Non-invasive tests for the diagnosis of helicobacter pylori: state of the art.

Usually, non-invasive tests are the first methods for diagnosing Helicobacter pylori (HP) infection. Among these, serological test, stool antigen research and urea breath test are the most used. Antibodies anti-HP are not recommended in low prevalence population, moreover they cannot reveal an ongoing infection, but they only prove a contact with the bacterium. Also, they can persist for a long time after the eradication of the infection, therefore, they should not be used to verify the success of eradication therapy. Stool antigen research and Urea Breath Test (UBT) are useful both in diagnosis and during follow-up after eradication treatment. The stool antigen test is cheaper than Urea breath test with similar sensitivity and specificity.  Non-invasive tests are not able to diagnose the associated complications to HP infection.

Introduction

Helicobacter pylori (HP) is a very motile, spiral or curved rod-shaped Gram-negative bacterium with multiple flagella that lives in the gastric mucous-coated lining and the gastric pits of the epithelial tissue of the stomach and/or duodenum. Usually, HP colonizes the human stomach during childhood (1) and survives in the human stomach for the lifetime of the carrier (2). In most of the individuals HP infection may be asymptomatic, causing chronic gastritis. Around 20 to 30% of the infected individuals may develop peptic ulcer disease, (3) and less than 2% may develop gastric cancer (4). Therefore, testing for HP infection has become a very important part of the diagnostic process for gastric and duodenal diseases, since the presence or absence of the infection determines the type of treatment to be applied. Testing is also useful for monitoring the effectiveness of anti-microbial treatment.

A number of different invasive and non-invasive diagnostic methods are currently available (1-3). Invasive tests include histological examination and culture of biopsy sample. These tests are considered to be highly specific, particularly histological examination, but its sensitivity is partly dependent on the accuracy of the biopsy procedure. Moreover, both histological examination and culture of biopsy samples are time-consuming and require specialized laboratory facilities with highly trained staff. For these reasons, several techniques for HP non-invasive diagnostic tests have been developed and are widely used. They belong to three main categories:

Laboratory Serological Assay

Helicobacter pylori Stool Antigen tests (HpSA)

Urea Breath Test (UBT)

The aim of the present review is to focus on the state of the art of non-invasive test for the diagnosis of HP.

1. Laboratory Serological Assay

Serological testing consists in the dosage of specific antibodies against HP or its toxins on serum samples.

Serological testing is the most widely available non-invasive test for the diagnosis of HP infection. These tests are rapid and cheap and may be helpful in screening populations or in confirming the presence of HP infection in case of equivocal results of the other diagnostic methods due to bleeding ulcers, antibiotic and/or antisecretory treatment (5). People infected with HP generally present specific circulating antibodies (IgG, IgA and IgM) and these can be detected by specific serological tests. At present, several commercially available tests have been developed, mostly based on IgG detection.

Infection by cytotoxin-associated gene A (CagA) positive strains (type I) is generally more likely to be associated with more serious gastroduodenal disease, as MALT lymphoma and gastric adenocarcinoma, compared with negative (type II) strains. The detection of a serological response to CagA antigen could, therefore, give clinically useful information about the infecting strain. This association, however, is not seen in all countries (6). Several different techniques are available for serum antibody detection, such as enzyme-linked immunosorbent assay (ELISA), latex agglutination techniques or immunochromatography (5, 7).

The ELISA test is based on sandwich enzyme immunoassay technique with purified H. pylori bacterial antigen adsorbed on micro well plate and detection antibody labelled with an enzyme (i.e. horse radish peroxidase). In the final step, a solution containing the enzyme’s substrate is added. The subsequent reaction produces a detectable signal, most commonly a color change in the micro well that is read using a spectrophotometer.

Rapid tests are also available. They can be applied at the point-of-care, either based on latex agglutination or on immunochromatographic technology.

The latex agglutination tests contain latex particles sensitized with H. pylori antigens. H. pylon if present in the serum samples will react with the sensitized latex resulting in visual detectable clumps. This assay is still used as a rapid test even if its interpretation (positive/negative result) is highly subjective. Latex agglutination tests are most suitable as near patient tests because they are technically simple to perform and provide a result within minutes rather than the hour or two for ELISA tests.

