Diagnostic Methods for Helicobacter pylori

Abstract

Helicobacter pylori infection is a significant global health concern. It cannot be diagnosed based solely on the patient’s medical history and symptoms, and laboratory and imaging tests are often required to confirm the diagnosis. Both noninvasive and invasive methods are available for diagnosing H. pylori infection, including conventional and advanced detection techniques. It is not uncommon for patients to present with false-negative results due to the use of inadequate investigation methodologies, which prevents the adoption of appropriate clinical management. Thus, an analysis of the literature regarding the methods of diagnosis of H. pylori, with its advantages and disadvantages, is necessary. Publications in specialized scientific journals will undoubtedly contribute to facilitating access by professionals interested in the topic providing greater knowledge and potentially clinically useful guidance. In this review, the authors have sought to analyze and summarize the invasive and noninvasive methods, their applications, limitations, and the conditions that affect the sensitivity of the tests used for diagnosing H. pylori, an essential step for the successful treatment of this infection. It is essential to treat all patients infected with H. pylori. This represents a significant change in the approach, as the treatment was recommended previously only for patients showing symptoms of infection. Therefore, it is crucial to understand the limitations of traditional diagnostic methods and help raise awareness among healthcare professionals about the latest advances in diagnosing this important bacterium.

Highlights of the Study

• H. pylori infection is associated with an increased risk of pathologies including extra gastric diseases and therefore its prompt diagnosis is crucial.

• The choice of method for detecting H. pylori infection depends on the patient’s clinical situation, age, and prevalence of this bacterium in the population.

• Comprehensive knowledge of advances in H. pylori diagnostics is essential for selecting more appropriate and reliable diagnostic tests.

Introduction

Helicobacter pylori is a helical, Gram-negative, microaerophilic bacterium that requires low concentrations of O₂ and infects the epithelial lining of the stomach [1]. It was one of the first bacterial species to have its genome fully sequenced showing a very high genetic diversity among H. pylori strains [2]. Although H. pylori infection remains asymptomatic in most people, it remains an important health problem worldwide and its presence is associated with an increased risk of several pathologies [1, 3]. It is transmitted from human to human and is responsible for disease regardless of an individual’s symptoms and the stage of the disease [4]. This bacterium constitutes the main cause of chronic gastritis, gastric cancer, and peptic ulcer [5] and may also be responsible for nonulcer dyspepsia, recurrent peptic ulcer bleeding, unexplained iron deficiency anemia, idiopathic thrombocytopenia purpura, and colorectal adenomas [6]. H. pylori is the first bacterium formally recognized to have carcinogenic potential. The relationship between H. pylori and diseases unrelated to the digestive system is mainly due to the release of various proinflammatory agents such as cytokines, eicosanoids, and acute-phase proteins, in addition to the possibility of microbial antigens mimicking those of the host resulting in the activation of autoimmunity [1].

According to the World Gastroenterology Organization (2021), H. pylori infection affects half of the world’s population, and its prevalence depends on geographic region, ethnicity, race, age, and socioeconomic factors, being higher in developing countries. There is wide variation in the prevalence of infection between and within countries, within a single city, and even in subgroups within a population [8]. In Brazil, both urban and rural areas have high rates of infection prevalence [9]. Among adults, a prevalence rate of 82% has been recorded, while for the age group of 10–19 years the rate is 72% [10]. The prevalence of H. pylori infection is highest in adults in Africa, Eastern Mediterranean regions, Russia, Central America, and South America. In children, the prevalence is lower than that in adults in Russia, Western Pacific regions, and Europe. However, the prevalence of H. pylori infection is similar in children and adults in Africa, Eastern Mediterranean regions, Central America, and South America [11]. Guidelines of the European Helicobacter Study Group (EHSG) Fourth Maastricht/Florence Consensus Conference recommend that people with a high risk of gastric cancer and those with dyspepsia (depending on the local incidence of gastric cancer) be tested for H. pylori, in addition to patients without characteristic symptoms or “alarm” signs, but with an increased risk of gastric cancer such as weight loss, dysphagia, upper gastrointestinal bleeding, abdominal mass, or iron deficiency anemia. People presenting with unexplained iron deficiency anemia, idiopathic thrombocytopenic purpura, and young patients with dyspepsia but from regions where the prevalence of H. pylori is high (≥20%) should also be tested [6, 12, 13].

