To investigate the gastric microbiota, tissue is generally obtained by an endoscopic biopsy. Slightly less invasive methods are available to obtain a specimen such as the use of a small bowel biopsy tube or capsule, or biopsy forceps that can be passed through a modified nasogastric tube positioned either in the gastric body or antrum. A biopsy is clinically unnecessary to diagnose H. pylori via microbiological methods unless one wishes to isolate the organism for antibiotic susceptibility testing. Recommendations to maximize the diagnostic yield of endoscopic biopsies include the use of large-cup biopsy forceps, obtaining at least two samples from the lesser curvature and the greater curvature (the prepyloric antrum and the body), and proper mounting and preparation of the samples. Special stains (H&E, Giemsa, and Warthin-Starry staining) are often used to help detect the presence of H. pylori (38).
The rapid urease test (by agar gel slide tests) involves placing a biopsy specimen from the antrum of the stomach on a test medium that contains urea (39). The biopsy specimens for the rapid urease test have to be removed from the sterilized biopsy forceps with a sterile toothpick, and have to be placed immediately into a tube. The urea is hydrolyzed by urease enzymes of H. pylori, and the ammonium formed increases the pH. A phenol indicator that changes the color from yellow at pH 6.8 to magenta at pH 8.4 can detect the pH alteration. The color change read off 1 hour after and 24 hours after the introduction of the gastric biopsy is an indication for the presence of H. pylori. Recommendations to maximize the rapidity and sensitivity of rapid urease tests are to warm the slide, and to use two regular or one jumbo biopsy specimen(s) (40). Increasing the number of biopsies to more than two biopsies from the antrum may increase the sensitivity, given that this probably increases the H. pylori load, and therefore the amount of urease. However, this will prolong the endoscopy time and add to the discomfort of the patient. The agar gel test may take up to 24 hours to turn positive, particularly in the presence of a low bacterial density. Recent use of antibiotics, bismuth, or proton pump inhibitors may render rapid urease tests falsely negative. Compared with histology as the gold standard in the diagnosis of H. pylori infection, the sensitivity of the rapid urease test is 70-99%, and the specificity is 92-100% in untreated patients (40). Mucosal biopsies can be fixed in neutral buffered formaldehyde, and if the rapid urease test is negative the biopsy can sent in the next day for histologic assessment. The presence or absence of H. pylori can be established by examining three sets of tissue levels within 12 consecutive sections. On microscopic examination of the tissue obtained by biopsy, the bacteria may be seen lining the surface epithelium. The sensitivity for histologic examination is 70-90%. Giemsa staining is required for H. pylori diagnosis. Culture for H. pylori is insensitive. Biopsies should be plated within 2 hours (or transported in a special medium) on nonselective media enriched with blood or serum, and incubated in a moist and microaerobic atmosphere. The identity of any colonies grown can be confirmed using Gram's stain and biochemical tests.
In order to sample gastric fluid a Shiner tube may be used. This is a polyvinyl tube with a stainless steel sampling capsule at the end with which the specimens are obtained by suction. This tube can be sterilized in the autoclave or by boiling (6). Sampling the luminal content of the stomach may lead to underestimation of the size or even misinterpretation of the composition of gastric microbial communities (29). Estimates per unit weight of material of the population levels of microbes attached to an epithelium surface made from samples of the mucosa itself have been found to be higher than estimates made from the luminal content in the region (29). This technique is not clinically relevant, and is hardly ever used in research models.
