The Electronic Journal of Pediatric 
Gastroenterology, Nutrition and Liver
Diseases
 

Diagnosis of Helicobacter pylori infection: with special reference to isotopic techniques.

Abstract

The interest in Helicobacter pylori has not declined in these years. H. pylori causes a chronic gastric infection which is usually life-long and many epidemiological studies have shown that this is probably one of the most common bacterial infections throughout the world involving 30% of the population in developed countries and up to 70-90% of the population in developing regions. Thus, it is clear that the diagnosis of H. pylori infection represents at least a key step in the management of many of the patients referred to the gastroentelogist. Additionally, due to the wide range and relevance of pathologies possibly related to infection, including malignancies, there is the potential for H. pylori to be a major health problem. Improved methods for the diagnosis and follow up treatment of the infection have been developed in the last decades. The use of stable isotopes in non-invasive diagnostic methods, as the breath tests, was the key to a new era of research about H. pylori epidemiology, diagnostic, criteria for the eradication treatment, etc. This paper focus on the different diagnostic methods employed, especially in those where isotopic techniques are applied. Key words: Nuclear techniques, Isotopes, Diagnosis, Helicobacter pylori.

Introduction

Helicobacter pylori is a Gram-negative, spiral shaped, microaerophilic bacterium isolated from human gastric mucosa. This bacterium causes chronic antral gastritis, peptic ulcer and is associated with stomach cancer ^{1, 2}. H. pylori carriers are up to 15 times more likely to develop duodenal ulcers than H. pylori negative individuals. The predominant infections and concomitant inflammation in duodenal and gastric ulcers tend to occur in the antrum and corpus, respectively. The most convincing evidence that H. pylori is a key pathogen in peptic ulcer disease is the observation that its eradication dramatically reduces the ulcer recurrence rate. The hypothetical cascade of events in the pathogenesis of duodenal ulcer initiates with the infection causing inflammation in the mucosa ². This leads to alterations of regulatory hormones with the net effect of acid hypersecretion ³. The duodenal mucosa reacts to acid hypersecretion with the formation of gastric metaplasia, which in turn allows H. pylori to colonize duodenum ³. The final event of mucosal breakdown is caused by several facultative factors. In the long time interval between initial infection and the occurrence of intestinal-type gastric carcinoma, data suggests that H. pylori-associated chronic gastritis evolves through atrophy, intestinal metaplasia and dysplasia ^{1, 3}.

As it is well known, there are two identified patterns of infection of H. Pylori ^{4, 5}. In the first case, developing countries have a large proportion of children infected during early life and almost all adults. Studies showed a prevalence of approximately 13-70% in subjects between 0-20 years of age and 70-94% in those older than 30. The second pattern is characterized by the increase in the prevalence of H. pylori infection from 20 years onwards, and it is typical of developed countries with values of 5-15% of prevalence in children and 20-65% in individuals between 30-75 years. Thus, it is accepted that H. pylori infection is wide world disseminated, with a prevalence of 30-50% and there is an inverse relationship between the prevalence of the infection and the socioeconomic level of the population concerned ^{1, 6}. The scarcity of sero-epidemiological studies of H. pylori in children because of difficulties of collecting serum and interpreting pediatric antibody titers have limited paediatric data ⁷. In addition, although maternally derived H. pylori antibody titers fall by 4-6 months, primary infection at this age has been reported. The use of nuclear techniques employing stable isotopes, as the 13C-urea breath test (13C-UBT) avoids these problems of studying prevalence and transmission in children. This is an important issue to investigate since several studies have found a higher incidence of infection in children less than 5 years of age compared with older children or adults, indicating either greater exposure or increased susceptibility to infection during early childhood ⁷. This may be related to the subsequent development of gastric cancer.