There are two types of diagnostic methods to identify H. pylori infection involving invasive approaches (endoscopy, histology, culture, and molecular methods) and noninvasive approaches (urea breath test [UBT], fecal antigen, serological and molecular methods) [11, 14–16]. Diagnostic accuracy for most methods is affected by medications to treat H. pylori, as they can reduce the sensitivity of the test [17]. It is important to evaluate the performance of laboratory tests for H. pylori at the local and regional levels. Test selection and interpretation should be based on disease prevalence, potential for therapeutic interference, and cost [18]. Some conditions affect the diagnostic accuracy of the tests used as shown in Table 1.

Table 1.

Factors that can affect the diagnosis and influence the accuracy of this test

Factors	Test
Consumption of PPIs, antibiotics, and N-acetyl cysteine (NAC) [14, 16]	Stool samples
Consumption of bismuth-containing products, and antibiotics [16, 67]	UBT/RUT/SAT
H2-receptor antagonists, PPIs, antibiotics, and the presence of blood [17]
PPI or bismuth-containing products [16, 67]	Serology
Alcohol drinking, bleeding, antibiotic, and PPIs [16, 67]	Culture
H2-receptor antagonists, PPIs, and antibiotics [17]
Bleeding [9]
Bleeding [16]	UBT/SAT
PPIs [9]
Bleeding [9]	PCR (endoscopic tests)

PPIs, proton-pump inhibitors; UBT, urea breath test; RUT, rapid urease test; SAT, stool antigen test; PCR, polymerase chain reaction.

An accurate diagnosis of H. pylori infection is the first step toward successful treatment of the patient with this infection [16]. For accurate diagnosis of this infection, the ideal is not only to use noninvasive methods but also to use invasive tests [14]. This review aims to explore invasive and noninvasive diagnostic tests for diagnosing H. pylori infection. In addition, the review seeks to identify appropriate and reliable diagnostic tests that are available to contribute to better patient management and eradication of this infection.

Methodology

We conducted a literature search based on the databases PubMed, Cochrane, Google Scholar, and Virtual Health Library (BVS). We used the following descriptors in English “Helicobacter pylori,” “Helicobacter infections”, and “diagnostic test” and in Portuguese “Helicobacter pylori,” “infecções por Helicobacter,” and “teste diagnóstico.” Only articles in Portuguese and English were chosen. In the first approach, potentially eligible articles were chosen by reading the title and abstract. The following are the main methods of diagnosis of H. pylori infection.

Methods for Detecting H. pylori

Conventional and advanced detection techniques (Fig. 1) have been successfully applied to the diagnosis of H. pylori infection that include noninvasive and invasive methods [11, 15]. Invasive methods are performed on tissues obtained during gastrointestinal endoscopy, while noninvasive methods are based on the presence of bacterial enzymes, antigens, antibodies, or DNA sequences [16]. Among the diagnostic methods to detect or confirm the eradication of H. pylori infection, the choice of method depends on several factors. Such factors include the availability of diagnostic tests, need for endoscopy, cost, accessibility, and advantages and disadvantages of each method, in addition to the age of the patients [19]. In patients with “alarm symptoms” that include dysphagia, odynophagia, weight loss, anemia, and bleeding, the so-called test-and-treat strategy has been advocated to detect and eradicate H. pylori [20]. Test-and-treat is a well-defined strategy and refers to noninvasive testing for H. pylori in patients with dyspeptic symptoms and eradication of the infection whenever detected [21]. In young patients with uninvestigated dyspepsia the “test-and-treat” strategy, noninvasive tests are chosen over referral for Oesophago-Gastro-Duodenoscopy, avoiding costs, inconvenience, and discomfort [4]. The “test-and-treat” strategy using a noninvasive approach is considered for adult patients aged <40 years and dyspepsia but no concern signs including weight loss, dysphagia, persistent vomiting, palpable mass in the abdomen, jaundice, gastrointestinal bleeding, and no history of gastric cancer in first-degree relatives [9]. The accuracy of diagnostic tests for H. pylori in patients with bleeding peptic ulcers indicated that combined index tests showed a trend toward higher sensitivity, while single index tests had higher specificity values when ranked [22].

Fig. 1.

