The Diagnostic Tests for Detection of Helicobacter pylori Infection

Abstract

Helicobacter pylori causes one of the most common infections in human populations. The role of this bacterium in chronic gastritis, gastric ulcer, gastric cancer, as well as extra-digestive diseases such as ischemic heart disease and chronic obstructive pulmonary diseases, is well known. Prevention and control of these diseases can occur by early diagnosis and eradication of H. pylori infection. At present, different methods have been established to detect H. pylori infection. The biopsy-based tests, which are known as invasive methods, such as rapid urease test and histology, have the highest specificity among the others. Similarly, culture of biopsy samples is used for diagnosis of H. pylori infection. It has a high specificity value, and also allows us to perform antibiotic sensitivity testing. On the contrary, polymerase chain reaction and other molecular methods have good sensitivity and specificity, and can be used for detection of H. pylori infection, its virulence factors, and eradication success after treatment. While serological tests are more appropriate for epidemiological studies, their main weakness for clinical use is low specificity. Overall, specificity and sensitivity, cost, usefulness, and limitation of tests should be considered for selection of detection methods of H. pylori in each country.

Introduction

Helicobacter pylori is a microaerophilic, Gram-negative, and flagellated bacterium, which was isolated from a human gastric biopsy specimen for the first time in 1983.⁽¹⁾ This organism causes one of the most common chronic bacterial infections in humans, present in almost half of the world population.^(2–6) H. pylori is etiologically related to chronic gastritis, gastric ulcer, duodenal ulcer as well as gastric mucosa-associated lymphoid tissue lymphoma and gastric cancer.^(5–14) Indeed, the role of H. pylori in extra-digestive diseases such as ischemic heart disease, chronic obstructive pulmonary diseases, iron deficiency anemia, idiopathic thrombocytopenic purpura, Alzheimer's disease, migraine diseases, and hyperemesis gravidarum has been studied in recent years.^(14–26) A number of independent studies have reported a strong association between H. pylori and gastric cancer. H. pylori has been classified as a class I human carcinogen by the International Agency for Research on Cancer of the World Health Organization.⁽⁹⁾ Our objective is to review the current methods that are used for the detection of H. pylori infection among people.

Literature search

A systematic literature search was conducted using PubMed, EMBASE, and Google Scholar for “H. pylori” and “Diagnosis” as well as “Invasive” and “Non-invasive tests.”

Evaluation of tests

The high clinical relevance of H. pylori infection has stimulated the development of numerous diagnostic methods. There seems to be no firm agreement as to which method should be used as the gold standard for the detection of H. pylori infection. On the contrary, choosing among them is not easy, and several issues need to be considered. We have to evaluate the sensitivity, specificity, and positive and negative predictive value (PV) of a given test.^(27–34)

Upon evaluating the result of a test, it is important to know how reliable the test is and how suitable it is for its intended purpose. The terms sensitivity and specificity were used to describe the characteristics of analytical methods. The specificity of a test is a measure of the incidence of negative results in persons known to be free of a disease; that is, true negative (TN). Sensitivity is a measure of the incidence of positive results in patients known to have a condition; that is, true positive (TP). A specificity of 90% implies that 10% of disease-free people would be classified as having the disease on the basis of the test result: they would have a false-positive (FP) result. A sensitivity of 90% implies that only 90% of people known to have the disease would be diagnosed as having it on the basis of that test alone: 10% would be false negative (FN).

Specificity and sensitivity are calculated as follows: \documentclass{aastex}\usepackage{amsbsy}\usepackage{amsfonts}\usepackage{amssymb}\usepackage{bm}\usepackage{mathrsfs}\usepackage{pifont}\usepackage{stmaryrd}\usepackage{textcomp}\usepackage{portland, xspace}\usepackage{amsmath, amsxtra}\usepackage{upgreek}\pagestyle{empty}\DeclareMathSizes{10}{9}{7}{6}\begin{document} \begin{align*} { \rm Specificity} = { \rm TN} \times 100 / { \rm all \ without \ the \ disease} \ ( { \rm FP} + { \rm TN} ) \end{align*} \end{document} \documentclass{aastex}\usepackage{amsbsy}\usepackage{amsfonts}\usepackage{amssymb}\usepackage{bm}\usepackage{mathrsfs}\usepackage{pifont}\usepackage{stmaryrd}\usepackage{textcomp}\usepackage{portland, xspace}\usepackage{amsmath, amsxtra}\usepackage{upgreek}\pagestyle{empty}\DeclareMathSizes{10}{9}{7}{6}\begin{document} \begin{align*} { \rm Sensitivity} = { \rm TP} \times 100 / { \rm all \ with \ the \ disease} \ ( { \rm TP} + { \rm FN} ). \end{align*} \end{document}

