INTRODUCTION
Helicobacter pylori (H. pylori) infection, a prevalent condition affecting over half the global adult population, stands as a significant etiological factor in numerous gastroduodenal diseases. These include chronic active gastritis, peptic ulcer disease, atrophic gastritis, mucosa-associated lymphoid tissue (MALT) lymphoma, and non-cardia gastric cancer. The prevalence of H. pylori infection exhibits considerable geographical variation, influenced by factors such as age, race, and socioeconomic status. While generally increasing with age, a notable decline in H. pylori prevalence has been observed in recent decades across several large populations [1]. Notably, H. pylori is implicated in over 80% of peptic ulcer diseases, with an estimated lifetime risk of approximately 15% for ulcer development in infected individuals [2]. Gastric cancer, the third leading cause of cancer-related deaths globally, is significantly linked to H. pylori infection, accounting for approximately 74.7% of non-cardia gastric cancer cases [3,4]. The combined impact of gastric cancer and peptic ulcer disease results in over a million deaths annually, underscoring H. pylori infection as a critical global health concern [5].
Effective management of these H. pylori-associated diseases hinges on accurate diagnosis. Diagnostic tests with high sensitivity and specificity, ideally exceeding 90%, are essential for reliable detection of H. pylori infection in clinical settings. While a range of diagnostic methods are currently available, each presents unique advantages, disadvantages, and limitations. The selection of a particular diagnostic approach is often dictated by factors such as test availability, laboratory capabilities, patient clinical status, and the pre-test probability of infection. Broadly, diagnostic tests are categorized as invasive (endoscopy-based) and non-invasive methods. Invasive tests encompass endoscopic imaging, histology, rapid urease test (RUT), culture, and molecular techniques. Non-invasive methods include the urea breath test (UBT), stool antigen test (SAT), serological assays, and molecular examinations. This article aims to provide a comprehensive review of current diagnostic options and emerging developments in H. pylori diagnosis, highlighting their applications in diverse clinical scenarios and guiding test selection based on specific clinical conditions (Table 1).
Table 1.
Diagnostic options for Helicobacter pylori infection in different clinical circumstances and special applications of diagnostic tests
| Gastroduodenal bleeding | Post gastrectomy | Post eradication therapy | Special applications |
|---|---|---|---|
| Rapid urease test | √ | ||
| Histology | √ | ||
| Culture | √Antibiotic sensitivity | Antibiotic sensitivity | |
| Polymerase chain reaction | √ | √ | √Antibiotic sensitivity√Virulence factors√Environmental/oral sample |
| Urea breath test | √ | √ | |
| Stool antigen test | √ | ||
| Serology1 | √ | √ | √Virulence factors |
1Although serology is not affected by local change in stomach, result of serology should be interpreted with caution before further management.
INVASIVE TESTS
Endoscopy
Conventional endoscopy plays a crucial role in diagnosing H. pylori-related diseases, including peptic ulcer disease, atrophic gastritis, MALT lymphoma, and gastric cancer. Beyond diagnosis, endoscopy is instrumental in obtaining gastric mucosal biopsy specimens for subsequent invasive diagnostic tests like RUT, histology, culture, and molecular analysis. While the antrum is typically the preferred biopsy site for H. pylori detection, corpus biopsies from the greater curvature are recommended, particularly in patients with antral atrophy or intestinal metaplasia, to mitigate the risk of false-negative results [6,7]. The patchy distribution of H. pylori within the stomach, influenced by clinical context, can lead to sampling errors in biopsy-based examinations. Consequently, efforts are underway to develop real-time diagnostic methods for H. pylori infection during endoscopic procedures.
Standard endoscopic findings such as mucosal redness, swelling, or nodularity lack the specificity required for accurate H. pylori diagnosis and offer limited diagnostic value [8]. While meticulous close-up observation of gastric mucosa patterns during standard endoscopy might improve diagnostic accuracy, it can be time-consuming and may not surpass the efficacy of other invasive tests [9]. Chromoendoscopy utilizing phenol red, leveraging H. pylori‘s urease activity, has also been investigated. However, its reliability is compromised by low sensitivity (73%-81%) and specificity (76%-81%) [10,11].
Magnifying endoscopy, which allows direct visualization of gastric mucosal surface microstructure, reveals high correlations between high-resolution endoscopic patterns and histopathological changes, including H. pylori infection. Indigo carmine staining with magnifying endoscopy demonstrated high sensitivity (97.6%) and specificity (100%) for predicting H. pylori-positive corporal gastritis. However, these values decreased to 88.4% and 75.0% respectively in H. pylori-positive antral gastritis [12]. Confocal laser endomicroscopy (CLE), another advanced endoscopic technique, enables subsurface analysis and in vivo histological assessment of gastric mucosa during endoscopy. Based on CLE findings, features such as white spots, neutrophils, and microabscesses have been used for H. pylori diagnosis, achieving accuracy, sensitivity, and specificity rates of 92.8%, 89.2%, and 95.7%, respectively [13]. Magnifying narrow band imaging and I-scan have also been explored for H. pylori detection, but results have varied [14–16]. Diagnostic accuracy using magnifying endoscopy is influenced by image classification systems and operator expertise, necessitating specialized training and equipment availability [17–20]. Furthermore, detailed examination using magnifying endoscopy, with or without image enhancement, can be time-consuming and potentially more uncomfortable for patients compared to biopsy-based tests. These factors often limit the routine clinical application of magnifying endoscopy for H. pylori detection.
