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Non-invasive test

Currently, there are two non-invasive tests that are becoming extremely reliable in detecting H. pylori infection. The more established one is the urea breath test and the other is the stool antigen test. Serology test is not recommended as diagnostic tool because of its poor sensitivity and test-to-test variability [33].

Urea breath test

The urea breath test utilizes the essential enzyme urease, which is produced by H. pylori. Urease converts urea to ammonia and CO2. If the CO2 is labeled with a stable isotope, this can be detected in the expired air (Fig. 2). In the non-infected individual, urea will leave the stomach unchanged. This test is essentially a detection of urease activity, which can also be produced by other bacteria in the oral cavity, as in the setting of bacterial overgrowth. 13C and 14C are the two isotopes that are well validated [66]. Only the 13C urea breath test has been extensively tested in children [67]. 14C is radioactive and is not acceptable to be used in children. The 13C test is more expensive than the 14C test, because it requires mass spectrometry or infrared spectroscopy equipment for analysis of the expired breath. The results of the urea breath test are reported as A/baseline (DOB), which is a measure of the ratio of 13C CO2, to 12C CO2. If the DOB exceeds a certain point, the patient is considered to be infected with H. pylori infection. The test is best performed when the patient has fasted

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Figure 2. Carbon in Urea is labeled with either 13C or 14C and exhaled after being converted to label CO2 by urease produced by Helicobacter pylori in the gastric mucosa.

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Figure 2. Carbon in Urea is labeled with either 13C or 14C and exhaled after being converted to label CO2 by urease produced by Helicobacter pylori in the gastric mucosa.

and has not been on a PPI for at least 2 weeks. The patient should not have taken antibiotics for 4 weeks prior to testing because this can reduce the H. pylori load. The use of an acid solution as part of the test solution, either citric acid or orange or apple juice, is ideal because urea activity is highest in an acid environment. Expired breath can be collected between 15 and 45 min depending on the laboratory. Despite the variability in the dosage of urea used and the different cut off points, this study consistently shows that the urea breath test has a sensitivity of over the 96% and a specificity of over 90% [68]. Infants and toddlers are much more likely to have a positive result in comparison to school-age children and adolescents [69]. Reducing the tracer dose and changing the DOB value increase the specificity of this test in younger children [70, 71]. Also, technically, it is much more difficult to collect reliable expired air from infants and toddlers compared to school age children.

Stool antigen test

This is a non-invasive test for H. pylori antigen in stool in the pediatric population. Unlike with the urea breath test, there is no collection difficulty, particularly in the younger age group. There are two types of stool antigen tests on the market: one is polyclonal, and the other a monoclonal, antibody enzyme immunoassay. In a recent meta-analysis of 22 studies including 2499 patients, the monoclonal stool antigen test was found to have sensitivity and specificity of 94% and 97%, respectively. In 13 of the studies in which the monoclonal was compared to the polyclonal stool antigen tests, the monoclonal test had a higher sensitivity of 95% versus 83% for the polyclonal test. In terms of eradication, analyzing 12 studies with 957 patients post treatment, the sensitivity and specificity for the monoclonal test were 93% and 96%, respectively. In 8 of the studies where both monoclonal and polyclonal tests were used, sensitivity was higher for the monoclonal (91%) than for the polyclonal test (76%), demonstrating that the monoclonal stool antigen test is a much more accurate, non-invasive test for both diagnosis and confirmation of eradication of H. pylori infection post treatment [72].