Many factors involved in H. Pylori virulence have been studied in detail, including urease, the vacuolating cytotoxin (Vac A), the product of cytotoxin-associated gene A (Cag A), the neutrophil-activating protein (NapA) and the lipopolysaccharide (8-10). Bacterial adhesion to the mucosal surface is an initial important step for colonization and infection. On the other hand, it is widely accepted that H. Pylori adheres to receptors in the gastric epithelium by means of specific adhesins ¹¹. This is another topic in research in order to clarify H. pylori colonization and possibly develop new strategies of treatment.

The production of high amounts of urease by H. pylori has been used in the development of diagnostic methods using either 14C-urea or 13C-urea for its detection ^{12, 13}. These methodologies have the advantage of being representative of the whole surface of the stomach and being non-invasive methods ^{12, 13}. Until now, several invasive methods have been used in the diagnosis of the gastric infection produced by H. pylori: histology, culture, rapid urease test and polymerase chain reaction. None of them can be considered as a reference method because the samples are obtained by an endoscopic biopsy, which is focal in nature and does not represent the whole surface of the stomach ^{1,12, 13}.

Besides its definite role as a gastroduodenal pathogen, H. pylori is now being actively investigated for possible involvement in various non-gastrointestinal conditions such as impaired growth, coronary heart disease, migraine headache, Reynaud’s phenomenon, diabetes and gallstone disease. On the other hand, future perspectives in research are also related to the non-conventional treatment of H. pylori by means of the use of probiotics ¹⁴ and the relationship of this pathogen with micronutrients deficiencies ¹⁵.

Diagnostic methods

Numerous tests, both invasive and non-invasive, are available for diagnosis of H. Pylori infection. Each test has unique features that offer additional information over others, but none is perfect ¹⁶. Nowadays, the discussion over the different methods adressed asking what is the best diagnostic tool in each definite situation. Diagnostic purposes may be as different as epidemiological screening, follow up treatment, diagnosing the H. pylori infection, search for susceptibility to antibodies, diagnosing the infection in a clinical setting, look for putative markers of increase virulence/pathogenicity of a strain, etc. The choice has also to take into account the cost of the diagnostic technique when is relevant as well as all the factors involved such as the need for the endoscopy, for a technician/nurse to assist the patient, the need for a dedicated laboratory, instrumentation/material to evaluate samples and facilities already available. Histology and culture of a sample from gastric mucosal obtained by endoscopy was considered the gold standard method for the detection of H. pylori infection. Alternative methods using this same sample are rapid urease test, the polymerase chain reaction (PCR) and molecular typing. However they all required and invasive approach in order to obtain the sample. Therefore they do not applied for healthy hosts. Additionally they only represent the local result of the stomach sample and not the whole organ, which implies a risk of false-negatives.

¹³C or ¹⁴C labelled urea breath tests (UBT) are two non-invasive methods widely available. They are based on the detection of ¹³CO₂ or ¹⁴CO₂ expired air as a result of H. pylori urease activity. Serology is also widely used and is based on the detection of a specific anti-H. pylori immune response, mostly by IgG antibodies in a patient serum. Because of their non-invasive nature, both UBT and serology , have been widely used in epidemiological studies to assess the prevalence of H. pylori infection in different populations. Non-invasive H. pylori testing can be successfully employed in two other settings: ¹ pre-endoscopic screening of patients to refer to a gastroenterology service for investigation of dyspepsia and ² therapeutic monitoring following eradication therapy to confirm the cure. In this case, UBT can be used 4 weeks after treatment, while serology requires waiting for a longer period to allow a fall in the antibody titer.

Therefore, UBT employing stable isotopes has become the new reference method and the evidence supporting this fact is described below.

Stable isotopes applied to diagnostic methods.

Isotopic tracers have been used to determine dynamic aspects of metabolism in both animals and humans. These tracers can be radioactive or stable, and the latter allows the use of tracers in vulnerable groups, such as pregnant women and children where radioactive tracers may pose an unacceptable medical risk. Stable isotope of an element differ in their neutron numbers, and are, as their name suggests non-radioactive. Thus, stable isotopes tracers have been used to determine quantitative aspects of substrates metabolism, such as rates of synthesis, transformation or degradation ¹⁷.