Histology
Histology is widely regarded as the gold standard for direct H. pylori detection and was the first method employed for its identification. However, several factors can impact its diagnostic accuracy, including biopsy site, size and number, staining methods, and the use of proton pump inhibitors (PPIs) or antibiotics, as well as the pathologist’s experience. PPI use can yield inconsistent histological results, and discontinuing PPIs for 2 weeks prior to testing is generally recommended [21]. Obtaining multiple biopsy samples from appropriate locations can minimize sampling errors and false-negative results in histology and other biopsy-based assays. Clinical guidelines typically recommend biopsies from both the antrum and corpus, with at least two specimens from each site considered optimal for maximizing diagnostic yield [22,23]. As previously mentioned, corpus biopsies are particularly crucial for H. pylori diagnosis in the presence of atrophic gastritis [7].
Staining is a critical step in histological examination. Various stains, such as routine hematoxylin and eosin (H&E), Giemsa, Warthin-Starry, H. pylori silver stain, toluidine blue, acridine orange, McMullen, Genta, Dieterle, and immunohistochemical stains, have been used for H. pylori detection. While immunohistochemical staining offers the highest sensitivity and specificity, H&E staining is often sufficient for routine clinical H. pylori diagnosis. Ancillary staining is usually recommended for biopsy specimens displaying moderate to severe chronic gastritis but lacking H. pylori identification on H&E staining. Immunohistochemical staining is the preferred ancillary stain for H. pylori detection [24,25]. If immunohistochemical stains are unavailable, Giemsa stain is a practical alternative due to its simplicity, high sensitivity, and lower cost [26].
Peptide nucleic acid fluorescent in situ hybridization (PNA-FISH), applicable to histological preparations, is a highly sensitive (97%) and specific (100%) technique for H. pylori diagnosis. PNA-FISH can identify coccoid forms of H. pylori, which are often missed by routine histology due to morphological ambiguity, thus mitigating subjective interpretation. Moreover, PNA-FISH is a rapid, accurate, and cost-effective method for detecting clarithromycin resistance in H. pylori from gastric biopsies [27–29]. FISH also holds potential for H. pylori detection in environmental samples, facilitating studies on transmission routes and environmental reservoirs [30,31]. Despite its advantages in simultaneously detecting H. pylori and clarithromycin resistance, PNA-FISH has limitations, including complex preparation, the need for a fluorescence microscope, and specialized expertise for slide interpretation, which may restrict its widespread adoption.
Caption: Histological section showing Helicobacter pylori bacteria stained purple.
RAPID UREASE TESTS
For routine clinical practice, the rapid urease test (RUT) stands out as a highly valuable invasive diagnostic tool for H. pylori infection. Its advantages include low cost, rapid turnaround time, ease of use, high specificity, and widespread availability. RUTs leverage H. pylori‘s urease enzyme activity. The presence of H. pylori in a biopsy specimen converts urea in the test reagent to ammonia, causing a pH increase and a corresponding color change on the pH indicator. Various commercial RUT kits are available, including gel-based (CLOtest, HpFast), paper-based (PyloriTek, ProntoDry), and liquid-based tests (UFT300, EndoscHp), each with varying reaction times. CLOtest typically requires 24 hours for accurate results, while PyloriTek takes 1 hour, and UFT 300 provides results within 5 minutes. Premature reading of urease tests can lead to false-negative results [32]. Beyond kit design, the bacterial load in the biopsy specimen also influences reaction time and diagnostic accuracy; a minimum of approximately 10,000 organisms is usually needed for a positive RUT result. Factors such as H2-receptor antagonists, PPIs, bismuth compounds, antibiotics, achlorhydria, and blood presence can also compromise RUT accuracy, increasing the likelihood of false-negative outcomes. Formalin contamination of biopsy specimens can also reduce RUT sensitivity [21,33–35].
Generally, commercial RUTs exhibit specificity exceeding 95%-100% and sensitivity ranging from 85%-95%. Increasing the number of antral biopsies can improve RUT sensitivity. Dual biopsy specimens from both the gastric corpus and antrum are preferred over antral specimens alone, as corpus biopsies enhance diagnostic accuracy and mitigate sampling bias due to uneven H. pylori distribution in the stomach. Combining antral and corpus specimens before RUT, rather than testing them separately, also increases RUT sensitivity and accelerates reaction time [32,36–39]. To minimize false-negative results, it is advisable to avoid medications affecting urease activity and bacterial density before RUT, such as PPIs for 2 weeks and antibiotics for 4 weeks. Bleeding significantly diminishes RUT sensitivity and specificity, making it less reliable than other tests in this clinical context [40]. A study evaluating biopsy number and site impact on RUT results in patients with peptic ulcer bleeding demonstrated that four antral biopsies or one corpus biopsy increased RUT sensitivity compared to a single antral biopsy. In this study, sensitivity for one antral biopsy was 64%, while sensitivities for four antral biopsies and one corpus biopsy were 74% and 73%, respectively [41]. If RUT is used in patients with gastrointestinal bleeding, biopsies from both antrum and corpus are recommended to improve diagnostic accuracy.