H. pylori detection methods that allow successful diagnostic strategies. On the left are invasive methods (endoscopy, culture, histopathology, rapid urease test, and molecular methods). On the right are noninvasive methods (respiratory tests, fecal antigens, serology, and molecular methods). These various techniques contribute to a comprehensive range of diagnostic options for H. pylori, enabling effective detection strategies.

Sensitivity and specificity are essential indicators of test accuracy and allow healthcare providers to determine the appropriateness of the diagnostic tool aiding in decision-making and improving healthcare for patients [23]. It is critical to use at least two diagnostic tests with high sensitivity and specificity based on the clinical situation to achieve an accurate diagnosis of the pathogen [24, 25]. Therefore, the sensitivity, specificity, and limitations of the H. pylori detection test, which plays a critical role in the accurate diagnosis and assessment of the presence of the bacteria, are presented in Table 2.

Table 2.

Sensitivity, specificity, and limitations of H. pylori detection

Tests	Sensitivity (95% CI)	Specificity (95% CI)	Limitations
Magnifying endoscopy	89 [29]	82	Higher costs [62]
LCI	82 [26]	8
WLI	53 [26]	8
Histology	89 [31]	100	Higher costs [8, 24]
	95.6 b [24]	77.8	Very operator-dependent [8]
Culture	91.2 [31]	100
	91.46 [24]	100	Specific transport conditions, laboratory skills, and equipment [8]
RUT	88.5 [23]	84.2	Positive results should be confirmed by a second test [23]
	95.6 [24]	100
	89.3 [68]	89.9
13C-UBT	97 [37]	96	Specific conditions (cool temperature, microaerophilic conditions) [31], high cost of commercial kits, lack of expertise [8], and restrictions by national authorities to acquire the substrate [9]
SAT	61.54 [66]	59.65
	55.6 [68]	98.9	Differences in antigens used in different geographic regions may cause heterogeneity of results [38]
Multiplex serology	89 [40]	82	Antigen preparations [40]
Serological test	88.4 [69]	93.4	Cannot distinguish between current and past infections [34]
FISH	95.7 [31]	98.6	High cost and skill requirement [44]. Poorly standardized requires experience in the interpretation of the results, and sensitivity is far below that of PCR in case of analysis from primary materials [52]
PCR	75–100 [70]	95.6–100	High cost and skill requirement, false-positive results due to detection of DNA fragments of dead bacteria [44]
MLST	50 [59]	86	High cost of DNA sequencing [55]

WLI, white light imaging; LCI, linked-color imaging; CI, confidence interval; RUT, rapid urease test; UBT, urea breath test; SAT, stool antigen test; FISH, fluorescence in situ hybridization; MLST, multilocus sequence typing.

Endoscopy

The main endoscopic methods for diagnosing H. pylori infection are conventional white light imaging and magnification narrow band imaging (NBI). The linked-color imaging technique is a recent endoscopic method, which clearly shows slight color differences caused by pathological changes in the mucosa of the digestive tract [26, 27]. Oesophago-Gastro-Duodenoscopy should be considered in patients with dyspeptic symptoms, particularly in populations with a low prevalence of H. pylori and when alarm symptoms are present such as weight loss, dysphagia, apparent gastrointestinal bleeding, abdominal mass, or iron deficiency anemia [4]. Endoscopic techniques such as LCI and blue laser imaging are being evaluated for the diagnosis of H. pylori gastritis or the detection of early gastric cancer [28]. In Asia, the prevalence of H. pylori infection and upper gastrointestinal malignancy is high. Alarm features and age have been of limited value in predicting malignancy and therefore immediate endoscopy should be the initial strategy for uninvestigated dyspepsia.

The optimal age limit for endoscopic screening should be 35 years [29]. Endoscopy with biopsy of the gastric mucosa for rapid urease testing and histopathological examination remains the standard for the diagnosis of H. pylori. A study compared site-specific biopsy using C-NBI system and standard biopsy. Sensitivity, specificity, and positive and negative predictive values were assessed for the diagnosis of H. pylori infection and premalignant gastric mucosa in daily clinical practice. Magnifying endoscopy for H. pylori infection enables a high level of diagnostic accuracy, clear visualization of the microsurface structure, and microvascular architecture of the gastric mucosa [30]. The sensitivity, specificity, and positive and negative predictive values for predicting H. pylori positivity have been higher on site-specific biopsy using C-NBI compared to standard biopsy [31].

