**4. Diagnostic techniques**

It is pretty challenging to diagnose or assess the IB function directly due to the invasiveness of intestinal tissue sampling. However, there are some methods or biomarkers that indirectly assess IB function. The measurement of biomarkers in urine, blood, or feces generally needs simple, non-invasive test methods.

#### **4.1 Tissue culture methods**

The tissue culture methods are based on the direct detection of intestinal bacteria in extraintestinal tissues. The mesenteric lymph nodes (MLN), extraintestinal tissues, swabs of the intestinal wall serosa or abdominal cavity, blood, and lymph are subjected to bacterial culture. Living bacteria are isolated, counted, and detected using microscopy. Direct detection has tremendous importance; however, many factors may interfere with the measurement [38]. For the culture of MLN, lymph nodes are suggested to be excised and be homogenized in saline. The homogenate can be inoculated onto Columbia blood agar and cysteine lactose electrolyte deficient media.

BT can also be measured by identifying intestinal bacteria from MLN, which is normally sterile [86]. For the culture of MLN, a lymph node from the mesentery of the terminal ileum is suggested to be excised at laparotomy and be homogenized in saline. Then, the homogenate can be inoculated onto Columbia blood agar and cysteine lactose electrolyte-free media.

#### **4.2 Use of labeled bacteria**

Radioactive tracers or plasmids are used to label bacteria for their in vivo detection. This method can detect the bacteria which is attached to intestinal mucosa-associated lymphoid tissue. However, this method can be used primarily in scientific research labs due to its cost and technical requirements.

Fluorescence and isotopes labeling methods are recently in use. PUC19 plasmid was constructed in the 1980s and was used extensively in molecular cloning and discrimination of gene recombination [87]. In this method, the plasmid vector carries the ampicillin resistance gene; the plasmid contains polyclonal restriction endonuclease sites, which discriminates against the positive bacteria. Thus, the PUC19 plasmid is introduced as an ideal tracer for demonstrating BT.

#### **4.3 PCR techniques**

Intestinal BT can be demonstrated by isolating bacterial DNA in patients' blood or body fluids and then amplifying and sequencing them. The PCR detection method is more sensitive, has a higher positive rate, and can specifically detect

*Intestinal Barrier Dysfunction, Bacterial Translocation and Inflammation: Deathly Triad… DOI: http://dx.doi.org/10.5772/intechopen.99554*

certain bacteria when compared to blood culture methods. PCR technic is also a valuable tool for detecting BT in patients with undetected infectious focus. The disadvantage of this method is that only the presence of bacterial debris can be detected, the viability and quantity of the bacteria cannot be determined, and drug sensitivity tests cannot be performed. The quantification of total bacterial 16S rDNA in plasma is used to assess human and animal systemic microbial translocation *in vivo* and thus is a great tool to study the role of systemic microbial products in disease pathogenesis and mucosal barrier function. The bacterial 16S rDNA assay can analyze 90% of bacterial strains, including Gram-positive and Gram-negative bacteria. However, the use of this assay is highly challenging because of its high technical demands and the risk of contamination [88].

#### **4.4 Detection of endotoxin**

Lipopolysaccharide (LPS), the integral component of the outer membrane of all gram-negative bacteria are shown to lead to BT. Since sepsis leads to intestinal submucosal edema and impairs the integrity of the mucosal barrier resulting in an imbalance of gut microflora and increased bacterial endotoxin-induced mucosal injury, endotoxin detection in the blood is a good marker of BT [38, 89].

Endotoxins can be detected in biological fluids by the Limulus amoebocyte lysate assay. Methods using fluorescence phage recombinant technology are also introduced [90].

#### **4.5 Measurement of IB function**

Several methods are used for the measurement and the evaluation of intestinal permeability and barrier function, such as the measurements of transepithelial resistance and assessment of macromolecular flux across isolated segments of GI tissue or colonic biopsies in Ussing chambers, measurement of permeability using fluorescein isothiocyanate (FITC)-dextran permeability in cell lines, morphological measurements of the TJ components, measurement of dilution potentials, and polyethylene glycol (PEG) profiling to assess the pore pathways [91, 92].

#### **4.6 Intestinal permeability**

Methods used to measure intestinal permeability are based on detecting the passage of the molecules across the intestinal epithelium. Several markers can be used alone or in combination to assess intestinal fluxes. Large molecules, lipophilic compounds, and nutrients generally prefer the transcellular route by passing through the intestinal epithelial cells by endocytosis, passive diffusion, or membrane transporters. On the other hand, ions and small hydrophilic molecules prefer the paracellular transport pathway. Thus, molecular size and structure are significant determinants of intestinal permeability [93].

Cell culture-based models using Caco-2 or HT-29 cell lines assess electrical resistance and intestinal flux in vivo. In contrast, differential urinary excretion tests with the use of chromium-labeled EDTA (51Cr-EDTA), polyethylene glycols (PEG), or non-metabolizable sugars such as lactulose and mannitol are frequently used to measure in vivo intestinal permeability [94]. Lactulose can cross the membrane via the paracellular pathway, while mannitol can easily cross the intestinal epithelium through transcellular and paracellular routes. Multi-sugar tests are offered to assess intestinal permeability simultaneously [28, 95]. Ovalbumin, horseradish peroxidase, dextrans, and fluorescently labeled microorganisms are used to measure the intestinal permeability in the blood [96]. Claudin protein levels can be measured in

urine samples for intestinal TJ loss since claudins play a critical role in regulating the paracellular barrier pathway [97].