An ideal diagnostic test would be 100% sensitive, giving positive results in all subjects with a particular disease, and also 100% specific, giving negative results in all subjects free of the disease.

Even a highly specific and sensitive test may not necessarily perform well in a clinical context. This is because the ability of a test to diagnose the disease depends on the prevalence of the conditions in the population being studied (prevalence is denoted by the number of people with the conditions in relation to the population). This ability is given by the PV. PV_+ve, the PV for a positive result, is the percentage of positive results that are TPs; that is, \documentclass{aastex}\usepackage{amsbsy}\usepackage{amsfonts}\usepackage{amssymb}\usepackage{bm}\usepackage{mathrsfs}\usepackage{pifont}\usepackage{stmaryrd}\usepackage{textcomp}\usepackage{portland, xspace}\usepackage{amsmath, amsxtra}\usepackage{upgreek}\pagestyle{empty}\DeclareMathSizes{10}{9}{7}{6}\begin{document} \begin{align*} { \rm PV}_{ + { \rm ve}} = { \rm TP} \times 100 / { \rm TP} + { \rm FP}. \end{align*} \end{document}

If a condition has a low prevalence and the test is <100% specific, many FPs will result and the PV will be low.

A high PV for a positive test is important if the appropriate management of a patient with a TP result would be potentially dangerous when applied to someone with a FP result. However, when a test is used for screening, the appropriate management is to perform further (diagnostic) testing, and although this may cause inconvenience for subjects with FP results, they are unlikely to be dangerous.

In order not to miss cases, a screening test should have a very high PV_−ve, the PV for a negative result; this is the percentage of all negative results that are TNs, that is, \documentclass{aastex}\usepackage{amsbsy}\usepackage{amsfonts}\usepackage{amssymb}\usepackage{bm}\usepackage{mathrsfs}\usepackage{pifont}\usepackage{stmaryrd}\usepackage{textcomp}\usepackage{portland, xspace}\usepackage{amsmath, amsxtra}\usepackage{upgreek}\pagestyle{empty}\DeclareMathSizes{10}{9}{7}{6}\begin{document} \begin{align*} { \rm PV}_{ - { \rm ve}} = { \rm TN} \times 100 / { \rm TN} + { \rm FN}. \end{align*} \end{document}

This conclusion follows directly from the fact that the test must be highly sensitive.^(35,36)

Nowadays, several tests and strategies are available for the detection of H. pylori infection, which can be classified as invasive, that is, those that detect H. pylori directly in gastric biopsy specimens, and noninvasive, that is, those based on the study of various samples (e.g., serum, urine, expired air), which indirectly indicate the presence of H. pylori.⁽³⁷⁾ On the contrary, recently, enzyme immunoassays (EIAs) for direct detection of H. pylori antigens in the stool have been developed and widely used because of their complete noninvasive nature.

Invasive Tests

Endoscopy as a first-line investigation in patients with dyspepsia can diagnose peptic ulcer disease, gastric malignancies, and H. pylori infection through biopsy-based tests such as the rapid urease test (RUT) and histology and culture. Although biopsy-based tests show a high diagnostic yield, they include disadvantages such as being expensive, the need for referral to a specialist, risk, and the discomfort experienced by patients.