Histopathology

Histology is one of the most commonly used diagnostic methods by regular staining or immunostaining [28]. It is considered the gold standard for the direct diagnosis of H. pylori. However, the accuracy of this diagnostic method depends on the load of microorganisms in the sample and the site of the gastric mucosa in which the biopsy was collected [14, 25]. Histological diagnosis is generally confirmed through four biopsies (two from the curves and two from the body of the stomach) [1]. Occasional disagreement in interpretations of review of biopsy slides and original histopathological diagnoses reflects that histopathology is not a perfect gold standard even though it is the best available reference standard. When histopathology is rectified by a consensus review by multiple pathologists, it offers the gold standard for diagnosis. Histopathological evaluation with routine hematoxylin eosin staining has reported a sensitivity of 70–95% and this can be increased by special stains such as silver-based Genta or H pylori immunohistochemistry [18].

Culture

Cultures of H. pylori from gastric biopsy samples are performed only in specialized laboratories, but it is not a routine method to detect this bacterium. This approach is mainly performed for scientific research and when previous treatments have failed to be effective [16]. The culture method is time-consuming and requires specific conditions, such as cool temperature, microaerophilic conditions, and enriched culture media that are not usually available in diagnostic centers [32]. Procedures less invasive than a biopsy, such as gastric juice sampling or string test, can be used for culture; however, the sensitivity is lower when biopsy samples are used [19]. A detectable number (median 1.75 × 10¹ cfu/mL) of viable H. pylori can be cultured from gastric juice. This may vary based on infection severity, and not all organisms may remain viable when shed into gastric juice [33].

Rapid Urease Test

Urease is an enzyme that hydrolyzes urea (carbamide) in ammonia and carbon dioxide and is produced by several bacterial species [34]. In clinical practice, when there is an indication for endoscopy and there is no contraindication for biopsy, the rapid urease test (RUT) is recommended as a first-line diagnostic test to detect the presence of H. pylori in the gastric mucosa [4]. After the biopsy, the sample is transferred to a solution containing urea and a pH indicator. The urease converts the urea into ammonia and CO₂, which leads to a change in the color of the indicator due to the increase in pH. To obtain better results, biopsy samples should be taken from the gastric antrum and body. Several organisms, such as Klebsiella pneumonia, Staphylococcus aureus, Proteus mirabilis, Enterobacter cloacae, and Citrobacter freundii, isolated from the oral cavity and/or stomach, also show urease activity and can induce false-positive results when present [16] leading to low test specificity. The urease produced by some bacteria of the microbiota in the oropharynx, which is swallowed in saliva, does not influence the results of this test, as it is rapidly denatured due to the high acidity of the stomach [19]. Another interesting aspect is that the gastric biopsy used for RUT can be reclaimed for other H. pylori tests such as PCR [28].

Urea Breath Test

The UBT is described as a gold standard noninvasive method for H. pylori diagnosis [9, 16]. The UBT evaluates urea conversion using a radioactive isotope of carbon [14, 35]. This test measures the activity of urease produced by H. pylori in the stomach following oral administration of isotopically labeled urea (¹³C or ¹⁴C), which is then hydrolyzed into ammonia and carbon dioxide. ¹³C-Labeled urea is stable and nonradioactive; thus, it is preferable to ¹⁴C. Despite the low cost and easy execution, the use of the ¹³C-UBT has not yet been incorporated into daily practice in Brazil, due to restrictions by national authorities regarding the acquisition of the substrate [9].

The isotope-labeled urea is consumed by the patient and then H. pylori breaks down this urea through urease in the stomach and the CO₂ that diffuses through the epithelial cells is absorbed by the blood and excreted through the lungs. CO₂ is detected through exhalation during breathing after 10 min and serves as an indicator of the presence of H. pylori [16, 35]. Conventional UBT uses ¹³C-urea diluted in orange juice, which can be influenced by the urease-producing oral flora, increasing the excretion of ¹³CO₂ in previous sample collections with false-positive results. The test using ¹³C-urea with a capsule has been presented with a lower dose than the conventional test with high sensitivity and specificity [36]. The results of the UBT film-coated [¹³C] urea tablets can be influenced by the presence of urease-positive non-H. pylori bacteria. It is likely that non-H. pylori urease-positive bacteria in the stomach rather than in the oral cavity can cause false-positive reactions, as UBT values at 20 min were significantly higher than those at 5 min. Urease-positive non-H. pylori bacteria in the oral cavity might be responsible for false-positivity after their translocation from the oral cavity to the gastric mucosa in a deficiency of stomach acid [37].