The global nutrition community first recognized the significance of nuclear and isotope techniques, especially of stable isotopes, for accurate and non-invasive measurements of nutrient utilization, nutrient status and related metabolism ¹⁸. As analytical tools, stable isotopes are now seen to be invaluable, since there is virtually no health risk involved in their use ¹⁸. They are therefore, preferred for working in humans, especially in infants and pregnant women. The application of these tools is increasingly recognized as commonplace, since naturally occurring elements exist as a mixture of two or more stable non-radioactive isotopic forms. Stable isotopes are thus used in measurements by determining the changes in the ratio of different isotopes. They can be administered either orally or intravenously and incorporated into metabolic products, such as body water, urea or CO₂ and they can be sampled in saliva, milk, breath, urine and stool ¹⁸.

In the case of the breath tests, they are based on the delivery of a ¹³C-labelled substrate into the body by oral ingestion or by injection. A specific enzyme in the target tissue then selectively metabolizes the substrate such that the tracer is irreversibly released as ¹³CO₂ into the body CO₂ pool. The tracer is then transported to the circulation and excreted in the breath, such that the pattern of breath enrichment over time can be obtained. The isotope analysis can be performed either by mass-spectrometry or by optical-spectrometry. In the form of nondispersive infrared spectrometry the latter method becomes increasingly important, because it implies simple devices which can be easily operated ¹⁹.

The breath test concept did not immediately take hold ²⁰. Two things, however, changed that in the past decade. The first was the development of a breath test that provided vital and virtually unique information to the investigator and the second is technological development. The ability to monitor the presence of Helicobacter pylori with a non-invasive breath test has made it possible to improve treatment and perform epidemiological studies in field situations. This development has redirected research in the field. The second factor that is now beginning to impact ¹³C breath tests is the development of a lower cost, easy to use, infrared spectrometer for the measurement of ¹³CO₂ ²⁰.

The enrichment of ¹³CO₂ in the breath may be subjected to pharmacokinetic modelling which correlates the input dose to the output of tracer and a numeric index of the metabolism of the labelled substrate can be obtained. However, is not able to provide more detailed metabolic data about the pathways of metabolism of the substrate prior to oxidation. Thus, detailed information about pool sizes of the substrate, or it fluxes into and out the pools cannot be obtained ¹⁷.

¹³CO₂ breath tests may be considered excellent investigation methods, as their scientific bases are sound and well-conceived, the results have been validated in an unequivocal way, and their applications are accepted by an increasing number of scientists ²¹.

Although these breath tests are under current evolution at the laboratory, they can be applied successfully in less privileged countries, as their simple form they are very reliable to investigate gastrointestinal functions. The tests, which can be presented in a test kit, can be used in all members of the population. The breath samples can be kept unaltered in exetainers at least for six months before being sent to a centralized analytical laboratory ²¹.

¹³C-breath tests are widely applied as a tool to investigate metabolic processes and infectious diseases, but most of them have not yet entered into clinical practice ¹⁹. (Table 1 describes some breath tests using ¹³C).

In terms of infections, the most common breath test used in the ¹³C-urea breath test, which is used to diagnose the Helicobacter pylori infection. In this test, the ¹³C-urea is administered orally as a solution to drink, and the urease that is secreted by the H. pylori in stomach acts on the labelled urea to release the labelled ¹³CO₂. The ¹³CO₂ then enters into the body pool to be excreted in the breath immediately. Therefore, an increase in breath enrichment of ¹³CO₂ immediately after ingestion of the ¹³C-urea is indicative of an infection with this bacteria ¹⁷.

Different methods employed in the diagnosis of H. pylori infection.

Invasive techniques are most used in patients who, for clinical reasons, have been referred to endoscopy, therefore they are based on a biopsy sample ^{22, 23}.