Caption: Rapid urease test showing a positive result, indicated by a distinct color change.
Culture
H. pylori culture from gastric biopsy specimens is a highly specific but less sensitive diagnostic method. While culture specificity approaches 100%, sensitivity varies considerably, ranging from 85% to 95%. Cultivating H. pylori in vitro is challenging due to its fastidious nature, requiring specific transport media, growth media, and incubation conditions. Biopsy specimens can be stored in transport media like Portagerm pylori or Stuart’s transport medium for up to 24 hours at 4 °C. Various agar types can support H. pylori isolation. Commonly used media include Pylori agar, Skirrow agar, Columbia blood agar, Brucella agar, Brain heart infusion, or Trypticase soy agar, supplemented with sheep or horse blood. Agar plates are incubated in a microaerobic environment (80%-90% N2, 5%-10% CO2, 5%-10% O2) at 35 to 37 °C for at least 5-7 days, reflecting H. pylori‘s traditional classification as a microaerophile. However, recent research suggests that atmospheric oxygen levels, in the presence of 10% CO2, may promote H. pylori growth, proposing a novel concept of H. pylori as a capnophilic aerobe [42]. H. pylori diagnosis from culture is based on morphological characteristics and positive urease, catalase, and oxidase reactions, necessitating well-equipped and trained microbiology laboratories.
Factors such as poor specimen quality, transport delays, exposure to aerobic conditions, or inexperienced microbiologists can negatively impact culture performance and reduce diagnostic accuracy [43]. A study across 26 hospitals examining transport time and temperature effects on culture rates showed a decrease in positive culture rate to 26.3% in the 48-hour transport group compared to 32.8% in the 24-hour transport group (P < 0.05) [44]. A recent advancement in transport media is the GESA transport medium, a semi-solid medium capable of preserving gastric biopsy specimens at 4 °C for up to 10 days with a quantifiable H. pylori recovery rate of 90.7% [45]. A novel biphasic test combining selective enrichment broth and a urea agar biochemical test in a single vessel has also been developed for H. pylori culture from gastric biopsies. In a small study of 55 biopsy specimens, this biphasic test demonstrated a 100% positive predictive value after 48 hours of incubation. Furthermore, it exhibited a lower false-positive rate and required a lower bacterial load (approximately 105 cfu/mL) compared to CLOtest. This method can also be used under aerobic conditions and facilitates both culture and antibiotic susceptibility testing [46].
Host factors such as high gastritis activity, low bacterial load, bleeding, alcohol consumption, and the use of H2-receptor antagonists, PPIs, or antibiotics can negatively affect culture positivity rates. These medications, except for antibiotics (which should be avoided for at least 4 weeks), are generally recommended to be avoided for 2 weeks before culture. To minimize sampling bias from patchy H. pylori distribution, at least two biopsy specimens from both the antrum and corpus are recommended [47,48].
While H. pylori culture is time-consuming, expensive, and labor-intensive for diagnosis, its ability to provide antibiotic susceptibility testing is a significant clinical advantage. The Maastricht IV Consensus Report recommends H. pylori culture and antibiotic susceptibility testing when primary clarithromycin resistance exceeds 20% in a region or after second-line treatment failure [21]. Culture also enables H. pylori isolation for further phenotypic and genotypic characterization, enhancing pathogen understanding and therapy evaluation. Given increasing antibiotic resistance, culture remains a reliable method for managing H. pylori treatment failure and monitoring antibiotic resistance in population-based studies, particularly before molecular tests become more widely accessible.
Polymerase Chain Reaction
Since the advent of polymerase chain reaction (PCR) for H. pylori detection, PCR has been widely used for diagnosis from gastric biopsy specimens, saliva, stool, gastric juice, and other samples. PCR boasts excellent sensitivity and specificity, exceeding 95% compared to conventional tests, and offers more accurate H. pylori detection in patients with bleeding. Several target genes, including UreA, glmM, UreC, 16S rRNA, 23S rRNA, HSP60, and VacA, have been used for H. pylori detection. Employing two different conserved target genes can enhance specificity, reducing false-positive results, especially in non-gastric biopsy samples. Other PCR advantages, including requiring fewer bacteria in samples, faster results, and eliminating specialized processing or transportation, enable quicker and more informed treatment decisions. Furthermore, PCR allows for simultaneous detection of antibiotic resistance mutations, such as macrolide and fluoroquinolone resistance, and virulence factors like CagA and VacA [49–51].