UBTs had high diagnostic accuracy when comparing serology, stool antigen tests, and UBTs in people without a history of gastrectomy and who had not recently used antibiotics or proton-pump inhibitors (PPIs). On the other hand, serology and fecal antigen tests were less accurate for the diagnosis of H. pylori infection [6]. Differences in the dose of ¹³C-urea and the respiratory sample collection procedure are the main sources of variation in the outcome. Adjusting the testing protocol for these factors could improve accuracy estimates [38]. In places with difficult access to endoscopy, the use of UBT could minimize the number of invasive procedures and related expenses for the health system, as well as the discomfort of patients [35].

Stool Antigen Test

This test is based on the detection of H. pylori antigens in the stools excreted from infected individuals [39]. There are two types of SATs used for the detection of H. pylori, namely enzyme immunoassay and immunochromatography assay (ICA), using either polyclonal antibodies or monoclonal antibodies [16]. SAT does not require the patient to fast, and when monoclonal antibodies are used, there is no need to discontinue PPIs. ICA has the advantage of providing rapid diagnosis of the infection and may be useful in developing countries with many remote areas that are difficult to access. Some of the advantages of the ICA-based SAT are the possibility of storage in many hospitals and the facility of carrying out the test in small laboratories as this test does not require special equipment or specialists for its execution [35]. The stool antigen test (SAT) by enzyme-linked immunosorbent assay (ELISA) is another good option when ¹³C-UBT is not available, as long as a monoclonal antibody is used as a reagent [9]. A meta-analysis on H. pylori SAT for the diagnosis of the infection in children comparing diagnostic accuracy of pretreatment and posttreatment conditions suggested that the sensitivity and specificity of SAT after eradication were like pretreatment conditions. The sensitivity and specificity of H. pylori were significantly higher in studies using at least two diagnostic methods than just one diagnostic method because false positives are much lesser when using two or more diagnostic methods [40].

Serology

Serological tests are mainly based on the estimation of antibodies against H. pylori. Specific IgG, IgA, and IgM can be measured in screening for H. pylori infection, and IgG shows more reliable results [14]. In this method, antibodies against H. pylori are detected by ELISA, immunoblotting, and enzyme immunoassays requiring the use of serum, saliva, or urine [16, 41]. The three main serological tests are ELISA, latex agglutination tests, and Western blotting, with ELISA being the most commonly used [6]. Serology can be used as the initial test for the diagnosis of H. pylori infection, especially in the presence of gastrointestinal bleeding, atrophic gastritis, gastric mucosa-associated lymphoid tissue lymphoma, and gastric cancer [9]. Serology has the advantage of being an exclusion test due to its high sensitivity and negative predictive value [18].

As H. pylori infection is chronic, IgG is the only appropriate antibody to be tested in patients with documented peptic ulcer disease and/or active bleeding. Testing for IgM and IgA is rarely advised, as IgM antibodies are elevated only in acute infections and may be missed in chronic H. pylori infections. However, testing for IgA antibodies can be useful when ordered along with IgG, as the use of both tests was shown to result in a high positive and negative predictive value. H. pylori IgM and IgA have limited clinical utility, as they are imprecise and costly especially when more accurate alternatives are available [13].

Serology is not recommended in areas with a low prevalence of H. pylori [35]. The low prevalence of H. pylori in a population increases the chance that a positive serological test for this bacterium is false. This suggests that the positive predictive value of the test decreases with decreasing prevalence of H. pylori. Some serological tests have high sensitivity and specificity, but these tests may perform differently in different geographic locations according to the antigenic composition of the circulating strains and only locally validated tests should be used [4]. Several serological test kits for H pylori are available, but the performance of some is influenced by patient factors, including age, sex, and ethnicity. Therefore, appropriate selection of kits and local validation of performance are important [18]. A validated serological test is useful for initial screening before the diagnosis is confirmed by histology or culture, especially in a country with a deficiency of endoscopy centers [19, 35]. The use of serological testing to screen patients with dyspepsia may save cost and reduce the number of endoscopies [35].

