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Helicobacter pylori Diagnosis
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Helicobacter pylori infection still remains one of the most prevalent infections worldwide, especially in low-resource countries, and the major risk factor for peptic ulcer and gastric cancer. The "test-and-treat" strategy is recommended by several guidelines and consensus. The choice of testing method is based on patient age, presence of alarm signs and/or symptoms, use of non-steroidal anti-inflammatory drugs, as well as local availability, test reliability and cost. 

Culture is the gold standard to detect H. pylori and, possibly, to perform susceptibility testing, however, it requires upper endoscopy and dedicate labs. Recent advances in molecular biology provide new strategies in detecting the infection and antimicrobial resistance without invasive tests. 

Helicobacter pylori testing antibiotic resistance molecular techniques artificial intelligence
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Table of Contents

    1. Helicobacter pylori infection

    The infection can be essentially detected by invasive and non-invasive tests. The choice of technique relies upon the patient needs. Presence of alarm symptoms, use of non-steroidal anti-inflammatory drugs (NSAIDs), advanced age (>45-50, years or >60 years) [1][2][3][4] history of premalignant conditions or follow up for a previous malignant disease dictates an upper endoscopy evaluation. The indication for esophago-gastric-duodenoscopy allows to directly observe the mucosa, to collect biopsy samples for histology examination, urease test, bacterial culture and molecular assay. In the absence of endoscopy recommendation, non-invasive tests such as urea breath testing or stool antigen assay are appropriate to confirm an active infection. Serology may be used in specific setting to assist the physician in the diagnosis of bacterial infection [5]. However, the diagnostic strategy cannot prescind from the local availability, costs of the test, labs reliability and patient preferences. As a rule of thumb, it is important for the physician to confirm the diagnosis, to evaluate the presence of gastric lesions induced by the infection according to the patient clinical history, to offer H. pylori eradication therapy and to check treatment success.

    2. Invasive Tests

    2.1. Endoscopy

    For patients in whom an upper endoscopy is required, where available, advanced endoscopy techniques should be preferred to conventional endoscopy, especially for patients/subjects with a high pretest probability to harbor premalignant lesions, such as those from countries or subpopulations with high prevalence of gastric cancer, or individuals with strong familiarity for gastric malignancy or those patients who need a strict endoscopy surveillance for previous diagnosed premalignant lesions.

    By standard white light endoscopy (WLE) the infection may be identified on the base of specific gastric mucosa features such as the presence of antral nodularity with a sensitivity and a specificity ranging from 39.8% to 96.4% and from 83.6% to 100%, respectively [6]. Additional reports identified the erythema, erosions, thickened folds or absence of rugae, mosaic appearance with or without hyperemia and visible submucosal vessels in the gastric mucosa as the hallmarks of H. pylori infection [7][8][9][10] or gastric black spots associated with H. pylori eradication [11], and mucosal swelling (77%) associated with mild atrophy [12]. However, the low interobserver agreement may be a limitation to translate gastric mucosal features into a diagnosis of specific gastritis with or without H. pylori infection.

    The results obtained with the narrow band imaging (NBI), which uses blue light from a laser source (415 nm) to highlight the vascular architecture of the gastric mucosa are more promising. Based on distinct patterns of the gastric mucosa the endoscopist may predict H. pylori infection by conventional NBI [13] and by the magnifying NBI technique the presence of intestinal metaplasia with a sensitivity and specificity greater than 95% [14]. Moreover, a high degree of concordance was observed between magnifying NBI and the operative link for gastritis and for gastric intestinal metaplasia assessment [15][16]. Interestingly, by this technique, specific morphological patterns, including reddish depressed lesions, are frequently observed in association with H. pylori eradication [14][17]. The magnifying endoscopy with NBI proved to be superior to WLE and chromoendoscopy in the diagnosis of early gastric cancer after H. pylori eradication [18]. The confocal laser endomicroscopy is more accurate than NBI for grading gastric premalignant lesions [19]. The blue laser imaging (BLI) and the linked color imaging (LCI) are also highly accurate in detecting H. pylori infection and premalignant lesions related to the infection [20][21][22][23][24][25]. Endocytoscopy (EC), an ultra-high magnification endoscopy, is able to provide histologic assessment in vivo [26]. Overall, high-definition endoscopy allows in real time the diagnosis of H. pylori infection, detection of premalignant and malignant gastric lesions and targeted mucosa biopsy sampling.