Immunochromatographic tests employ a combination of anti-human immunoglobulin dye conjugate and highly purified H. pylori proteins. As the sample flows through the adsorbent device, the anti-human immunoglobulin dyed conjugate bind to the human IgG antibodies present in positive sample forming an antigen antibody complex. This complex binds to H. pylori proteins fixed in the adsorbent device and produces a colored band. At present, this test is little used as laboratory serological assay for H. pylori.

Performance Characteristics of Serological Assays

A meta-analysis evaluated the performance of several commercially available quantitative serological assays and found an overall sensitivity and specificity of 85% and 79%, respectively, with no significant differences among assays (4). Three of the qualitative whole blood antibody kits were compared in another study demonstrating sensitivities ranging from 76% to 84% and specificities from 79 to 90 (5). In general, performance characteristics of qualitative tests have been more variable than those of the qualitative tests, which are more standardized.

Limitations of serology for Helicobacter pylori

Several factors limit the usefulness of antibody testing in clinical practice. Firstly, serological testing cannot be used to monitor the effectiveness of antimicrobial therapy, since patients may continue to carry serum antibodies specific to HP for several months after eradication. Qualitative tests remain positive for up 3 years after successful treatment and quantitative antibodies levels do not decline significantly for 6 to 12 months after treatment (7). Furthermore, false positive serology tests are more common in low prevalence population, since the positive-predictive-value of antibody testing is greatly influenced by the prevalence of HP infection in the considered area (6). Also the American College of Gastroenterology does not recommend use of serology in low prevalence populations. Generally, the prevalence of elevated IgG in the population tends to be higher in developing countries than in developed ones (8). In case of positivity of a serological test for HP in low-prevalence populations, that positive result should be confirmed with a more reliable test such as the histological examination and culture of biopsy sample (invasive tests) or the urea breath test and the fecal antigen test (non-invasive tests) (2). Finally, antibody tests developed using antigens from one region of the world may not perform well when applied to patients in another part of the world, suggesting that local validation may be necessary (9, 10).

2. Helicobacter pylori Stool Antigen Tests (HpSA)

The evidence that HP is present in stools was the prerequisite for the development of non-invasive diagnostic immunoassay tests using mono or polyclonal antibodies, based on the direct identification of the bacterium antigen in stools (15, 16). Unlike other tests normally used for the diagnosis of the infection, HP stool antigen test (HpSA) detects the antigen of the bacterium and not the antibodies against it. HpSA is able to diagnose an ongoing infection, while the serological tests are limited to diagnose a contact with the bacterium, which can be current or lifetime. The stool antigen test has many positive aspects: it is non-invasive, quick, has good sensitivity, specificity and reliability (presents good replication standards). This test can be used both for diagnosis of the infection and for monitoring therapy effectiveness, already four weeks after the end of treatment. Its low cost, easy use and the possibility to collect samples and perform the test at home have increasingly widespread the use of this method.

HPsA tests can be divided in and .

HpSA ELISA Test

HpSA ELISA test uses polyclonal or, more recently, monoclonal antibodies for anti-HP adsorbed in micro-wells (17-19). According to several studies and to the International Consensus Report, Urea Breath Test (UBT) and the stool antigen research are considered the first-line diagnostic methods with sensitivity and specificity above 90%. In the diagnosis of the infection, HpSA presents values of sensitivity and specificity only modestly lower than UBT, 93.3% and 93.2% respectively (15, 20-22). However, it is important to underline that a proper collection and storage of the sample are necessary, considering that the test sensitivity drops to 69% if the sample is kept at room temperature for 48-72 hours. Moreover, the method should not be applied on watery stools or diarrhea. In addition, the method loses in sensitivity in the early stage post-eradication therapy for the presence of false-positive responses, with the sensitivity varying from 88 to 92% and the specificity from 87 to 88%. It has been reported that the presence of a relatively higher number of false positives, after eradication therapy, can limit the use in this field for a poor positive predictive value (69% vs. 95% than UBT). It is hypothesized that the presence of false positives immediately after the eradication therapy could be linked to the physiological elimination of gastric cells containing the HP without real infectious capacity, but still recognized positive at the antigenic research (23, 24).