Several tests for detecting antibodies to H. pylori in urine and saliva samples have shown high sensitivity and specificity. It should be noted that urine and saliva samples can be obtained easily, without the need for special skills or tools, being cheaper than serum samples [35]. Assessment of H. pylori antibodies in urine or saliva is a relatively new diagnostic method. However, lower concentrations of antibodies in these samples compared to serum-based diagnostic methods may impede the correct identification of H. pylori infection [14, 35]. The main disadvantage of the serological approach is its inability to distinguish between current infection and previous exposure [14, 18]. The H. pylori infection may be recent, and the antibody levels may not yet be high enough for detection considering that IgG antibodies appear approximately 21 days after the H. pylori infection. After successful treatment for the eradication of H. pylori, IgG antibodies to this bacterium persist for several months [35]. This leads to misinterpretation, as a positive result can be demonstrated even after bacterial clearance [16].

In addition to conventional serological diagnosis of infections, multiplex serology is available for the simultaneous estimation of antibodies to protein antigen arrays. Multiplex serology for H. pylori is based on a glutathione-S-transferase capture immunosorbent assay combined with fluorescent bead technology [41]. Multiplex serology for H. pylori was carried out using 12 H. pylori antigens [42] or 15 proteins from 3 different strains [41, 43]. Utilizing H. pylori multiplex serology as a noninvasive test with high-throughput capacities may allow risk stratification for targeted prevention strategies when combined with other predictive factors [44].

Molecular Methods

Molecular techniques based on DNA/RNA are used in the invasive and noninvasive diagnosis of H. pylori for improvement of the conventional methods, especially the sensitivity. Molecular methods include polymerase chain reaction (PCR), real-time PCR, digital droplet PCR, fluorescence in situ hybridization (FISH), and next-generation sequencing (NGS) including 16S rRNA amplicon sequencing, transcriptomics, and metagenomics [45].

PCR tests have high sensitivity to detect low numbers of organisms in clinical samples, which can amplify the target DNA of coccoid forms of H. pylori that are difficult to cultivate and identify histologically [19, 46]. PCR-based detection of H. pylori could be classified as both invasive and noninvasive. This method detects specific DNA regions of H. pylori genes such as vacA, cagA, ureA, ureC (glmM), hsp60, 16SrRNA, 23S rRNA, and ureC (glmM), found in gastric biopsies, gastric juice, saliva, dental plaque, and feces collected from patients [14, 16]. PCR with analysis directed to the 16S rRNA gene was confirmed to be a much more sensitive method for the detection of H. pylori in gastric biopsies compared to other methods [28]. The accuracy of PCR is limited to the number of H. pylori genes (vacA, cagA, ureA, glmM, 16S rRNA, and 23S rRNA) tested. It is recommended that more than one gene should be included to minimize the chance of recognizing some closely related species, whose limitation also applies to 16S rRNA sequencing [45]. PCR has not been adopted as a routine test due to some requirements of the technique, in addition to the higher price compared to conventional tests. When nonspecific primers are used, there is a risk of false-negative results due to mutations and false-positives. The use of inappropriate, nonspecific primers and the constant use of outdated primers amplify the DNA of other biological species. The difference between positive cases identified by conventional or nested PCR indicates that even these molecular methods are not sensitive enough to detect all cases of low-density infection [47]. An important disadvantage of PCR is that the reactions can detect DNA segments of dead bacteria in the stomach lining of patients after treatment; consequently, they can produce false-positive results [19]. The prevalence of H. pylori in asymptomatic healthy persons, comparing saliva and stool samples, would have been even higher when used for the detection of H. pylori indirect immunofluorescence assay and molecular methods, including semi-nested PCR (vacA) and real-time PCR (16S rRNA) [3].