    All recent developments of high-definition endoscopy for the diagnosis of H. pylori infection and detection of pre-malignant and malignant gastric lesions, allowing a real-time decision-making, prompted the revision of Kyoto endoscopic classification [27]

    In the last years there was also an attempt to use more sophisticated tools to diagnose H. pylori by using artificial intelligence approach mimicking the brain neural network [28]. In a recent meta-analysis, the artificial intelligence algorithm demonstrated to be an accurate tool for the prediction of H. pylori infection during endoscopic procedures, although the real application needs to be evaluated in clinical studies [29].

    2.2. Histology

    The examination of gastric mucosal biopsy specimens remains the gold standard for detection of H. pylori, with a sensitivity of 95% and a specificity of 98%. In addition, it enables the visualization of gastric morphology at any time. In order to obtain an accurate diagnosis, two antral biopsies including one from the gastric angulus, and two biopsies from the corpus are necessary [30]. The widespread use of proton pump inhibitors (PPIs) may result in atypical presentation of gastritis or in density variation of bacteria at different sites [31], the accuracy of histologic diagnosis of H. pylori infection can be improved by using special staining techniques, specific immune stain or digital pathology [32][33]. Gastric biopsy specimens obtained by high-definition or conventional endoscopy can be used for molecular testing to assess the presence of H. pylori and its antibiotic susceptibility profile also in patients under PPI treatment. This is particularly useful for those patients who cannot stop the PPI treatment (for instance because of double antiplatelet treatments, or with a Zollinger Ellison syndrome or similar circumstances).

    2.3. Rapid Urease Test

    Upper endoscopy enables to collect also biopsy specimens for urease testing. The method is based on the presence of pre-formed urease by the bacteria and, in media containing urea, the enzyme releases ammonia increasing the pH and resulting in a color change of the medium.

    The urease test is rapid (RUT), easy to perform, highly specific and inexpensive for H. pylori diagnosis, although it requires at least a 104 bacterial load in the gastric specimens [34]. False-negative results may occur with recent use of antibiotics, bismuth-containing compounds, PPIs, especially omeprazole and lansoprazole, and in children younger than five years [35]. To collect biopsies from the corpus rather than from the antrum, or combining antral and corpus biopsies has demonstrate to enhance RUT sensitivity [36][37]. In addition to false negative, also false-positive RUT may occur in presence of urease positive bacteria [34]. The gastric samples used for RUT can be re-used for molecular testing in order to identify bacterial resistance. However, compared with histology, RUT does not allow to plan a correct follow up for the patient.

    2.4. Culture

    In addition to histological examination and rapid urease testing, upper endoscopy offers the opportunity to collect gastric specimens for bacterial culture, susceptibility testing and even organism genotyping. Although culture is highly specific, it has a low sensitivity as H. pylori is difficult to grow and experienced laboratories are required. Sensitivity may be improved by sending the specimen to the laboratory within 30 minutes from collection, using a warm and non-selective culture medium, a longer incubation and the addition of hydrogen in the atmosphere, or by treating specimens with trypsin [38][39][40][41].

    3. Non-invasive tests

    Non-invasive tests can be divided into those able to detect an active infection, such as the urea breath test and stool antigen test, and those able to provide information on current or prior H. pylori infection without discrimination.

    3.1. Urea breath test

    The 13C-urea breath test (UBT) is the non-invasive method of choice to determine H. pylori status when available. Similarly to RUT, the test takes advantage from the urease produced by the bacteria, which hydrolyzes urea generating CO2 and ammonia. The urea substrate is enriched with a labeled carbon isotope that may be non-radioactive (13C) or radioactive (14C) and ingested, usually, with a test meal to prolong the permanence of urea in the stomach. Breath exhaled samples are collected in proper tubes before and after urea ingestion. Even though the dose of radiation is small in the 14C-UBT, the non-radioactive 13C test is routinely preferred. The test is also used to ascertain the eradication and it is recommended for the “test-and-treat” strategy in dyspeptic patients [1]. The test could also be successfully applied to patients with partial gastrectomy, especially when performed with the patient in the right position [42]. The 13C-UBT shows high sensitivity (95%) and specificity (95% to 100%) [43].