Rapid urease test

The urease test is based on the presence of bacterial urease and its enzymatic activity that could be visualized. The H. pylori urease enzyme splits the urea substrate to form ammonia and carbon dioxide. Then, ammonia increases pH, which is detected by the indicator phenol red: \documentclass{aastex}\usepackage{amsbsy}\usepackage{amsfonts}\usepackage{amssymb}\usepackage{bm}\usepackage{mathrsfs}\usepackage{pifont}\usepackage{stmaryrd}\usepackage{textcomp}\usepackage{portland, xspace}\usepackage{amsmath, amsxtra}\usepackage{upgreek}\pagestyle{empty}\DeclareMathSizes{10}{9}{7}{6}\begin{document} \begin{align*} { \rm{N}}{{ \rm{H}}_2} - { \rm{CO}} - { \rm{N}}{{ \rm{H}}_2} \; + \;{{ \rm{H}}_2}{ \rm{O}} \; \to \;{ \rm{C}}{{ \rm{O}}_2} \; + \;2{ \rm{N}}{{ \rm{H}}_3} \end{align*} \end{document} \documentclass{aastex}\usepackage{amsbsy}\usepackage{amsfonts}\usepackage{amssymb}\usepackage{bm}\usepackage{mathrsfs}\usepackage{pifont}\usepackage{stmaryrd}\usepackage{textcomp}\usepackage{portland, xspace}\usepackage{amsmath, amsxtra}\usepackage{upgreek}\pagestyle{empty}\DeclareMathSizes{10}{9}{7}{6}\begin{document} \begin{align*} { \rm{N}}{{ \rm{H}}_3} \; + \;{{ \rm{H}}_2}{ \rm{O}} \; \to \;{ \rm{N}}{{ \rm{H}}_4}^ + \; + \;{ \rm{O}}{{ \rm{H}}^ - } \end{align*} \end{document}

A lot of commercial urease tests are available, including gel-based tests (CLOtest, HpFast), paper-based tests (PyloriTek, ProntoDry HpOne), and liquid-based tests (CPtest, EndoscHp). The sensitivity and specificity of RUT have been shown to be 85%–95% and 95%–100%, respectively.^{(31,35,38–40)} The method in comparison with other invasive tests is simple, inexpensive, and rapid.

Culture

Culture as a traditional method has been used for the detection of H. pylori and for determination of antimicrobial resistance pattern. There are different kinds of liquid and plating media for cultivation of H. pylori. The agar plates are cultured immediately in a microaerophilic environment, which is obtained by using a jar with a gas-generating kit for a microaerophilic atmosphere (5% oxygen and 5%–10% carbon dioxide). The plates are incubated for at least 5 days at 37°C, even though Helicobacter colonies sometimes appear after just 3 days. Then, the colonies are identified by Gram stain and biochemical tests. The colonies are Gram negative and urease, oxidase, and catalase positive. However, definitive identification of bacteria can be made using a culture method, but the rate of isolation from infected subjects varies widely between laboratories and makes it the most technically demanding of the H. pylori diagnostic tests. By definition, the specificity of culture for detecting H. pylori is 100%, while its sensitivity can widely vary between laboratories due to microbial contamination or local expertise. However, the sensitivity of the method has been reported to be <100% and >90%.^{(31–35,41,42)} It seems that the emerging multiresistant strains of H. pylori lead to increased prerequisite of culture and antibiotic sensitivity testing for patients with persistent infection after initial or repeated treatment failure.

Histology

Histology was the original technique for the diagnosis of H. pylori infection as described and introduced by Marshal and Warren.⁽⁴³⁾ Several stains such as Warthin–Starry, Hp silver stain, Dieterle, Giemsa, Gimenez, hematoxylin and eosin, acridine orange, McMullen, and immunostaining have been used for detection of H. pylori infection. This is the only method that can show both the extent of H. pylori infection and the degree of mucosal damage.⁽⁴⁴⁾ In addition, this is the only method that can detect H. pylori, lesions associated with infection, and other causes of the patient's symptoms.⁽⁴⁵⁾ The sensitivity and specificity of the histology have been reported to be 90%–95% and 95%–98%, respectively.^{(31,41,45,46)}

Molecular methods

Molecular techniques based on polymerase chain reaction (PCR) are used for the detection of specific regions of H. pylori DNA that exist in gastric biopsies, gastric juice, saliva, dental plaques, and stool of patients. Various PCR techniques have been developed, such as real-time PCR, restriction fragment length polymorphism-PCR, and nested PCR.^(33,47–49) Typically, PCR is useful in determining the effectiveness of eradication therapies, identifying antibiotic resistance mutations, and molecular typing of H. pylori by virulence-genes.^(42–57) Therefore, various genes have been used for designing primers such as 16S rRNA and 23S rRNA, Cag A, Vac A as well as fla A and ure genes (A-H).^(34,35)