• Urease tests are rapid and quite specific, but when early reading is performed the sensitivity is poor. Different methods have been proposed to speed the color development but sensitivity remained low.
• Histological examination is the only method that can show both the extent of the H. pylori infection and the degree of mucosal damage. However, a limit to histological diagnosis is the inter-observer variation.
• Culture is theoretically one of the best diagnostic method, but problems occur with the strict transportation requirements. Only culture allows testing for antimicrobial susceptibility and it also allows typing of strains by many methods. When culture has to be performed, it is mandatory for optimal recovery to use a transport medium to carry the biopsy to the laboratory, in order to maintain the viability of isolates. On the other hand, it is very difficult to obtain the appropriate atmosphere in which to grow H. pylori. A candle jar is a good method to use, however, the growth of the organism is slow and the cells may appear in coccoidal forms.
• DNA techniques, specifically the polymerase chain reaction (PCR), can also be used to detect H. Pylori in biopsy specimens, but there is still potential for increasing its sensitivity. PCR has the advantage of a quick result and does not need strict transport conditions. PCR-derived techniques have also been used for molecular typing. They showed the great genomic diversity of H. pylori strains among different individuals and the frequent occurrence of similar genotypes within families, indicating intrafamilial transmission. The choice of target DNA and the improvement of detection methods of amplified products may also contribute to the sensitivity of the reaction. When PCR is used to detect H. pylori specimens other than gastric biopsies, the recommendation is to use two sets of primers and sequence the product. PCR can also be used on cultivated isolates or directly on an H. pylori positive biopsy specimen to detect the Cag A gene marker of pathogenic strains.

Non-invasive tests are mainly based on samples of blood or expired air. They are particularly valuable because they provide a rapid diagnostic ^{22, 23}.

• UBT. The ¹³C and ¹⁴C-urea breath tests are highly sensitive and specific for detection of H. pylori infection. In contrast to biopsy-based methods, they assess the global presence of H. pylori in the stomach even when the bacteria are distributed in a patchy way. Breath testing can also be used for follow-up after therapy. The most important deficiency is that it does not allow further studies of H. pylori such as antimicrobial resistance testing or typing. The used of the urea breath test (UBT) has been standardized. The assays are based on the detection of ¹³C or ¹⁴C-carbon dioxide in expired air. This is a global test and the only one that allows determination of eradication of H. pylori within 1 month after therapy without performing endoscopy. The sensitivity of the UBT has been reported to be influenced by the administration of omeprazole, lanzoprazole and ranitidine. Some studies have been performed to evaluate and clarify the effects of these agents and found that patients under treatment with lanzoprazole or roxatidine may show negative UBT results. Therefore, the re-examination after the cessation of these drugs to confirm the true negativity of H. pylori infection is advisable. A modification for the UBT was proposed by Zubillaga et al. (1997) ¹², which consists in the administration of the urea labelled with ¹⁴C and a colloid labelled with ^99mTc. The colloid, which is not absorbed in the gastrointestinal tract, allows the visualization of the urea solution inside the gastrointestinal tract using a gamma camera. As a consequence of the described above, the exact location of urea hydrolysis by H. pylori and then the production of ¹⁴CO₂ may be established. This combination allowed increasing the sensitivity and specificity of the UBT up to 90% and 96%, respectively.
• Serological tests were also validated for diagnostic purposes. Monitoring of H. pylori eradication after antimicrobial treatment is also possible by serology if the test is performed not sooner than 6 months after the end of the treatment and is compared with the pre-treatment sample. The difference in test accuracy can be explained by the use of various antigen preparations, but also differences in strains that result in different immune responses. Detection of specific IgM antibodies does not seem to be of major value even in children. Another main use for serological tests is the detection of antibody response to virulence markers such as the cytotoxin-associated gene protein A (Cag A) which is linked with the peptic ulcer and gastric cancer. The ELISA method has the advantage of the low cost and then it can be used for epidemiological purposes.
• Stool assays. Despite the difficulties encountered in stool culture because viable organisms are present in only a small percentage of cases, there is the possibility of a diagnostic test based on the detection of bacterial antigen in stool. This assay was initially approved by the food and drug administration (FDA) for two indications: diagnosis of H. pylori infection in symptomatic adult patients and monitoring response to therapy in adult patients. This is another non-invasive test, which also is accurate, simple and cost effective. Therefore, the stool antigen test is another potential diagnostic tool to be employed in many different clinical settings from epidemiological studies to paediatric investigation, from pre-endoscopic screening strategies to post-monitoring.