Compared to agar dilution methods (Etest), often considered the gold standard for antibiotic susceptibility testing, real-time PCR (RT-PCR) offers several advantages. Firstly, RT-PCR using formalin-fixed paraffin-embedded gastric tissue is more convenient, rapid, and sensitive than Etest using fresh biopsy specimens. RT-PCR has also demonstrated non-inferior results to Etest in antibiotic susceptibility testing. Additionally, PCR is more reliable in detecting heteroresistance, which can lead to false-negative results in Etest, providing clinicians with more accurate pre-treatment information [52]. A recent study using RT-PCR on formalin-fixed paraffin-embedded samples to detect H. pylori infection and clarithromycin resistance investigated the efficacy of genotype-guided quadruple therapy as first-line treatment for 385 patients with functional dyspepsia. In this study, 136 patients (35.3%) were diagnosed with H. pylori infection, with RT-PCR sensitivity of 95.6% and histology sensitivity of 69.9%. Genotype-sensitive patients received quadruple therapy with bismuth potassium citrate, rabeprazole, amoxicillin, and clarithromycin, while genotype-resistant patients received bismuth potassium citrate, rabeprazole, amoxicillin, and furazolidone. Eradication rates were 100% for clarithromycin-susceptible H. pylori and 94% for clarithromycin-resistant H. pylori in per-protocol analysis [53]. Secondly, RT-PCR is a convenient tool for epidemiological studies of regional antibiotic resistance rates, guiding empirical first-line treatment. RT-PCR can also detect point mutations causing antibiotic resistance and track mutation changes or new mutation emergence, providing valuable data for epidemiological and molecular research on genotype-phenotype relationships. Due to the potential for temporal mutation changes, defining more than 5 point mutations is crucial for accurate antibiotic resistance detection using PCR-based methods [54–56].
Genetic mutations conferring resistance to clarithromycin (23S rRNA), quinolones (gyrA gene), tetracycline (16S rRNA), rifabutin (rpoB gene), and amoxicillin (pbp-1a gene) have been identified. Several commercial kits, such as MutaREAL H. pylori kit, ClariRes real-time PCR assay, and Seeplex ClaR-H. pylori ACE detection system, are available for clarithromycin resistance detection [57]. However, the metronidazole resistance mechanism is less clear, with susceptibility genes like rdxA and frxA implicated with debated results. A recent study using Illumina next-generation sequencing identified mutations in the rdxA gene as a major contributor to metronidazole resistance, and frxA mutations enhancing resistance only in the presence of rdxA mutations. A novel finding of mutations in the rpsU gene may explain metronidazole resistance in strains lacking rdxA and frxA mutations [58]. GenoType HelicoDR assay, a molecular test combining PCR and hybridization, enables H. pylori detection and clarithromycin and fluoroquinolone resistance determination within 6 hours. Studies using bacterial strains or gastric biopsy specimens have shown high accuracy for clarithromycin resistance (94%-100% sensitivity, 86%-99% specificity) and fluoroquinolone resistance (83%-87% sensitivity, 95%-98.5% specificity) compared to culture-based methods [59,60]. However, a Korean study evaluating GenoType HelicoDR’s clinical utility reported lower sensitivity and specificity for clarithromycin resistance (55.0% and 80.0%, respectively) and fluoroquinolone resistance (74.4% and 70.0%, respectively), suggesting potential limitations in clinical applicability requiring further evaluation [61]. RT-PCR, conventionally used for H. pylori DNA quantification, can be expensive due to thermocycler costs. A dual-priming oligonucleotide (DPO)-based multiplex PCR was developed for H. pylori detection and clarithromycin resistance detection, compatible with conventional thermocyclers. Using DPO primers to amplify H. pylori 23S rDNA and detect common clarithromycin resistance mutations (A2142G and A2143G), DPO-PCR has proven rapid and accurate for H. pylori diagnosis and clarithromycin susceptibility determination using gastric biopsies [62,63]. Furthermore, a study using RUT-processed tissue samples demonstrated that DPO-PCR had higher sensitivity than RUT and histology, detecting H. pylori in RUT-negative samples, potentially reducing false negatives and the need for repeat endoscopy. DPO-PCR concordance between gastric biopsies and RUT-processed samples was 94.4% [64].
PCR-based virulence factor detection aids in assessing genetic variations and understanding clinical discrepancies among patients infected with different H. pylori strains. Studies have linked virulence factors like CagA and VacA genes to more severe gastric inflammation and higher peptic ulcer disease and gastric cancer prevalence [65–67]. Duodenal ulcer promoter gene A (DupA) was also proposed to be associated with H. pylori-induced ulcer formation, but inconsistent results, potentially due to primer mismatches, have been reported. A newly designed RT-PCR with a specific primer based on 221 DupA gene sequences was recently introduced to improve DupA gene detection, increasing the detection rate to 64.2%, compared to 29.9% to 37.8% with commonly used PCRs. This highlights the significant impact of PCR design on virulence factor detection and the distinction between detecting specific DupA alleles versus the actual DupA gene [68].
PCR is also valuable for detecting H. pylori in environmental samples for epidemiological studies. High H. pylori detection rates in drinking water samples by PCR support waterborne transmission [69]. Higher H. pylori detection in unwashed vegetables suggests thorough vegetable washing reduces contamination [70]. PCR-based H. pylori genotyping in vegetables and high genotype similarity between vegetable and human samples suggest vegetables as potential bacterial sources [71].