However, PCR detects only specific gene fragments, not viable bacteria as well as the number of microorganisms/human cell. The quantification method with the real-time quantitative PCR can be applied to clinical samples and research samples. This assay is rapid, sensitive, and quantitative, which can reliably measure 10³ organisms. Therefore, it could facilitate monitoring of therapy, and enable more accurate epidemiological studies on the acquisition and spread of H. pylori infection.^(33,49)

Fluorescence in situ hybridization (FISH) as a molecular cytogenetic technique is a highly sensitive and specific technique for the diagnosis of H. pylori infection. In this method, fluorescent probes bind to only those parts of the DNA or RNA with a high degree of sequence complementarity. It can be a method of identification when a patient is infected with coccoid forms of H. pylori. Indeed, the ability to determine clarithromycin resistance is a considerable advantage of this method.⁽³²⁾

Noninvasive Tests

Noninvasive tests are mainly based on specimens, such as blood, expired air, saliva, and feces, for diagnosis of H. pylori infection. These tests fall into two categories: direct and indirect; the former looks for direct evidence of the presence of H. pylori, as invasive tests do. On the contrary, these tests can distinguish between active infection and a previous contact. For these reasons, these are called active tests, including the stool antigen tests, which assess the presence of H. pylori antigens in stool, and urea breath test (UBT), which assesses H. pylori urease activity.

Indirect tests detect the H. pylori infection by evaluating indirect evidence, such as the presence of serum, saliva, and urine antibodies against H. pylori, or the amount of labeled CO₂ in the breath, an expression of the urease activity of the bacterium.^(58,59) Indeed, potential indications for the use of noninvasive tests should therefore be based on (1) screening of patients not necessarily requiring histologic examination; (2) follow-up of treatment success, evaluation of infections associated with a low pathogen density; (3) investigation of the association between H. pylori and extra-gastric disorders; and (4) comparative epidemiology and vaccination against H. pylori. ^(37,60) In the following, discussion of the most important noninvasive methods and their current status has been summarized.

Urea breath test

The UBT is the most accurate of noninvasive tests developed by Graham in year 1987 only a few years after the description of H. pylori.^(60,61) The basis of test relies on the urease activity, which is found in the stomach of persons infected with H. pylori. Therefore, it can distinguish active infection. Patients ingest urea labeled ¹³C or ¹⁴C. Hydrolysis of urea occurs within the mucus layer and results in the production of labeled CO₂. This is close to the epithelial blood supply and the concentration gradient will pass to it, and within a few minutes the isotopic carbon dioxide will appear in the breath. The sensitivity and specificity of the UBT are 97% and 95%, respectively.^(59,62–65) At the moment, UBT has been used as the gold standard test by many clinicians.

Serological tests (tests based on antibodies)

The host immune system produces antibodies against H. pylori soon after gastric colonization by the bacterium. The antibody-based tests are also called immunologic tests developed for the detection of specific anti-H. pylori immunoglobulin g (IgG) antibodies in the serum of patients. Indeed, several commercial kits are able to detect immunoglobulin A (IgA) in the saliva or IgG in the urine.^(66–80) In this regard, different techniques are available which include the following tests.

Enzyme-linked immunosorbent assay

After the successful cultivation of H. pylori, serological tests became available.⁽⁴²⁾ The types of enzyme-linked immunosorbent assay (ELISA) are more appropriate than a diagnostic test for detecting H. pylori infection in epidemiological studies of populations. Indeed, these techniques use EIAs in ELISA format for the detection of antibodies against H. pylori antigens in serum, saliva, and urine samples.^(66–77) Various antigen preparations have been done in coating ELISA wells in these methods: crude antigens, such as whole cell extracts and sonicated cell extracts, glycine extracts, heat-stable antigens and recombinant antigens.^(81,82)

