD14-ST in blood serum 
1 hour before surgery, 24 
and 72 hours after it and 
16s rRNA in MLN 

ABO- acute bowel obstruction; HIV - human immunodeficiency virus; LBP - lipopolysaccharide-binding protein; 
MLN - mesenteric lymph nodes; N – number of participants; sCD14-ST - Soluble CD14 subtype (presepsin);  SIRS - 
systemic inflammatory response syndrome. 
 
Primary Outcome Measures: 
Postoperative infectious and inflammatory complications.  
 
Secondary Outcome Measures: 
- LBP level in serum blood. 
LBP levels (1 hour before surgery, 24 and 72 hours after it) will be compared between groups/ 
subgroups and in each group/subgroup in dynamic.  
- sCD14-ST level in serum blood. 
sCD14-ST levels (1 hour before surgery, 24 and 72 hours after it) will be compared between 
groups/ subgroups and in each group/subgroup in dynamic.  
-16s rRNA in mesenteric lymph nodes. 
Presence or absence of 16s rRNA in mesenteric lymph nodes will be compared between 
groups/subgroups. 
 
Study plan 
Scope and summary of the work 
Deadline 
Theoretical work 
Preparation of articles (abstracts) for publication 
with a literature review and analysis of the data 
obtained on the problem under study 
During the execution of research work 
Experimental work 
Set of material for research 
During the execution of research work 
Statistical analysis  
During the execution of research work 
Design of research work 
Completion of the writing of the main sections of 
the research work, registration of the study, 
publication  
November 2021 - October 2023 
 
Data analysis and monitoring 
The statistical analysis will be carried out using IBM SPSS Statistics 20.0 program. The data will 
be presented as mean (M), standard deviation (SD), median (Me) and interquartile range (IQR). 
Testing of statistical hypotheses for dependent groups (between marker values before and after 
surgery on the 1st and 3rd day in each of the groups) will be carried out using the nonparametric 
Wilcoxon T-test. For independent groups, statistical hypotheses will be tested using the 
nonparametric Kruskal-Wallis test. To compare the decrease or the increase in markers levels in 
dynamic, depending on the presence of SIRS and postoperative complications, the Fisher exact 
test will be used. To determine the critical values of biomarkers and determine the sensitivity and 
specificity of the tests, ROC analysis will be applied. To identify the correlation relationship, the 
Spearman correlation coefficient will be calculated. Results at p <0.05 will be considered 
statistically significant. Moreover, α = 0.05, 1-β = 80%. 
 
Data storage and confidentiality 
Confidentiality will be ensured by encrypting the personal data of the participants with a digital 
code. The storage of information about the participants will be carried out in writing in the 
registration journal of the research topic, in electronic form on a computer in the databases of 
Microsoft Excel and SPSS Statistics 20.0. The supervisor and performer of the research will have 
access to the data. The research results, without specifying personal data, will be used for writing 
a doctoral dissertation, the research results will be published in print and electronic publications 

and reported at conferences.  
 
IV. Assessment of the risk / benefit ratio 
1. Degree of risk. In a study, the benefit outweighs the risk. 
2. Potential risk. In a study, the benefit outweighs the risk. 
3. Protection against risk. In a study, the benefit outweighs the risk. 
4. Potential benefits for the participant. The results of the study can be applied in surgical practice 
in predicting infectious and inflammatory complications in acute bowel obstruction. 
5. Alternatives for the participant. The patient has the right to refuse the proposed diagnostic 
method. The patient cannot be included in the study without written consent. 
 
V. Identification of research participants, recruitment and consent 
Before each examination, the participant is explained the goals and objectives of the examination, 
the examination procedure is brought up, his right to refuse the examination is given, and a written 
consent to the examination is obtained. 
1. Methods for identifying participants and their recruitment. Study participants will be sampled 
according to the inclusion / exclusion criteria. 
2. The process of obtaining consent. The process of obtaining consent will be carried out by the 
researcher in the form of written consent after explaining to the subject the objectives and methods 
of the study, as well as all potential risks. 
3. The state of the participant. If not all participants are able to give informed consent, then they 
will not be included in the study. 
4. Understanding. Before each examination, the patient is explained the goals and methods of the 
study, potential risks, the examination procedure and his right to refuse the examination are 
explained, and written consent to the study is obtained. 
5. Consent Forms. An informed consent form will be drawn up according to the recommendations 
of the Bioethics Committee. 
6. Documenting consent. The responsible executor is responsible for obtaining and documenting 
IP from all entities. 
7. Participation price. The examinations are free of charge for the participants, since this study is 
carried out within the framework of the research work granted by the Science Committee of the 
Ministry of Education and Science of the Republic of Kazakhstan (Grant No. AP09260597). 
8. Participation fee is not provided. 
 
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