While PCR offers rapid, highly accurate H. pylori detection and antibiotic resistance strain identification, cost, equipment availability, and molecular expertise can limit its feasibility in local laboratories.
NONINVASIVE TESTS
Noninvasive diagnostic methods are preferred to avoid the discomfort and risks associated with endoscopy, particularly for patients with comorbidities or contraindications. Endoscopy and related disposables and anesthesia also contribute to higher healthcare costs. Furthermore, biopsy-based methods are susceptible to sampling bias due to the uneven distribution of H. pylori in the stomach.
UREA BREATH TEST
The urea breath test (UBT), used for approximately 30 years, remains a widely used and highly accurate noninvasive method for H. pylori diagnosis. UBT leverages H. pylori‘s urease activity. Patients ingest 13C- or 14C-labeled urea, which H. pylori urease hydrolyzes into labeled CO2 in the stomach. This labeled CO2 is absorbed into the bloodstream, exhaled, and measured in breath samples. Despite factors related to the patient, bacteria, and test procedure influencing UBT results, it is a highly accurate and reproducible test, achieving approximately 95% sensitivity and specificity under standardized conditions. A recent meta-analysis evaluating UBT accuracy in adults with dyspeptic symptoms reported a pooled sensitivity of 96% (95%CI: 0.95-0.97) and pooled specificity of 93% (95%CI: 0.91-0.94) [72]. UBT is also valuable for epidemiological studies and assessing eradication therapy efficacy [21,73]. Patients should discontinue PPIs for 2 weeks and antibiotics for 4 weeks before UBT to prevent false-negative results [74]. Bleeding can also compromise UBT accuracy, necessitating delayed testing after bleeding resolution to minimize false negatives [75]. Rarely, urease-producing pathogens in the stomach can cause false-positive UBT results.
UBT is a suitable, simple, noninvasive, and safe method for H. pylori detection in pediatric patients, although its accuracy may be slightly lower than in adults, particularly in children under 6 years old (75% to 100% sensitivity and specificity) [76].
13C-UBT is preferred over 14C-UBT to avoid radiation exposure, although 14C-UBT is considered safe for children and pregnant women due to low radiation levels, even lower than natural background radiation. However, 14C-UBT remains more common in developing countries due to lower equipment and reagent costs. Diagnostic accuracy is comparable between 13C-UBT and 14C-UBT, and both are considered gold standards among noninvasive H. pylori diagnostic tests [77]. Two 14C-urea administration protocols exist: non-encapsulated and encapsulated. Encapsulated 14C-UBT was initially developed to prevent 14C-urea hydrolysis by oral urease-producing flora, avoiding early false-positive breath samples [78]. However, rapid capsule transit from the stomach or incomplete capsule dissolution during breath collection can limit encapsulated 14C-UBT’s superiority over the non-encapsulated protocol [79]. A study using dynamic scintigraphy to monitor capsule gastric fate compared non-encapsulated and encapsulated protocol sensitivity in 100 dyspeptic patients. Non-encapsulated protocol demonstrated higher sensitivity. Encapsulated 14C-UBT sensitivity was 90.5% at 10 minutes and 91.8% at 15 minutes, while non-encapsulated sensitivity was 98.6% at 10 minutes and 97.2% at 15 minutes. Incomplete capsule dissolution observed by scintigraphy explained the lower encapsulated 14C-UBT sensitivity [80].
The optimal delta over baseline (DOB) cut-off value for differentiating H. pylori-positive and -negative results remains debated. A DOB value of 5.0‰ was initially recommended and widely adopted, while lower values (3.0 or 3.5‰) have been proposed to improve accuracy without compromising sensitivity and specificity. Previous studies have described a “grey zone” of inconclusive UBT results, suggesting cautious interpretation of borderline DOB values near the selected cut-off [81]. A recent study introducing a novel UBT method using an optical cavity-enhanced integrated cavity output spectroscopy system aimed to determine an optimal diagnostic cut-off. This preliminary study defined a diagnostic cut-off of cumulative percentage of 13C dose recovered (c-PDR) = 1.47% at 60 minutes, achieving 100% sensitivity, 100% specificity, and 100% accuracy compared to invasive endoscopic tests. However, the small sample size necessitates larger studies to confirm these findings [82].
Caption: A urea breath test device used for non-invasive Helicobacter pylori diagnosis.