The use of the NGS technique leads to faster characterization of virulence factors that can result in higher levels of personalized treatment and management of H. pylori-associated infections. NGS is capable of answering questions such as the determination of virulence factors, prediction of antibiotic resistance, phylogenetic analysis, and epidemiological monitoring [2]. The decrease in genetic sequencing costs allows the inclusion of NGS in the arsenal to detect H. pylori. It is possible to use NGS to detect H pylori mutations in the gyrA, 23S rRNA, and 16S rRNA genes, which are also known to confer antibiotic resistance [28]. The 16S rRNA amplicon sequencing approach is a useful tool for diagnosing H. pylori infection, especially among those who present with abnormal gastric histopathology and negative results using routine diagnostic methods [48]. NGS for the bacterial 16S rRNA gene has high sensitivity and specificity (95–100%) and presents quantitative data from the microbiome showing interaction between species, and prediction of antibiotic resistance. This method has some limitations such as high cost, requires skills and false positives due to the cutoff value and lack of negative controls, is also unable to distinguish between live and dead bacteria, and may not separate H. pylori from closely related species [45]. The 16S rRNA gene includes nine hypervariable regions (V1–V9) [49, 50] with more conservative regions being useful for determining higher-ranking taxa, while regions of fast-evolving can help to identify the genus or species [49]. The choice of the 16S rRNA region can significantly affect estimates of taxonomic diversity. While the full V1–V9 region consistently yields the best results (<99% identity), the V2–V3 region and the V3–V4 region provide a better picture of species or genus diversity resolution. V3–V4 fragments that detect genus with greater sensitivity than species (e.g., Helicobacter genus vs. H. pylori) may not allow distinguishing some level-specific OTUs, erroneously merging them into a single species [48].

A recently developed reagent for H. pylori genes and the clarithromycin resistance mutation showed performance comparable to that of real-time PCR and sequencing analysis. This detection performance of a reagent for H. pylori genes using a stool specimen was achieved using Smart Gene™, which is based on point-of-care genetic testing. The detection rate for H. pylori genes using Smart Gene™ was 95.7%, whereas real-time PCR using the same samples had a detection rate of 92.8%, and sequencing analysis had a detection rate of 89.2%. In terms of detection performance for a clarithromycin resistance mutation-resistant mutation, the overall concordance rate between Smart Gene™ and sequencing analysis was 96.7% [51]. The use of molecular techniques such as FISH and multilocus sequence typing (MLST) has become popular and widely available for microbiologists. FISH is suitable for providing spatial information, which is useful in identifying key species in mixed infections and studying biofilm architecture in microbial communities-associated infections [52, 53]. It is a suitable alternative to phenotypic methods and can also detect point mutations associated with antibiotic resistance [54]. More than half of antibiotic-resistant cases were found to have antibiotic heteroresistance, with a high prevalence of intraniche-type heteroresistance often occurring in both the antral and corpus regions of the stomach. Therefore, it is recommended to take multiple samples and test the clarithromycin susceptibility of every H. pylori-positive antral and corpus biopsy specimen to avoid false-susceptible results [55]. The FISH method has the highest percentage of consistency with the gold standard. The combination of histology with a molecular technique such as FISH can lead to a reduction in false-negative results and improve the accuracy of diagnosing H. pylori infection [32].

MLST is a method for identifying bacterial isolates by sequencing seven housekeeping genes [56–58]. The capacity of H. pylori to acquire and randomly incorporate DNA into its own genome (natural transformation competence) makes RAPD test results unreliable for this bacterium. Two methods are currently able to distinguish H. pylori strains from one another. One of the methods is MLST, which relies on the stability of seven highly conserved genes in the genome of H. pylori, and the other one is whole-genome sequencing [59]. This approach can help identify the evolutionary connections between isolates obtained from a single patient who has H. pylori mixed infections defined as an individual with two or more H. pylori isolates with different antibiotic sensitivity characteristics. Additionally, the geographical origin of the human host can be determined through the MLST outcomes [58]. MLST updates the data on the prevalence and resistance of H. pylori to antibiotics, performs a phylogeographic characterization of the isolated strains [60], and explores the association between H. pylori virulence genes and chronic gastritis [61].

Eradication Test

The recommended duration of H. pylori therapy is 14 days. The treatment plan should be based on susceptibility testing or high local success rates. After treating every patient, it is essential to confirm the success using a test of cure. This test will also provide an indirect measurement of resistance or susceptibility in the population [62]. The detection of H. pylori after eradication therapy requires special attention due to the possible loss of sensitivity of the tests in some cases [9]. It is important to evaluate the prognosis after eradication therapy to detect recurrence and treatment failure, which is common due to the organism’s resistance to multiple drugs [7]. The choice of test to assess the eradication of H. pylori should be based on low invasiveness and high accuracy [9].