According to the results of other studies, it was shown that IgA and immunoglobulin M (IgM) antibodies against H. pylori are not useful for the diagnosis of H. pylori infection.^{(2,37,81,83,84)} In other words, IgA and IgM detection only provides a little additional sensitivity to the method. Furthermore, the methods based on antibody detection are not able to distinguish between active infection (the presence of live H. pylori in the gastric tissue) and previous contact. Antibody levels persist in the blood for long periods of time.^{(58,59,66,83)} Another key issue in these tests is the choice of antigens for coating the ELISA wells. The highest performance of these tests is based on specificity for the detection of local H. pylori strains in each country.^(49,70) However, according to the meta-analysis studies, the sensitivity and specificity of commercial ELISA tests have been 54%–94% and 59%–97%, respectively.^(85,86) Many studies considered urine-based ELISA as accurate as serum-based ELISA, but salivary ELISA did not show appropriate sensitivity and specificity.^(2,58) However, many studies recommended the serological tests for epidemiological studies based on the following reasons: inexpensive, availability, rapid, and ease of use. But it is not recommended for the confirmation of H. pylori eradication after treatment.

Immunodot blot assays

The principle of these tests is based on the Western blotting technique for detection of antibody in serum, urine, stool, and saliva against specific proteins of H. pylori, such as CagA and VacA.^(41,58) In this regard, several studies have reported the production and application of monoclonal antibodies (MAbs) specific for H. pylori antigens. Species-specific interaction of a MAb with a surface-exposed protein leads to rapid and highly specific identification of H. pylori infection. In addition, the dot blotting test eliminates the need for biochemical tests for typing of bacterial isolates with properties similar to those of H. pylori, and can therefore be regarded as a more specific method for identification after the culture of samples. Furthermore, other applications with MAbs may be developed; that is, detection of H. pylori antigen in gastric fluid or biopsies by inhibition ELISA or immunofluorescence microscopy. Many studies have found its sensitivity and specificity as those measured for the serum ELISA.^(2,41,70)

Immunochromatography

RAPIRUN Kit as a point-of-care testing is an immunochromatographic assay that is rapidly able to detect antibodies against H. pylori in urine. Its sensitivity and specificity are similar to those measured for the serum ELISA.^(2,49,78) Since this test can be performed at the physician's office and it quickly detects contamination, it is superior to the ELISA method. In recent years, H. pylori IgG antibody from maternal and cord serum was evaluated using a commercial immunochromatographic test kit. The principle of this test is based on the transmission of H. pylori IgG antibody through the placenta into the fetal circulation. However, the results of the test can be affected by a low level of antibody titer. Therefore, accuracy of the test kit needs to be evaluated before utilization in screening.⁽³⁰⁾

Stool antigen tests

These methods are similar to invasive tests, which are directly looking for evidence of the presence or absence of H. pylori. Therefore, those are classified as active tests that are able to detect active infection of H. pylori. In fact, these tests are based on the detection of H. pylori antigens in the patient's stool in ELISA format. In these methods, an antibody against H. pylori antigens is coated into ELISA wells. Just a small amount of stool suspension is needed for performing the test.^(87–90)

During this year, different kinds of stool antigen tests such as stool-PCR have been developed. According to the results, the sensitivity and specificity of stool-PCR were 62.5% and 92.3%, respectively. This finding indicates that the stool-PCR test as a simple and noninvasive method can be useful for the detection of H. pylori active infection as well as virulence genes in stool samples of patients, especially among children.⁽⁹¹⁾

Conclusion

Although there are several available diagnostic methods for the detection of H. pylori infection, each one of them has its own convenience and limitations. It seems that the stool antigen test and PCR may be slightly superior to other diagnostic tests for the detection of H. pylori from different clinical samples but are expensive. Indeed, serological tests are appropriate for epidemiological screening studies because of their reproducibility, ease of use, lower cost, and availability. Although the culture method is assumed to be a more specific test, a golden standard test has not yet been developed for the detection of H. pylori. Accordingly, the most reliable diagnosis can be achieved by a combination of two or more tests in routine clinical practice.

Footnotes

Acknowledgment

This work was supported by the Department of Microbiology, Shahr-e-Qods-Branch, Islamic Azad University, Tehran, Iran.

Author Contributions

T.M. contributed to this article with conception and design of the study, literature review and analysis, drafting and final approval of the final version. L.G. contributed to this article with critical revision and editing, and final approval of the final version.

Author Disclosure Statement

No competing financial interests exist.

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