Methods are described in table 2.

Pitfalls of diagnostic methods

Tests for the detection of H. pylori infection range from direct visualization in culture and on biopsy samples to measurement of specific bacterial metabolic products, as well as the systemic immune response. The biological samples on which these measurements can be performed include gastric mucosal specimens, gastric juice, breath samples, blood, stool and urine. The general aspects to be taken into account for the correct interpretation of the results are ^{24 1} the H. pylori prevalence in the population studied, ² the type of medication taken by the patient prior to and at the time of testing and ³ the selection of the best suited for the clinical situation.

Several drugs employed in the treatment of H. pylori infection are either bactericidal or suppress the growth of the bacteria in vitro, or may interfere with urease activity. Thus, these medications need to be stopped for a sufficient time before testing. The time for optimizing the test accuracy also needs to take into consideration the class and dose of the drug as well as duration of treatment. The selection of proper testing in relation to the clinical problem becomes more important as new strategies for the management of this infection are established. In young patients with dyspepsia and without alarm symptoms or a family history of malignancy, some guidelines recommend to test and treat. For this purpose, non-invasive testing such as serology, UBT and stool antigen tests provide the option. In areas with a high prevalence of gastric cancer, endoscopy is required for primary diagnosis. In all other conditions, and in patients older than 45 years of age, an endoscopy based diagnosis is better, because of the ability to detect the degree and type of mucosal damage, especially with respect to premalignant and malignant conditions ²⁴.

The biopsy-based methods include the rapid urease test, histology and culture and the samples processed are obtained during endoscopy. The main disadvantage is that they are prone to sampling error thus, the recommendation is to take 4-5 biopsies for histology assessment, 2 for urease rapid test and 2 for culture, all of them from definite sites of the stomach in the antrum and the body ²⁴.

Serology methods have many advantages such as precision, rapid results, low cost and wide availability ^{25, 26}. A multitude of serological tests has been introduced for routine use in the past few years. The most common is conventional ELISA tests, rapid whole blood/serum tests and immunoblot methods. While the former give quantitative results the rest of the tests only provide a qualitative result. Immunoblot kits have a defined antigen preparation and are ready to incubation. This shortens the performance time and reduces the need for special equipment. However, they are still not recommended for routine serology because they are less accurate than conventional ELISA, interpretation of results is tricky since no cut-off is defined and their cost is high ²⁴. Consequently, immunoblot assays for H. pylori are recommended at this time only for scientific studies or as a second-line assay for results after first-line testing with ELISA ²⁴. Rapid blood tests offer the advantage of very quick results and can be performed by general practitioners without any special equipment. The problem with these tests is their limited accuracy although recent data from the U.S. suggest accuracy comparable to conventional ELISA ^27-29. Nevertheless, when using these tests as a sole means of testing H. pylori, clinicians should be particularly aware of these limitations. Several aspects must be considered when using these tests. First, the reading time of rapid blood tests is critical and increasing it increases sensitivity and decreases false-negative rate ^30-32. Second, subjectivity in the interpretation of the test is a problem ³⁰. Third, a very weak colour change should be regarded with caution ³⁰. Increasing the grey zone in order to increase the accuracy of the test is at the expense of less certain results. Salive antibody test showed a low sensitivity and specificity and has the same problems as conventional serological tests in definition of the cut-off point ^{33, 34}. Even the combination of two different serological tests does not always result in detection of H. pylori infection with high sensitivity and specificity. The result of a serological test must be considered in relation to the clinical application ²⁴. Serological results might be influenced by the patient sample, the size of the group, age and ethnic background, prevalence of H. pylori infection in the test population, underlying diseases of the test group and their medical treatment, and finally the titer in the individual patient after eradication. The definition of the cut-off values is a common problem with ELISA tests ³⁵. By lowering the cut-off values, sensitivity is increased but specificity is decreased. In the elderly it may be recommended to reduce the cut-off due to a decreased immunoreactivity. In other populations the definition of a larger grey zone leads to better sensitivity and specificity of the method. The definition of the cut-off by the manufacturer sometimes needs adaptation to the local conditions. Extending the reading time has showed an increase in sensitivity but false-positive rate may increase.