STOOL ANTIGEN TEST
The stool antigen test (SAT) is another noninvasive method with good sensitivity and specificity (94% and 97%, respectively, in a global meta-analysis) for H. pylori diagnosis [83]. SAT detects H. pylori antigens in stool samples. Two main SAT types exist: enzyme immunoassay (EIA) and immunochromatography assay (ICA) based methods, utilizing polyclonal or monoclonal antibodies. Numerous SATs are commercially available, with varying diagnostic accuracy reported across studies due to different SATs and study designs. Generally, monoclonal antibody-based tests are more accurate than polyclonal antibody-based tests [83], and EIA-based tests provide more reliable results than ICA-based tests [84,85]. A recent study evaluating the Tesmate pylori antigen (TPAg) EIA, using a monoclonal antibody to detect native H. pylori catalase, demonstrated 92.4% sensitivity and 100% specificity in adults compared to RT-PCR, with 94.9% accuracy [86]. Premier Platinum HpSA Plus test, another monoclonal EIA-based test, also showed reliable diagnostic results with 92.2% sensitivity, 94.4% specificity, and 93.4% accuracy for H. pylori diagnosis compared to four other SATs (1 monoclonal EIA-based, 2 monoclonal ICA-based, and 1 polyclonal ICA-based), all of which had accuracy below 90% [84]. However, ICA-based tests are easy to perform and do not require specialized equipment, making them suitable for in-office testing and developing countries. A new monoclonal ICA-based SAT, Atlas H. pylori Antigen Test, has been introduced, showing improved results compared to previous monoclonal ICA-based SATs, with 91.7% sensitivity, 100% specificity, and 96.6% accuracy [87].
Similar to UBT, monoclonal EIA-based SAT is a reliable test recommended by guidelines for assessing H. pylori eradication therapy efficacy. Testing should be performed at least 4 weeks post-treatment [21,88]. Meta-analyses have reported pooled sensitivity and specificity of 93% and 96%, respectively, for monoclonal SAT in confirming eradication post-therapy [83]. Recent studies have confirmed monoclonal EIA-based SATs as accurate tools for determining H. pylori eradication outcomes, with 91.6%-100% sensitivity and 93.6%-98.4% specificity [89,90]. Furthermore, monoclonal ICA-based SATs, RAPID Hp StAR and ImmunoCard STAT! HpSA, also show promising results with 90.0%-100% sensitivity and 93.6%-94.9% specificity.
In addition to eradication assessment, monoclonal SAT is a convenient, noninvasive, and useful test for H. pylori diagnosis in pediatric patients [91]. A study applying SAT in children aged 6-30 months showed reliable results for H. pylori diagnosis in very young children [92]. A meta-analysis of 45 studies with 5931 patients evaluating SAT performance in children reported pooled sensitivity and specificity of 92.1% and 94.1%, respectively. Subgroup analysis showed monoclonal SAT sensitivity and specificity of 96.2% and 94.7%, polyclonal SAT of 88.0% and 93.0%, and one-step rapid monoclonal SAT of 88.1% and 94.2%. Monoclonal SAT is considered a reliable H. pylori diagnostic test for children [93]. SAT is also a valuable tool for epidemiological studies and screening programs [94,95]. SAT is more cost-effective and equipment-friendly than UBT for large-scale surveys. Compared to serological tests, often used for screening, SAT appears to provide more reliable H. pylori diagnostic results. However, a previous study found SAT less accurate than serological tests in patients with severe atrophic gastritis, and the implications of this finding for SAT’s role in screening H. pylori-associated diseases, like gastric cancer, require further evaluation [96]. Conversely, another study using a new polyclonal EIA-based SAT (EZ-STEP H. pylori) found that atrophic gastritis and/or intestinal metaplasia did not significantly affect SAT results [97].
SAT accuracy is influenced by factors such as antibiotics, PPIs, N-acetylcysteine, bowel movement frequency, and upper gastrointestinal bleeding. Specimen preservation conditions (temperature and transport time) and cut-off values also impact diagnostic accuracy [98–100].
ANTIBODY-BASED TESTS
Numerous serological tests detecting anti-H. pylori IgG antibodies are widely available for H. pylori diagnosis. EIA is the most common and accurate technique among them. Serological tests are frequently used for epidemiological screening due to their low cost, rapid results, and patient acceptability. Serology is also useful for H. pylori infection evaluation in children. A recent study using E-Plate, a commercial serum antibody kit, comparing serology and SAT performance in 73 children, reported serological test sensitivity, specificity, and positive likelihood ratio of 91.2%, 97.4%, and 35.6%, respectively, using adult cut-off values in children [101]. Serological test accuracy depends on the antigen used in commercial kits and the prevalence of specific H. pylori strains used as the antigen source. Locally validating appropriate antigens, either using local strains or pooling antigens from diverse strains, and establishing reliable cut-off values is essential before population-based investigations [102,103]. Several immunogenic proteins, including CagA, VacA, UreA, Omp, and GroEL, have been used as antigens for infection detection. The H. pylori FliD protein, crucial for flagella assembly, is also recognized as a novel serological marker for H. pylori infection, with 99% sensitivity and 97% specificity [104]. A novel line immunoassay, recomLine H. pylori IgG, utilizing six highly immunogenic virulence factors (CagA, VacA, GroEL, gGT, HcpC, and UreA), has been recently introduced for serological H. pylori diagnosis. RecomLine, unlike EIA and immunoblot, allows identification of specific antibody responses to distinct H. pylori antigens, increasing discriminatory power. Compared to histology, recomLine showed 97.6% sensitivity and 96.2% specificity. RecomLine is also useful for identifying specific H. pylori virulence factors [105,106].
Another serological test advantage is that its accuracy is not affected by ulcer bleeding, gastric atrophy, or PPI or antibiotic use, which can cause false negatives in other tests. However, serology is not reliable for assessing eradication therapy due to persistent antibody levels even after successful eradication [21]. Because serological tests cannot distinguish between active infection and past exposure, confirmation with other tests is needed before eradication therapy.
