Detection of Clarithromycin Resistance in Helicobacter pylori Using the AllplexTM H. pylori & ClariR Assay and the Ezplex® HP-CLA Real-Time PCR Kit
Article information
Abstract
Objectives
The success of Helicobacter pylori eradication using clarithromycin-based triple therapy relies on the bacteria being sensitive to clarithromycin. This study evaluated the diagnostic performance of two frequently used polymerase chain reaction (PCR) methods (AllplexTM H. pylori & ClariR Assay [Seegene] and Ezplex® HP-CLA Real-time PCR [SML Genetree]) to detect H. pylori infection and identify point mutations associated with clarithromycin resistance.
Methods
Patients who underwent esophagogastroduodenoscopy between August 2023 and April 2024 at Incheon St. Mary’s Hospital were enrolled in this study. The diagnostic performance of the Allplex method was evaluated against the rapid urease test (RUT), culture, and Ezplex HP-CLA methods. Point mutation detection using the Allplex and Ezplex methods was compared with the results of gene sequencing. The rates of H. pylori eradication following Ezplex-based tailored therapy were also analyzed.
Results
Eighty-seven gastric biopsy specimens were analyzed. For diagnosing H. pylori infections, Allplex demonstrated kappa values of 0.670 compared with RUT, 0.468 compared with culture, and 0.880 compared with Ezplex. Among the 87 bacterial isolates subjected to gene sequencing to detect clarithromycin resistance-associated point mutations, the Allplex and Ezplex methods demonstrated 74 and 76 concordant results, respectively. The H. pylori eradication rate using Ezplex-based tailored therapy was 90.7%.
Conclusions
This study demonstrated that both the Allplex and Ezplex methods are helpful for diagnosing H. pylori infections and detecting clarithromycin resistance. Furthermore, the Ezplex method was clinically effective for guiding tailored therapy to yield successful H. pylori eradication.
INTRODUCTION
Helicobacter pylori is the primary cause of peptic ulcers and gastric cancer; thus, international guidelines recommend eradicating this bacteria [1-3]. Clarithromycin-based triple therapy is the traditional regimen used for H. pylori eradication [2,3]; however, recent increases in clarithromycin resistance have led to a decline in the effectiveness of triple therapy [4]. Consequently, recent guidelines recommend avoiding clarithromycin-based triple therapy in populations with high clarithromycin resistance [3,4].
The primary cause of clarithromycin resistance in H. pylori is a point mutation in the 23S ribosomal RNA (rRNA) gene that ultimately prevents clarithromycin from binding to its target. The most frequently reported point mutation is the adenine-to-guanine transition in 23S rRNA at positions 2142 and 2143, accounting for 81.5% of clarithromycin resistance in H. pylori isolates [5].
Polymerase chain reaction (PCR) is commonly used to detect clarithromycin resistance. Several PCR methods are currently employed to diagnose H. pylori infections and identify point mutations related to clarithromycin resistance. AllplexTM (Seegene) is a quantitative multiplex PCR method that detects the A2142G, A2143G, and A2142C mutations. Ezplex® (SML Genetree) is a real-time PCR method designed to detect H. pylori and the A2142G and A2143G mutations associated with clarithromycin resistance. To date, limited research has evaluated the diagnostic performance of PCR methods for detecting H. pylori and clarithromycin resistance mutations. One study involving 142 gastric biopsies found that AllplexTM exhibited a sensitivity of 97.6%, a specificity of 96%, a positive predictive value of 91.1%, and a negative predictive value of 99% for detecting A2143G mutations compared with gene sequencing [6]. However, few studies have directly compared the performance of Allplex with other PCR methods. Therefore, this study assessed the diagnostic performance of Allplex for H. pylori detection and compared it with that of Ezplex for detecting clarithromycin resistance in gastric biopsy samples.