The UBT is a non-invasive test of choice for detecting H. pylori infection with the greatest accuracy under pre and post-treatment conditions ^36-38. The interpretation of the results needs to take into consideration ¹ modifications of the test procedure, ² gastric physiology and ³ the influence of drugs. The correct performance of the UBT is essential and should guarantee the collection of breath samples before and at a definite time point following administration of the urea labeled with ¹³C or ¹⁴C. For this purpose, technicians need to be carefully instructed in how to perform and collect breath samples. Changing the dose of urea labeled, or the timing for taking the second breath sample, leads to different cut-off values and this need to be taken into account ²⁴. The UBT is commonly based on a test meal or test drink given before the substrate ¹³C-urea, but the substrate can also be dissolved in the test drink and administered simultaneously ³⁶. The composition of the test meal has an important impact on the diagnostic accuracy of the UBT ²⁴. In conditions of low urease activity and borderline results the use of orange juice appears to provide a better sensitivity than other test meals. The aim of the test meal or drink is to provide a prolonged reaction time for ¹³C-urea and H. pylori urease. The test meal is also crucial for the delivery of urea solution to the gastric body and fundus. Without the test meal, UBT results may reflect only the urease activity of bacteria colonizing the antrum, and because of the lower density of H. pylori in this location after therapy with antisecretory drugs, the test may give a false-negative result. The test meal provides an equal distribution of the substrate and measurement of global urease activity in the stomach becomes more predictable. In contrast to serology, once a UBT test is validated there is no need for further local validation.

This test is influenced by gastric physiology since rapid gastric emptying with only a short contact time of ¹³C-urea with H. pylori leads to false-negative results ³⁹. False-positive results may also occur with intestinal colonization by other urease-producing bacteria, which are also detectable in the oropharynx, thus activity measured in breath samples collected within the first 10 minutes after ingestion of ¹³C-urea may be confounded with H. pylori infection ⁴⁰.

The use of antibiotics, bismuth salts and proton pump inhibitors in the short or long-term treatment of eradication of H. pylori may provide false-negative results. With regard to proton pump inhibitors (PPI) in short-term treatment, the dose of omeprazole seems to play an important role. Nevertheless, even lower doses lead to an increase in false-negative when treatment is long-term. For practical purposes, when using the UBT, it is necessary to know the class, dose and duration of drug intake in order to interpret negative results. In clinical practice the standard recommendation to wait 4 weeks after the end of the eradication treatment is still valid ²⁴.

Testing for H. pylori infection may be affronted with a wide range of invasive and non-invasive methods. Their accuracy is influenced by several factors such as backgound prevalence of H. pylori infection as well as specific individual, technical and methodological aspects. Additionally, drugs for H. pylori eradication treatment may exert an important influence on test accuracy and interpretation.

Table 1: substrates used for breath tests (Ghoos et al, Fisher et al)

Table 2: Diagnostic methods for H. pylori.

References

1. José Boccio and Marcela Zubillaga. Helicobacter pylori. (1997). Current concepts. Acta Physiologica Pharmacologica et Therapeutica Latinoamericana 47: 194-196.

2. Hernandez Triana. (2001). Helicobacter pylori. La bacteria que más infecta al ser humano. Rev Cubana Aliment Nutr 15: 42-54.