Rapid HpSA Test

Recently, a rapid, mono-phase test for the detection of HP bacteria in stools is available on the market, called Quick test. It consists of a reactive support (“card”) utilizing an immunochromatographic technology able to determine the presence of antigens of HP in human feces in a rapid, high quality and easy to perform method (23, 25). To evaluate the performance of the test, the results were compared with the diagnosis of HP infection by the reference tests, UBT and by means of a true gold standard of reference, represented by 500 patients who underwent gastroscopy with multiple biopsies. The Quick test on stool is, when compared to the true gold standard (gastroscopy with multiple biopsies), not only accurate for the diagnosis of HP, but also to control the effectiveness of eradication therapy. Four weeks after discontinuation of treatment, all patients underwent gastroscopy again to have a true gold standard of reference (26, 27). Studies published by Yang HR et al. (28) and Vaira D et al (29) show that, after 7 days of treatment, the majority of patients continue to experience symptoms and in theory have to wait the standard 4 weeks before performing the fecal test or the UBT (continuing to suffer for the symptoms). In contrast, if the rapid fecal test made after 7 days of therapy were positive, it would not be clinically appropriate to wait for the standard 4 weeks, and therefore the patient may either be submitted to another eradication therapy or be subjected to gastroscopy for antibiogram (28, 29).

Subsequent studies have shown the effectiveness of the HpSA Immuno-Card test both before and after eradication therapy (Table 1) (25).

Table 1.

Comparison of sensitivity and specificity for diagnosis and eradication between all the non-invasive tests for HP infection

Test	Sensitivity for diagnosis (%)	Specificity for diagnosis (%)	Sensitivity for eradication (%)	Specificity for eradication (%)
Serological assay (4)	85	79	/	/
HpSA ELISA (15, 20-22)	93.3	93.2	88-92	87-88
Rapid HpSA (25)	91.3	93.5	92	100
UBT (46)	96	93	100	89

As for all diagnostic tests, all results should be interpreted after an accurate clinical evaluation of the patient. If the test result is negative and clinical symptoms persist, it is recommended to investigate further. A negative result does not exclude the possibility of HP infection. As for the UBT, the positivity for HpSA can be affected by ongoing bleeding of the gastrointestinal tract, by the presence of atrophic gastritis or by use of proton-pump inhibitor (PPI) drugs, antibiotics, and preparations of bismuth that inhibit the growth of HP, for which the sample collection must be performed not earlier than two weeks after the last intake of inhibitors and/or preparations of bismuth and, in the case of taking antibiotics, four weeks after the end of treatment.

The test can be used, 30 days after the completion of eradication therapy, to evaluate the outcome.

It should be noted that the search of the fecal antigen does not allow any screening in depth about the heterogeneity of the strains, and their different pathogenicity, as the target antigens of the tests in use are common to all HP subtypes.

Compared with other non-invasive methods (Table 1), Rapid HpSA has been proven simple to perform, particularly useful for patients who have difficulty to undergo the breath test such as children and elderly patients, patients with asthma, after gastrectomy or in case of achlorhydria (28). These tests seem to be a valuable aid, immediate and precise, in guiding diagnosis.