Similar to SAT, EIA-based serological tests generally have better accuracy than ICA-based tests. A study comparing 29 commercial serological tests (17 EIA-based and 12 ICA-based) showed that 9 of 17 EIA-based tests had accuracy above 90%, while only 1 of 12 ICA-based tests exceeded 90% accuracy. Heterogeneous performance was observed across different serological tests, with EIA-based tests showing sensitivity ranging from 57.8% to 100% and specificity from 58.7% to 96.8%; ICA-based tests showed sensitivity from 55.6% to 97.8% and specificity from 60.3% to 96.8%. Serological tests should be carefully selected based on specific performance parameters for intended purposes, such as screening, initial diagnosis, or test confirmation [107].
Serological tests also play a role in pathogenesis and virulence factor studies, as immunological techniques can detect various antigenic proteins, providing additional diagnostic value. Attempts have been made to identify biomarkers for high-risk H. pylori strain infections using serology. Pepsinogen (PG) I, PG II levels, and PG I/II ratio, combined with H. pylori antibodies, have been widely used to predict atrophic gastritis and gastric cancer risk [108,109]. PG I/II ratio may also be useful for gastric cancer surveillance post-eradication therapy [110]. However, clinical applications of these serological markers have yielded conflicting results. A recent study evaluating GastroPanel accuracy, which measures gastrin-17, H. pylori antibody, PG I, and PG II, for atrophic gastritis detection reported only 50% sensitivity and 80% specificity, lower than previous studies [111]. Pepsinogen tests were also not sufficiently accurate for gastric cancer diagnosis, with 71.0% sensitivity and 69.2% specificity [112]. Some virulence factors have been evaluated for predicting H. pylori-associated disease prognosis. Serum CagA, VacA, and GroEL antibodies in H. pylori-infected patients are associated with gastric precancerous lesions and gastric cancer, potentially serving as predictors for high-risk strain infections related to gastric cancer development [106,113]. While epidemiological studies have linked virulence factors and clinical presentations, serological tests remain insufficiently reliable for gastric cancer diagnosis. A meta-analysis of CagA antibody for gastric cancer diagnosis reported pooled sensitivity and specificity of 71% and 40%, respectively, and a diagnostic odds ratio of 2.11 [114].
Urine H. pylori IgG detection has been evaluated in children, but results have varied [115,116]. Similarly, EIA-based salivary H. pylori IgG detection has not demonstrated sufficient accuracy for reliable testing [117,118]. Antibody detection in urine or saliva is less accurate than other tests and is not recommended for patient management [119].
DIAGNOSIS OF H. PYLORI IN OTHER SPECIMENS
PCR-based H. pylori detection in stool is a reliable and rapid noninvasive technique, particularly attractive for children. Stool PCR also enables genotype and antibiotic resistance identification [120,121]. The oral cavity has been suggested as an extra-gastric H. pylori reservoir, although its role in reinfection or transmission remains unclear. Saliva and dental plaque are common specimens for oral H. pylori detection, with PCR being the most reliable test in recent studies. RUT and culture were also used in earlier oral H. pylori detection studies. Oral H. pylori prevalence varies widely (0%-100%), with lower prevalence typically found in saliva compared to dental plaque [122]. Prevalence variation may stem from methodological differences, population variations, and primer selection in studies. Recent research focuses on primer modification to improve diagnostic accuracy or novel methods to overcome PCR limitations. A novel PCR system using H. pylori-specific primers based on highly conserved sequences from 48 H. pylori genomes was recently developed to enhance PCR diagnostic accuracy in oral samples [123]. Loop-mediated isothermal amplification (LAMP), a highly specific and sensitive DNA amplification method, was compared to PCR for H. pylori detection in dental plaque in a small study of 45 participants. LAMP showed a higher detection rate than PCR (66.67% vs 44%) [124].
DIAGNOSIS OF H. PYLORI IN SPECIFIC CLINICAL CIRCUMSTANCES
Upper gastrointestinal bleeding (UGIB) reduces the diagnostic accuracy of many H. pylori detection tests, both invasive and noninvasive. Meta-analyses have shown low sensitivity and high specificity for RUT, histology, and culture in UGIB patients. UBT remains relatively reliable, while SAT accuracy decreases in this setting. Serology is unaffected by UGIB but is not recommended as a primary diagnostic test [40]. Histology is less affected by bleeding than CLO test and culture, potentially remaining reliable even with blood presence [125]. PCR demonstrates significantly higher sensitivity than RUT, histology, and culture (91%, 66%, 43%, and 37%, respectively) and similar sensitivity to serology and UBT (94% and 94%, respectively). PCR and UBT show comparable diagnostic accuracy for H. pylori detection in bleeding peptic ulcers. However, PCR specificity (100%) is superior only to serology (65%) and similar to other tests (RUT: 95%, histology: 95%, culture: 100%, UBT: 85%) [126]. A study also demonstrated RT-PCR’s ability to detect H. pylori infection in formalin-fixed paraffin-embedded biopsy specimens that were histology-negative in peptic ulcer bleeding patients [127]. H. pylori eradication is crucial in managing H. pylori-associated ulcer bleeding to prevent rebleeding, and successful eradication is more effective than long-term PPI maintenance therapy in reducing rebleeding risk. Biopsy-based H. pylori testing is generally recommended during UGIB endoscopy, despite bleeding reducing biopsy-based test sensitivity. Meta-regression analysis suggests delayed testing (4 weeks post-UGIB episode) improves H. pylori detection rates in UGIB patients. Accurate bleeding ulcer etiology determination is crucial for management, and guidelines recommend confirming negative results with subsequent noninvasive tests [22,128,129]. Low negative predictive value has also been observed for UBT performed immediately after emergent endoscopy, necessitating delayed testing for all early UBT negatives [75]. Despite H. pylori testing importance in UGIB patients, direct H. pylori testing rates remain low (12%-60% in previous studies). Concerns about reduced sensitivity due to bleeding or PPI use, increased biopsy-related adverse event risk, and longer procedure times may influence clinician decisions regarding H. pylori testing [130].