The urea breath test is a noninvasive test that detects radio-labeled carbon dioxide excreted in the breath of persons with H. pylori infection; orally administered urea is hydrolyzed to carbon dioxide and ammonium in the presence of the enzyme urease, which is present in H. pylori. In non-infected subjects, urea leaves the stomach unchanged, unless there is urease activity from bacteria in the oral cavity or in situations of gastric bacterial overgrowth. The urea breath test is a highly sensitive (93.3%) and specific (98.1%) method (41). The two breath tests available are the 14C urea (radioactive), and 13C urea (stable isotope) breath tests. The 13C urea breath test avoids radioactivity, and is the test of choice for children and pregnant women. The major limitation is the need for a gas isotope mass spectrometer to analyze the breath samples and calculate the ratio of 12C to 13C. A 4-hour fast is generally recommended before the urea breath test, and a test meal is given before the solution of labeled urea. This test meal delays gastric emptying, and increases contact time with the bacterial urease. It is relatively inexpensive compared to the "gold standard" of endoscopy with biopsy, and histological examination described above. The urea breath test avoids sampling errors that can occur with random biopsy of the antrum. False positive results can occur if gastric bacterial overgrowth with urease-producing bacteria other than H. pylori are present. False positive results can also occur if the measurements are taken too soon after the urea ingestion because the action of the oral microbiota on the urea may be measured. False negative results can be obtained if the patients were recently treated with antibiotics, bismuth preparations or acid suppression therapy, because the test is dependent on the numbers of H. pylori (42). Performance of the urea breath test has been associated with several disadvantages especially in infants, toddlers or handicapped children because one needs active collaboration. False positive results in infants affect the accuracy of the test, but correction for the carbon dioxide production of the tested individual will improve the specificity (43,44).
Other tests that do not require a mucosal biopsy include serologic tests and stool antigen tests. Chronic H. pylori infection elicits a circulating IgG antibody response that can be quantitatively measured by enzyme-linked immunosorbent assay (ELISA tests). The ELISA is based on a specific anti-H. pylori immune response, and this serologic test is as sensitive (95.6%) and specific (92.6%) as biopsy-based methods (41). The presence of IgG does not indicate an active infection. IgG antibody titers may decrease over time (6-12 months) in patients who have been successfully treated. ELISA or immuno-chromatographic methods can be performed on the fecal samples to detect H. pylori antigen. The limit of sensitivity of the test is 105 H. pylori cells per g of feces (45). Sensitivities and specificities of 88-97% and 76-100% have been reported (41,44-47). The stool antigen test is not used for follow-up evaluation of the H. pylori eradication as it gives false positive results. In conclusion, the noninvasive tests are sufficiently accurate for the diagnosis of H. pylori infection.
SMALL INTESTINE: MICROBIOTA AND SAMPLING TECHNIQUES Normal Microbiota
The small intestine comprises the proximal, mid, and distal areas, which are designated the duodenum, jejunum, and ileum. The velocity of the intraluminal content of the small intestine decreases from the duodenum to the ileum. The microbes isolated from the small intestine include those descending from habitats above the small intestine such as the mouth, and ingested food. The microbes pass through the intestine with the chyme, and in the fasting state by the MMC. The MMC interdigestive motility prevents colonic microbiota from entering the proximal small intestine which would cause SBBO. The microbial species isolated from the small intestine are listed in Table 2. The density of microbiota increases towards the distal small intestine. The upper two thirds of the small intestine (duodenum and jejunum) contain only low numbers of roughly the same microorganisms, which range from 103 to 105 bacteria/ml (2). Culturing studies indicated that acid- and aero-tolerant Gram-positive species such as lactobacilli and streptococci dominate in the proximal part, while distally anaerobic, and more Gram-negative bacteria increasingly dominate. Whipple's disease is a rare multisystemic bacterial infection caused by Tropheryma whipplei. T. whipplei could not be cultured from the small intestine for decades, and was diagnosed by histopathology. Nowadays T. whipplei can be detected using polymerase chain reaction (PCR) or ribosomal RNA techniques on duodenal biopsies or fecal samples (48). The rich microbiota of the initial section of the large intestine (cecum) find their way through the ileocecal valve back into the ileum. The microbiota of the ileum begins to resemble that of the colon with around 107 to 108 bacteria/ml of the intestinal contents. With decreased intraluminal transit, decreased acidity, and lower oxidation-reduction potentials, the ileum maintains a more diverse and numerous microbial community (29). Factors that compromise the oxidation-reduction potential within the tissues are obstruction and stasis, tissue anoxia, trauma to tissues, vascular insufficiency, and foreign bodies (49). Decreased oxidation-reduction potential specifically predisposes to infection with anaerobes (50).