METHODS
Study design and patients
This study prospectively recruited 87 patients who underwent upper gastrointestinal endoscopy at Incheon St. Mary’s Hospital between August 2023 and April 2024 to diagnose H. pylori infections. All participants consented to participation in the study. Biopsy samples were taken, and Allplex, Ezplex, rapid urease test (RUT), and culture methods were performed to detect H. pylori infection. Allplex, Ezplex, and gene sequencing were also used to identify the presence of point mutations associated with clarithromycin resistance. This study was approved by the Institutional Review Board of Incheon St. Mary’s Hospital (OC22DIST0100).
Endoscopic procedure
Endoscopy was performed using standard procedures, and sedation was provided upon the patient’s request. Two biopsy samples, one each from the antrum and corpus of the stomach, were subjected to PCR testing and RUT. One biopsy sample from each location was also placed in an Eppendorf tube and stored on dry ice or at -80°C for culture and antibiotic susceptibility testing.
H. pylori DNA extraction and PCR studies
DNA was extracted using the QIAamp DNA Mini Kit (Qiagen) according to the manufacturer’s protocol [7]. First, gastric biopsy tissue was collected and transferred into a microcentrifuge tube containing 180 μL of buffer and 20 μL of proteinase K. The tube was vortexed and then incubated at 56°C until the tissue was completely lysed. Next, 200 μL of another buffer was added, and the mixture was incubated at 70°C for 10 minutes. Then, 240 μL of 100% ethanol was added and vortexed for 15 seconds. Each tube was placed in a QIAamp spin column and centrifuged at 8000 rpm for one minute. The columns were then rinsed with 500 μL of AW1 buffer and centrifuged at the same speed for another minute. Afterward, 500 μL of AW2 buffer was applied to each column, and the samples were centrifuged at 14000 rpm for 3 minutes. Thereafter, 200 μL of AE buffer was transferred to each sample and incubated at room temperature for one minute before being centrifuged at 8000 rpm to elute the DNA. The eluted DNA was used for molecular analysis. To assess the conservation of nucleotides in the 23S rRNA regions at positions 2142 and 2143, a 546 bp segment was amplified using PCR primers (5'–GTCCCTCCCGA CTGTTTACC–3' and 5'–AACCGCAATGAGCCAACC–3') [6]. H. pylori and its mutations were identified using the Allplex and Ezplex methods, in accordance with the manufacturer’s instructions.
Agar dilution method
The techniques for culturing H. pylori and evaluating its antibiotic resistance have been described previously [8]. The agar dilution method for assessing the resistance of H. pylori isolates was performed following the guidelines established by the Clinical and Laboratory Standards Institute (CLSI). The results were analyzed based on the CLSI breakpoints for clarithromycin (susceptible: ≤0.25 μg/mL, intermediate: 0.5 μg/mL, resistant: >0.5 μg/mL) [8].
Tailored therapy
Patients without genotypic resistance, based on Ezplex results, received clarithromycin-based triple therapy for 7 days. Patients with genotypic resistance received a bismuth-based quadruple therapy. The clarithromycin-based triple therapy consisted of twice-daily doses of esomeprazole (40 mg), amoxicillin (1 g), and clarithromycin (500 mg). Bismuth quadruple therapy included twice-daily doses of esomeprazole (40 mg), thrice-daily doses of metronidazole (500 mg), and four-times daily doses of bismuth (300 mg) and tetracycline (500 mg). A 13C-urea breath test (UBT) was used to confirm eradication at least 4 weeks after therapy. Patients discontinued histamine 2 blockers or proton pump inhibitors for at least 2 weeks before the UBT.
Outcomes
The primary outcome was the diagnostic performance of Allplex compared with RUT, culture, and Ezplex for detecting H. pylori. The secondary outcome was the diagnostic performance of Allplex and Ezplex for identifying point mutations related to clarithromycin resistance, with gene sequencing serving as the gold standard.
Additionally, the correlation between genotypic and phenotypic clarithromycin resistance was evaluated. Genotypic resistance refers to the presence of point mutations expected to confer resistance, whereas phenotypic resistance refers to the presence or absence of culture-based clarithromycin resistance. A point mutation (genotypic resistance) does not necessarily indicate resistance in culture. Furthermore, the H. pylori eradication rates of the tailored therapies, based on Ezplex results, were also assessed.