3. Deltenre Michel. Helicobacter pylori. An atlas. 5 Gastric Physiology. Sience Press Limited, London, UK. Pp 5.1-5.6.

4. Punder RE, Ng D. (1995). The prevalence of Helicobacter pylori in different countries. Aliment Pharmacol Ther 9 Suppl: 33-39.

5. Parsonnet J. (1995). The incidence of Helicobacter pylori infection. Aliment Pharmacol Ther 9 Suppl: 45-51.

6. Gasbarrini G, Pretolani S, Bonvicini F, Gatto MR, Tonelli E, Megraud F, et al. (1995). A population based study of Helicobacter pylori infection in an European country: the San Marino Study. Relations with gastrointestinal diseases. Gut 36: 838-844.

7. Logan RPH, Hirschl AH. (1996). Epidemiology of Helicobacter pylori infection. Current Opinion in Gastroenterology 12 Suppl 1: 1-5.

8. Wadström T, Hirmo S, Borén T. (1996). Biochemical aspects of Helicobacter pylori colonization of the human gastric mucosa. Aliment pharmacol Ther 10 Suppl: 17-28.

9. Valkonen KH, Wadström T, Moran AP. (1994). Interaction of lipopolysaccharides of Helicobacter pylori with basement membrane protein laminin. Ifect Immun 62: 3640-3648.

10. Crabtree JE, Farmery SM, Lindley IJD, Figura N, Peichl P, Tompkins DS. (1994). Cag A/cytotoxic strains of Helicobacter pylori and interleukin 8 in gastric epithelial cells. J Clin Pathol 47: 945-950.

11. Wadström T, Felipe A, Ljungh A, Lelwala-Guruge J, Ringnér M, Utt M, Valkonen K. Helicobacter pylori adhesins. (1993). Eur J Gastroenterol Hepatol 5 suppl: s12-s15.

12. Zubillaga M, Oliveri P, Calcagno ML, Goldman C, Caro R, Mitta A, Degrossi O, Boccio J. (1997). Min ¹⁴C UBT: A combination of gastric basal transit and ¹⁴C-urea test for the detection of Helicobacter pylori infection in human beings. Nuclear Medicine and Biology 24: 565-569.

13. Zubillaga M., Oliveri P., Panarello H., Buzurro M., Adami J., Goldman C., Alak M., Degrossi O., Caro R., Calmanovici G. and Boccio J. (1999). Stable isotope techniques for the detection of Helicobacter pylori infection in clinical practice. 13C-Urea breath test in different experimental conditions. Acta Physiologica Pharmacologica et Therapeutica Latinoamericana. 49 (2): 101-107.

14. Zubillaga M, Weill R, Postaire E, Goldman C, Caro R, Boccio J. (2001). Effect of probiotics and functional foods and their use in different diseases. Nutr Res 21: 569-579.

15. Annibale B, Capurso G, Delle Fave G. (2002). Sequences of Helicobacter pylori infection on the absorption of mocronutrients. Digest Liver Dis 34 suppl: s72-s77.

16. Mégraud F. (1996). Advantages and disadvantages of Helicobacter pylori diagnostic tests. Scand J Gastroenterol 31 suppl: 57-62.

17. Kurpad AV, Ajami A, Young VR. (2002). ¹³C breath tests in infections and beyond. Food and Nutr Bull 23: 21-29.

18. Iyengar V, Kastens RF. (2002). Preface. Food and Nutr Bull 23: 5-6.

19. Fisher H, Wetzel K. (2002). The future of ¹³C-breath tests. Food and Nutr Bull 23: 53-56.

20. Schoeller DA. (2002). Uses of stable isotopes in the assessment of nutrient status and metabolism. Food and Nutr Bull 23: 17-20.

21. Ghoos Y, Geypens B, Rutgeerts P. (2002). Stable isotopes and ¹³CO₂ breath tests for investigating gastrointestinal functions. Food and Nutr Bull 23: 166-168.