H. pylori diagnosis in partial gastrectomy patients is another area requiring consideration, though less studied due to this population’s smaller size. Meta-analysis comparing histology, RUT, and UBT in partial gastrectomy patients showed histology performing best, followed by RUT, with UBT exhibiting poorer diagnostic accuracy. Studies showed high heterogeneity, with pooled sensitivity and specificity for histology, RUT, and UBT of 93% and 85%; 79% and 94%; 77% and 89%, respectively. RUT is suggested as the initial test of choice in these patients, with biopsies from the gastric fundus or upper remnant stomach body recommended. Histology is recommended for RUT-negative cases [131]. SAT may be another reliable test in distal gastrectomy patients. A small study using HpSA test in 59 distal gastrectomy patients for gastric cancer demonstrated SAT sensitivity, specificity, and accuracy of 100%, 90.5%, and 96.6%, respectively [132]. UBT’s potentially inadequate performance in distal gastrectomy patients may be due to insufficient urea retention time in the gastric stump to interact with H. pylori urease. BreathID, a rapid continuous-real-time UBT, appeared to overcome this limitation, showing better accuracy than RUT (87% vs 72%). However, lower RUT sensitivity and specificity (82% and 71%, respectively) compared to previous studies were also observed, possibly due to biopsy location (gastric body slightly distal to the fundus) influencing RUT performance [133]. A recent study also found discordant results between UBT and biopsy-based tests in partial gastrectomy patients post-H. pylori eradication therapy. Authors suggested additional endoscopic biopsy-based tests to avoid unnecessary treatment due to high false-positive rates and low positive predictive value of UBT (19.1% and 44.7%, respectively) in these patients post-eradication [134].
Accurate H. pylori status determination post-eradication therapy is critical, and UBT and SAT are guideline-recommended for assessing eradication efficacy, typically performed at least 4 weeks post-therapy [21,88]. However, high false-positive rates (52.9%) have been observed with 13C-UBT using current cut-off values (2.5‰), especially in patients with more than two prior eradication attempts and moderate to severe gastric intestinal metaplasia [135]. A recent study using nested PCR to detect H. pylori from gastric biopsies post-eradication showed nested PCR to be more sensitive than RUT, histology, and culture. PCR-based methods can also differentiate between reinfection and recrudescence post-eradication [136].
CONCLUSION
Advances in diagnostic methods have significantly improved the accuracy of H. pylori infection diagnosis, leading to better management of H. pylori-associated diseases. While a single gold standard test may not exist, test selection depends on H. pylori prevalence and strains in endemic areas, test accessibility, method advantages and disadvantages, and individual patient clinical circumstances. Combining two or more tests can be a reasonable strategy in routine clinical practice to achieve the most reliable results. Ongoing efforts are expected to further enhance H. pylori diagnostic yield for diverse clinical purposes, specific populations, and genotypic characterizations, leading to more reliable and feasible diagnostic modalities in the future.
Footnotes
Supported by (in part) Grants from the Kaohsiung Medical University “Aim for the Top Universities Grant”, grant No. KMU-TP104G00, No. KMU-TP104G03 and No. KMU-TP104E25; and Ten Chan General Hospital, Chung-Li and KMU Joint Research Project, No. ST102004; and Kaohsiung Medical University Hospital, No. KMUH100-0R01.
Conflict-of-interest statement: The authors have no conflict of interest to disclose.
Open-Access: This article is an open-access article which was selected by an in-house editor and fully peer-reviewed by external reviewers. It is distributed in accordance with the Creative Commons Attribution Non Commercial (CC BY-NC 4.0) license, which permits others to distribute, remix, adapt, build upon this work non-commercially, and license their derivative works on different terms, provided the original work is properly cited and the use is non-commercial. See: http://creativecommons.org/licenses/by-nc/4.0/
Peer-review started: May 12, 2015
First decision: July 13, 2015
Article in press: September 30, 2015
P- Reviewer: Tosetti C S- Editor: Ma YJ L- Editor: A E- Editor: Zhang DN