    The 13C-urea is available in the market in different formulations such as a powder, capsules and tablets ranging between 50 and 100 mg. The test meal containing citric acid or malic acid enhance 13C-UBT performance increasing urease activity in the presence of bacteria [44]. However, quantitative results may be influenced by sex, age, body mass index, especially obesity, smoking, gastric atrophy and intestinal metaplasia and even by the socioeconomic status [45][46][47]. The most used cutoffs, expressed as delta over baseline (DOB), are 2‰, 2.4‰, 2.5‰ and 5‰ [48]. To analyze labeled 13CO2 several detector devices are available in the market [49][50].

    3.2. Stool antigen test

    To culture H. pylori from feces is very difficult and time consuming [51], on the contrary non-invasive tests able to detect H. pylori antigen in stool specimens are simple to perform and large head-to-head comparisons with other tests demonstrated high diagnostic accuracy of this approach [52]. Nowadays several assays are available, the more recent ones are listed in table 1.

    Table 1. Most recent stool antigen tests and their reported sensitivity and specificity.


    Based on





    LIAISON H. pylori SA assay (DiaSorin, Saluggia, Italy)

    chemiluminescent immunoassay






    Ramirez-Lazaro et al., 2016 [53]

    Genx H. pylori card test (Genx Bioresearch, Kocaeli, Turkey)

    monoclonal immunochromatographic assay



    Korkmaz et al., 2015 [54]

    Uni-Gold™ H. pylori Antigen (Trinity Biotech, Bray, Ireland)

    monoclonal lateral flow immunochromatographic assays



    Lario et al., 2016


    RAPID Hp StAR (Oxoid Ltd., UK)

    monoclonal lateral flow immunochromatographic assays


    77.1 to 84.7

    Lario et al., 2016


    ImmunoCard STAT! HpSA (Meridian Diagnostics, USA)

    monoclonal lateral flow immunochromatographic assays



    Lario et al., 2016


    IDEIA HpStAR®; ThermoFisher Sc., Waltham, USA

    monoclonal antibodies and the ELISA technique

    Before Hp treatment 93.6

    After Hp treatment 100

    Before Hp treatment 100

    After Hp treatment 92.8

    Moubri et al., 2018 [56]

    Quick Chaser H. pylori®, QCP, Misuho Medy, Tosu, Japan)




    Kakiuchi et al., 2019 [57]

    Vstrip®HpSA, (Meridian),




    Fang et al., 2020 [58]

    ImmunoCard STAT!® Campy (Meridian)




    Fang et al., 2020 [58]

    Overall, stool monoclonal antibody tests are superior to polyclonal antibody tests and demonstrated a pooled sensitivity and specificity of 93% and 96%, respectively [59][60]. They also show an excellent diagnostic accuracy in pediatric setting, especially when tests are ELISA based rather than immunochromatography based [61]. The use of stool antigen test (or UBT) for the initial diagnosis of H. pylori infection and post-treatment (when endoscopy is not required), is recommended by the majority of guidelines and consensus [1][2][3][4].

    The advantage of the UBT and of stool antigen test is that they assess the overall content of the stomach whereas hystology and RUT test only the tiny biopsy specimen. Theoretically and practically, the UBT and stool antigen test are the best methods for detection of active H. pylori infection. However, any drug that will diminish H. pylori load below the detection threshold of can cause false negative tests, particularly recent use of PPIs, bismuth-containing compounds or antibiotics. 

    3.3. Molecular Testing

    Molecular techniques should be preferred when available. The traditional or modified real-time (RT) PCR allows to detect the bacteria, and to screen for antibiotic sensitivity [62][63][64]. Moreover, real-time PCR is more accurate compared with other techniques for the detection of H. pylori in patients exposed to PPI [65], and is able to detect as low as 10 copies in adult [66] and children [67]. In addition to gastric biopsies, molecular testing can be applied to the gastric mucus present on biopsy forceps placed into water or into the RUT gel [68]. Alternatively, molecular tests to detect H. pylori and its susceptibility to antibiotics can be performed on gastric juice [69][70][71]. A droplet-digital PCR may also be applied to formalin-fixed, paraffin-embedded gastric tissue to determine the presence of clarithromycin resistance [72] or by next generation sequencing to determine levofloxacin and tetracycline resistance [73] (Figure 1).