Pathogenic bacteria of the small intestine, which cause severe diarrhea, are enterotoxic Escherichia coli (ETEC) and Vibrio cholerae. V. cholerae is diagnosed when it is present in fecal material. ETEC produces enterotoxins that cause intestinal secretion and diarrhea, and is a common cause of traveler's diarrhea. In SBBO, the proximal small intestine is populated by a substantially higher number of microorganisms than usual. These are frequently anaerobic bacteria that are normally not present in large numbers in the duodenum and the proximal jejunum. A total count of microorganisms exceeding 105 colony forming units/ml in a duodenal or jejunal aspirate is generally accepted as SBBO (51). Some gastroenterologists
Table 2 Microorganisms Isolated from the Small Intestine by Culturing
Most prevalent microbes in duodenum and proximal jejunum
Most prevalent microbes in distal jejunum and ileum
Bifidobacteria Clostridia nonsporing anaerobes Staphylococci Actinobacilli Yeasts
Candida albicans Haemophilus
Note: The most prevalent bacterial types are italicized. Source: From Refs. 2, 15, 22.
also accept a concentration of colonic microorganisms above 103 CFU/ml as positive for SBBO. A profound suppression of gastric acid may facilitate the colonization of the upper small intestine (20). To diagnose SBBO, the quantitative culture of a small intestine is used, and considered to be the gold standard. Fluid aspirated from the descending part of the duodenum may be cultured in order to detect bacterial overgrowth in diffuse small bowel disorders.
To obtain biopsy samples from the small intestine upper endoscopy has to be performed. Upper endoscopy is performed after an overnight fast of at least 10 hours. An endoscope has a length of approximately 1 meter, and has a biopsy channel. During endoscopy the esophagus, stomach, and duodenal wall can be systematically inspected. To allow a good view air insufflation is required; the patient may complain of bloating during the endoscopy. When the endoscope reaches the site of interest, the biopsy from the small intestinal mucosa is rapidly taken by standard biopsy forceps. Figure 2 shows the size (in centimeters) of the tip of an endoscope, and a biopsy forceps. The distal part of the jejunum and the ileum cannot be reached using a standard endoscope, and therefore is not sampled. Endoscopic biopsies are an adequate substitute for jejunal suction biopsies. The advantage over capsule biopsy is that the site of interest can be inspected before the biopsy is taken (52-54). Adequacy of mucosal biopsies is a function of size and numbers of biopsies obtained (54). Alligator-type forceps obtain larger specimen pieces than oval-shaped forceps (55). Forceps with a needle, or the multibite forceps, allow more biopsies to be taken per passage, and improve the quality of tissue obtained (55). Biopsy forceps without a needle can be used to obtain two samples per passage through the endoscope that are quantitatively as good as when only one sample is collected. This approach can save time, and causes no significant damage to the biopsy specimens. Because air insufflation may distort the intraluminal anaerobic environment, nitrogen could be used as a substitute if the intention is to culture anaerobic bacteria. There is also the risk of contamination with microbiota from more proximal habitats that were passed along via the endoscope.
Figure 2 Tip of a standard endoscope and biopsy forceps with needle (tape measure in centimeters).
The biopsies have to be taken at a certain distance from the endoscope to prevent sampling contaminated parts of the intestine.
Intestinal biopsies taken from living persons may not yield satisfactory results because the biopsies are only a minimal part of the total intestinal wall (56). The number of persons sampled must be large to generate reliable results. The best source of information on microbiota in the small intestine so far has been achieved with sampling from autopsy studies of accident victims. As slow cooling of the gastrointestinal tract can cause alterations in bacterial localization the samples have to be taken immediately after death (57), and the number of individuals sampled must still be quite large.
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