A recent European multi-centered study comparing urea breath tests with stool and serology tests, as well as antibody detection in urine, found that the urea breath test is the most sensitivity and specific. The stool antigen test used in this study was polyclonal (Meridian). The urea breath test had a sensitivity and a specificity exceeding 96%, whereas the stool test has sensitivity and specificity of 92%. This study only had a small number of children under the age of 6 (48 children accounted for 15% of the study population) [73]. However, another study performed in Egypt compared the urea breath test, monoclonal stool antigen test, and serology test to endoscopy with biopsy and rapid urease test. In this population 53 of the 108 children tested were under the age of 6. Overall, the sensitivity and specificity of the urea breath test was 98% and 89%, respectively, and those of the monoclonal stool antigen test were 94% and 81%, respectively. Interestingly, the urea breath test sensitivity was not affected by age but the specificity was lower in those under the age of 6 (86% versus 95%). With regard to the monoclonal stool antigen test, those performed on children under the age of 6 showed a sensitivity of 94% and specificity of 81%. However, above the age of 6, the sensitivity remained about the same at 92% but the specificity increased to 100%. Serology had the worst outcome in this study, giving a sensitivity of 50% and specificity of 80% [69]. Overall, the urea breath test remained the most sensitive and specific non-invasive test for H. pylori. There is still some conflicting data on how reliable this test is in the younger age group. The monoclonal stool antigen test is also proving to be quite a sensitive and specific test , and, again, its reliability in the younger age group requires further study. The use of these diagnostic tests needs to be interpreted in the local context, particularly whether these tests have been validated to the population that is under investigation, in particular with regard to the population age group as well as ethnicity and geography.

Invasive tests

Upper gastrointestinal endoscopy with mucosal biopsy remains the gold standard for the diagnosis of H. pylori infection in children. It has the advan tage of being able to detect upper gastrointestinal pathology including the complications of H. pylori infection such as nodular gastritis, peptic ulcer disease, gastric cancer, and MALT lymphoma. In pediatrics, the primary indication for upper GI endoscopy is the presence of persistent, severe upper abdominal symptoms and not simply the presence of H. pylori [33]. It is difficult to differentiate symptoms secondary to the complication of H. pylori infection such as peptic ulcer disease and functional dyspepsia. The most common endoscopic finding in children with H. pylori infection is nodular gastritis, which is seen most commonly in the antrum with an irregular (cobblestone) appearance, which is highlighted with blood from a bleeding biopsy site. When nodular gastritis is found, it has high specificity (98%) for H. pylori infection, and therefore a high predictive indicator for H. pylori infection, but it has low sensitivities (44%) [17, 74]. In naive patients, antral biopsy had the highest yield, particularly in the mid antrum region of the lesser curvature [75]. For a patient who has been on acid suppression therapy or antibiotics, a biopsy from the transitional zone and body are also required to improve the yield [76, 77]. For patients who have developed complications from H. pylori infection such as peptic ulcer disease, multiple biopsies from different regions of the stomach are required. H. pylori can often be seen using hematoxylin and eosin staining; immunohistochemistry using polyclonal and H. pylori antibodies is likely to be the most reliable detection method on biopsy sections, although this method is expensive and time consuming. Among other stains that are often used is the Giemsa stain, which is less reliable but widely available and affordable [78]. The optimal staining method is often guided by local expertise. With a biopsy of gastric mucosa, a rapid urease test can also be used. This test essentially detects the presence of urease produced by H. pylori. The test is highly specific and sensitivity in adults, but less so in children [79]. The accuracy of this test also depends on the number of biopsies taken, site of biopsy and the use of antibiotics and PPIs. However, one of the major advantages of biopsy in H. pylori infection is the ability to culture this bacterium. In clinical trials, the success rate is up to 80% of infected children [80]. Bacterial culturing is time consuming and expensive, but it does allow for antibiotic sensitivity to be determined. This is particularly useful for those who failed previous eradication therapy. In addition, for a positive culture, the genotype of clinical isolate can be investigated for specific bacterial virulent factors. There are now a number of molecular techniques that can be used to detect the presence of H. pylori and the presence of a number of point mutations in the bacterial genome that determines antibiotic resistance genotypes. For example, the predominant cause of clarithromycin resistance is a point mutation in the peptidyl transferase of the 23S rRNA gene. There are also a number of inactivating mutations involving some reductase genes that convert Metronidazole from a harmless product to an anti-bacterial agent, inactivating the gene responsible for a portion of H. pylori resistance to Metronidazole [81].

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