    Figure 1. Invasive, non-invasive methods and molecular testing to detect H. pylori and its antibiotic resistance.

    Several molecular tests have been developed in the last years to detect specific H. pylori antigens and/or resistance pattern in the stool (Table 2).

    Table 2. Recent molecular assays available to detected H. pylori and its antibiotic resistance.

    Molecular test

    H. pylori DNA target


    multiple genetic analysis system (MGAS)

    16S rDNA and ureC

    Zhou et al., 2015 [74]

    allele-specific PCR

    N87I mutation in the gyrA

    Trespalacios et al., 2015 [75]

    droplet-digital PCR (ddPCR)

    cagA and its EPIYA phosphorylation motifs

    Talarico et al., 2016 [64]

    loop-mediated isothermal amplification (LAMP)

    ureC gene

    Yari et al., 2016 [76]

    TaqMan RT-PCR

    A2142C, A2142G and A2143G mutations

    Beckman et al., 2017 [77]

    droplet-digital PCR (ddPCR)


    16S rDNA

    Talarico et al., 2018 [72]

    real-time PCR (THD fecal test®)

    23S ribosomal RNA

    Iannone et al., 2018 [78]

    MagNA Pure 96 (Roche)


    Clines et al., 2019 [79]

    Amplidiag® H. pylori + ClariR

    H. pylori and CLA resistance mutations

    Pichon et al., 2020 [80]

    3.4. Serology

    Unlike UBT and stool antigen testing, serology does not distinguish between an active or past infection, although in a recent study antibody response to H. pylori proteins such as VacA, GroEl, HcpC, CagA, Tip-α, HP1564 and HP0175 indicates an active H. pylori infection with a high diagnostic accuracy [81][82].

    Detection of serum IgG against H. pylori is usually based on the enzyme-linked immunosorbent assays (ELISA). The latex immunoassay may be employed with the advantage to save time [83]. Several kits are available in the market and, overall, they are highly sensitive and specific; however, to maintain a high diagnostic accuracy, serologic tests need to be validated locally [84], especially when the kit uses strains’ antigens from different geographic areas [85]. Because IgG titers decline slowly (over around six months) the test is not recommended to evaluate bacterial eradication after treatment. Serology testing does not have the ability to distinguish active infections from past infections. In addition, the positive predictive value of antibody testing is affected by the local prevalence of H. pylori, especially in those areas where the H. pylori prevalence is inferior to 20%.

    3.5. Tests on plasma, blood, saliva and urine

    The GastroPanel, especially the new-generation test, assesses simultaneously H. pylori antibodies and pepsinogen (PG) I plus PG II and gastrin-17 in the plasma, predicting H. pylori infection and the presence of atrophic gastritis with a likelihood of 94-95% [86]. The test is the most comprehensive non-invasive diagnostic test as it avoids false negative results respect to conventional tests [86][87][88][89][90]. A decreased PG1/2 ratio is associated with chronic atrophic gastritis and intestinal metaplasia (p < 0.001) and, inversely, an increased ratio correlates with gastritis [87]. The GastroPanel is useful under specific conditions. For example, in very old or fragile or with severe comorbidities patients, or healthy subjects from regions at low gastric cancer prevalence, but with gastric cancer familiarity who refuse to undergo upper endoscopy, evaluation with the GastroPanel may offer a comprehensive overview of the H. pylori and gastric mucosa status. 

    A plasma sample also offers the opportunity to detect circulating microRNAs (miRNAs) by molecular techniques. For example, the expression of four miR-28-3p, miR-143-3p, miR-151a-3p, and miR-148a-3p were found to be associated with H. pylori infection [91].

    IgG antibodies against H. pylori may also be detected in dried blood spots, saliva and urine by ELISA with a reported good accuracy [92][93][94].


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