3. Urea Breath Test (UBT)

Urea Breath Test (UBT) is a widely available test with high sensitivity and specificity (from 90 to 100%) for diagnosing HP infection. Moreover, its non-invasiveness, the simplicity of execution and safety, make it elective in the suspicion of the infection in adults, children, and in pregnancy (30-32). However, the test specificity of UBT decreases in young children (<6 years old) because it requires active cooperation of the patient (33, 34). HP is the only bacterium capable to resist to the gastric acidity, it is able to hide within the gastric mucosa and replicate therein. This characteristic is given by the distinct ability to produce urease, an enzyme that breaks down the urea in the stomach releasing carbonic acid and ammonia. Then, the urease neutralizes gastric acid, creating a favorable micro-environment for the replication of the bacterium. UBT uses the urease activity to detect the infection. Actually, it is based on the administration of urea labeled with a carbon isotope (13C or 14C) which, once ingested, is hydrolyzed by the urease produced by the bacterium into ammonia and carbon dioxide, that is subsequently absorbed across the lining of the stomach and into the blood. Then, this labeled molecule reaches the lungs through the bloodstream and is excreted in the breath. Samples of exhaled breath are collected, and the isotopic carbon in the exhaled carbon dioxide is measured. Urease activity is missing in the stomach of healthy subjects, therefore urea administered is absorbed and eliminated by urine. Instead, in HP-infected subjects, an amount of labelled carbon dioxide will be exhaled by the patients a few minutes after the ingestion. This indicates the presence of HP in the stomach. UBT should be performed at least 4 weeks after the use of antibiotics (the use of a single antibiotic seems not to interfere on the exam) and 2 weeks after the suspension of drugs such as PPIs and H2-receptor antagonists and sucralfate. If the contact time of urea with the gastric mucosa is short, then the hydrolysis doesn’t occur and false negative results will be obtained. For this reason several meals have been proposed to administer together with the labelled urea trying to delay the gastric emptying. Therefore, the oral administration of citric acid 10 minutes before the urea administration seems to be the best procedure. UBT is an accurate test for diagnosing HP infection in patients with a healthy stomach, but the sensitivity and specificity of the UBT in subjects who underwent partial gastrectomy are variable because of the lower bacterial load (35-39).

Conclusion

Several non-invasive HP tests are established in clinical routine, but at present, there is no single method, among the non-invasive tests, that can be considered as the gold standard for the diagnosis of HP infection. Clinical conditions, availability and costs should be considered in choosing the most suitable test. Serological testing is the most available test used for the diagnosis of HP infection. In patients treated with PPIs, if it is not possible to stop them for at least 2 weeks, a validated IgG serology test may be used. Antibodies against HP and especially against its most specific antigen CagA, remain elevated despite transient decreases of the bacterial load and even for long periods (months, even years) after the eradication. In recent years, new formats of the HpSA using monoclonal antibodies instead of polyclonal antibodies, which lead to a constant quality of the reagents have been developed. A systematic review by Leal et al. demonstrated that stool antigen test using monoclonal antibodies is an efficient non-invasive test for the diagnosis of HP infection also in children (41). UBT is similar to gastroscopy and biopsies for diagnosing HP infection in terms of sensitivity and specificity, but it is not able to show the associated complications such as gastroduodenal ulcers/erosions, gastric intestinal metaplasia nor neoplastic lesions. The 13C-UBT has a high accuracy, is easy to perform, and remains the best test to diagnose HP infection, although it has shown a variable level of accuracy in pediatric age, mainly in young children (<6 years old) as confirmed in a meta-analysis by Leal and colleagues (42). In special cases, such as gastric ulcer or gastric MALT lymphoma, follow-up is necessary with upper digestive endoscopy and then biopsy-based tests should be performed for confirmation of HP eradication. In other situations, a non-invasive test is used. As the HP antibodies remain for months after suppression and even eradication of theinfection, serology is not recommended in follow-up. However, stopping PPIs 2 weeks before testing, allows the bacteria to repopulate the stomach, preventing false negatives with UBT, HpSA, rapid urease test, histology and culture. Furthermore, no study has evaluated the washout period necessary after long-term PPI treatment. Regarding UBT, a study claimed that the use of an acidic test meal would overcome the problem of false-negative tests. Anti-H2 drugs may also lead to false-negative results but to a much lesser extent (43, 44) and it is not necessary to stop them before testing if using citric acid. The monoclonal HpSA tests are appropriate and widely available for the primary as well as for post-treatment diagnosis of HP infection (40), but there is now overwhelming evidence that the best test in order to assess the efficacy of eradication of HP is UBT (Table 1) (46). In fact the guidelines of the European group of HP recommend the UBT as the ideal method to confirm the eradication of infection and to ascertain the infection state in patients with recurrent symptoms after eradication treatment (45).