Testing for eradication of H. pylori is generally recommended in patients with peptic ulcer or MALT lymphoma associated with this bacterium, resection of early gastric cancer, or persistent symptoms. All tests for confirmation of eradication are considered more accurate when performed at least 4 weeks after the end of antibiotic therapy [17]. Two classes of drugs, potassium-competitive acid blockers and PPIs, are used in clinical practice [21, 63]. Effective acid suppression is an essential component of H. pylori eradication. More potent and quicker acid inhibition stimulates the growth of H. pylori, which in turn enhances the bactericidal effects of growth-dependent antibiotics. Eradication with Vonoprazan (the most studied potassium-competitive acid blockers) is more effective than PPI-based first-line therapy and was similar and not inferior to PPI for second-line and third-line treatments [64]. In patients taking bismuth-containing compounds or PPIs, testing should be performed after 1–2 weeks to avoid false negatives [17].

UBTs and stool tests used to confirm eradication of infection should be performed at least 1 month after the conclusion of eradication therapy [8, 19]. To minimize false-negative results, the patient should not take antibiotics or bismuth compounds for at least 1 month before the test. The use of PPIs should preferably be avoided for 2 weeks. The superiority of the monoclonal SAT compared to the polyclonal test occurs both for early diagnosis of infection and for validation of H. pylori eradication [19]. Therefore, the use of RUT is not recommended after bacterial eradication because the sensitivity decreases significantly. However, the SAT is a consistent test for both diagnosis and eradication control [9, 65].

Serology is not useful for assessing treatment outcomes, as antibody levels often persist for years after therapy [8]. As serological tests cannot distinguish between active infection and previous exposure, they cannot be used to confirm eradication. As antibodies to H. pylori can remain detectable in serum long after eradication, a positive serology for H. pylori can be a false positive, resulting in unnecessary treatment in patients who no longer have an active infection or who have completed eradication therapy. Due to the false-positive rates associated with antibody-based tests, UBT or SAT when ordered in patients with a positive H. pylori antibody may have an associated twofold cost [13].

Molecular tests can be used to assess the antimicrobial resistance of H. pylori after the second or third treatment failure. However, the lack of availability of bacterial cultures and antibiograms limits the use of these tests [9]. According to the World Gastroenterology Organization Global Guidelines, validation and implementation of rapid and inexpensive diagnostic PCR tests and antimicrobial resistance tests promise to be a major advance in the management of H. pylori infection [8].

Conclusions

It is essential to treat all patients infected with H. pylori. This represents a significant change in approach, as the treatment was recommended previously only for patients showing symptoms of infection. Therefore, it is crucial to understand the limitations of traditional diagnostic methods and to promote the updating of health professionals to the most recent advances in the diagnosis of this bacterium. Selecting the most reliable test is crucial for accurate diagnostic results. The choice of methods for detecting H. pylori should be based on regional prevalence (in terms of the country and city), the patient’s clinical status, age, effectiveness of eradication, cost, and the clinical reality of each location so that therapeutic interventions bring benefits to the patient. It is also worth noting that successful diagnosis of H. pylori is particularly important in the context of diseases associated with the presence of this bacterium in the human body.

Acknowledgments

The authors are grateful to Capes for the financial support for doctoral internships abroad, and to CNPq and FAPEMIG for several other types of support.

Statement of Ethics

This review article does not require any ethical authorization because no unpublished data related to experiments carried out on humans or animals are presented.

Conflict of Interest Statement

The authors have no conflicts of interest to declare.

Funding Sources

This study was supported by the Coordenação de Aperfeiçoamento de Pessoal de Nível Superior – Brazil (CAPES) under Grant Finance Code 001 and National Council for Scientific and Technological Development – CNPq under Grant Research Fellowship MGC 311185/2019-3.

Author Contributions

Liana Cristina Melo Carneiro Costa wrote the original manuscript and designed the outline of the topics to be covered in the review. She designed Figure 1 and tables. Liana Cristina Melo Carneiro Costa and Ana Cristina La Guárdia Custódio Pereira were responsible for the acquisition of the articles. Liana Cristina Melo Carneiro Costa analyzed the selected articles. Maria das Graças Carvalho and Melina Barros-Pinheiro revised the manuscript after initial and final writing. Melina Barros-Pinheiro, Rafael Gonçalves Teixeira Neto, and Lívia Carolina Andrade Figueiredo contributed to the finalization of the manuscript. All authors approved the final manuscript.

Funding Statement

This study was supported by the Coordenação de Aperfeiçoamento de Pessoal de Nível Superior – Brazil (CAPES) under Grant Finance Code 001 and National Council for Scientific and Technological Development – CNPq under Grant Research Fellowship MGC 311185/2019-3.