22. Lehn N, Mégraud F. (1996). Diagnosis of Helicobacter pylor. Current Opinion in Gastroenterology 12 suppl: 6-10.

23. Mégraud F, Hirschl A. Helicobacter pylori. An atlas. 3 Diagnosis. Sience Press Limited, London, UK. Pp 3.1-3.6.

24. Malfertheiner P, Leodolter A, Gerards C. (2000). Pitfalls in Helicobacter pylori diagnosis. Helicobacter pylori. Basic mechanisms to clinical cure 2000. Kluwer Academic Publishers, Dordrecht, The Netherlands pp 123-140.

25. Mégraud F. (1997). How should Helicobacter pylori infection be diagnosed. Gastroenterology 113 suppl: s93-s98.

26. Mégraud F. (1997). The most important diagnostic modalities for Helicobacter pylori, now and in the future. Eur J Gastroenterol Hepatol 9 suppl: s13-s15.

27. Laine L, Knigge K, Faige D et al. (1999). Fgerstick Helicobacter pylori antibody test: better than laboratory serological testing? Am J Gastroenterol 94: 3464-3467.

28. Faigel DO, Magaret N, Corless C, Lieberman DA, Fennerty MB. (2000). Evaluations of rapid antibody tests for the diagnosis of Helicobacter pylori infection. Am J Gastroenterol 95: 72-77.

29. Chey WD, Murthy U, Shaw S, et al. (1999). A comparison of three fingerstick, whole blood antibody tests for Helicobacter pylori infection: a United States multicenter trial. Am J Gastroenterol 94: 1512-1516.

30. Stone MA, Mayberry JF, Wicks AC, et al. (1997). Near patient testing for Helicobacter pylori: a detailed evaluation of the Cortecs Helisal Rapid Blood test. Eur J Gastroenterol Hepatol 9: 257-260.

31. Wilcox MH, Dent TH, Hunter JO, et al. (1996). Accuracy of serology for the diagnosis of Helicobacter pylori infection - a comparison of eight kits. J Clin Pathol 49: 373-376.

32. Duggan A, Logan R, Knifton A. (1996). Accuracy of near-patient blood tests for Helicobacter pylori . Lancet 348-617.

33. Reilly TG, Poxon V, Sanders DS, Elliot TS, Walt RP. (1997). Comparison of serum, salivary, and rapid whole blood diagnostic tests for Helicobacter pylori and their validation against endoscopy based tests. Gut 40: 454-458.

34. Feldman RA, Deeks JJ, Evans SJ. (1995). Multi-laboratory comparison of eight commercially available Helicobacter pylori serology kits. Helicobacter pylori Serology Study Group. Eur j Clin Microbiol Infest Dis 14: 428-433.

35. Van de Wouw BA, de Boer WA, Jansz AR, Staals AP, Roymans RT. (1995). Serodiagnosis of Helicobacter pylori infection: an evaluation of a commercially available ELISA Ig-G. Neth J Med 47: 272-277.

36. Bazzoli F, Zagari M, Fossi S et al. (1997). Urea breath tests for the detection of Helicobacter pylori infection. Helicobacter 2 suppl: s34-s37.

37. Logan RP. (1998). Urea breath tests in the management of Helicobacter pylori infection. Gut 43 suppl: s47-s50.

38. Metz DC, Furth EE, Faigel DO, et al. (1998). Realities of diagnosing Helicobacter pylori infection in clinical practice: a case for non-invasive indirect methodologies. Yale J Biol Med 71: 81-90.

39. Atherton JC, Washington N, Blackshaw PE, et al. (1995). Effect of a test meal on the intragastric distribution of urea in the ¹³C-urea breath test for Helicobacter pylori . Gut 36: 337-340.

40. Lotterer E, Ludtke FE, Tegeler R, Lepsien G, Bauer FE. (1993). The ¹³C-urea breath test-detection of Helicobacter pylori infection in patients with partial gastrectomy. Z Gastroenterol 31: 115-119.