Statistical analysis
Categorical variables are presented as numbers (percentages). The agreement of the Allplex results with those obtained using RUT, culture, and Ezplex were assessed using the kappa coefficient. All statistical analyses were performed using SPSS software (version 24.0; IBM Corp.).
RESULTS
Patient characteristics
The mean age of the participants was 61.2 years. Nearly half of the patients were males (n=49, 50.5%). Endoscopic submucosal dissection was the most common cause of a gastric neoplasm, accounting for 86.2% of the cases (Table 1).
Baseline characteristics of the study population
Diagnostic performance (H. pylori)
Among the 87 subjects, RUT and culture detected H. pylori infection in 48 and 29 patients, respectively (Table 2). Allplex and Ezplex identified infections in 54 and 49 patients, respectively (Table 2). For H. pylori detection, Allplex had a kappa value of 0.670 compared with RUT, 0.468 compared with culture, and 0.880 compared with Ezplex (Table 2). Overall, both PCR methods demonstrated high accuracy and agreement with RUT and culture methods for diagnosing H. pylori infection.
Performance of AllplexTM in H. pylori detection compared with RUT, culture, and Ezplexa®
Diagnostic performance (clarithromycin resistance)
Gene sequencing identified 14 patients with the A2143G mutation. One strain had both the A2143G and A2142G mutations (Fig. 1). Of the 87 samples tested, Allplex produced 74 results concordant with gene sequencing for detecting clarithromycin resistance, whereas Ezplex yielded 76 (Fig. 2). Gene sequencing, Ezplex, and Allplex resulted in 13 cases with discordant results for clarithromycin resistance; Ezplex disagreed with gene sequencing in 11 cases, whereas Allplex disagreed in 10 (Table 3).
Performances of the Allplex™ and Ezplex® methods for detecting point mutations associated with clarithromycin resistance. Inner circle, gene sequencing; middle circle, Ezplex®; outer circle, Allplex™.
Summary of antibiotic resistance rates. C+M, clarithromycin+ metronidazole; C+L, clarithromycin+levofloxacin; M+L, metronidazole+ levofloxacin.
Discordance between the PCR methods
Antibiotic resistance rates
Culture results successfully detected clarithromycin resistance in 9 of the 17 isolates determined to be resistant using gene sequencing. All patients with genotypic clarithromycin resistance also exhibited phenotypic resistance. Antibiotic resistance testing of H. pylori isolates revealed that 27.3% were clarithromycin-resistant, 51.5% were metronidazole-resistant, 42.4% were levofloxacin-resistant, and 12.1% were resistant to both clarithromycin and metronidazole (Fig. 2).
Tailored therapy eradication rates
At the time of this study, Allplex was not approved for use in Korea; thus, tailoring therapy based on the Allplex results would have been unethical. Hence, the tailored therapy was based on the Ezplex results; tailored H. pylori eradication was based on the genotypic resistance detected using the Ezplex results. The eradication rates for clarithromycin-susceptible and resistant cases were 91.7% and 89.2%, respectively. Overall, Ezplex-based tailored therapy achieved an eradication rate of 90.7% (Fig. 3).
Eradication rates following tailored therapy based on the Ezplex® genotypic resistance results. CLA-S, clarithromycin-sensitive; CLA-R, clarithromycin-resistant.
DISCUSSION
Antibiotic susceptibility is crucial for successful H. pylori eradication, making resistance detection essential for successful treatment. Although culture-based methods are considered the gold standard for identifying antibiotic resistance, they have several limitations, including a time requirement of 20–72 hours, low efficiency, and restricted accessibility owing to specific laboratory requirements. Molecular biological methods, such as PCR, are increasingly used as alternatives [9]. Modern PCR techniques are simpler, faster, and less expensive than conventional culture methods [10,11]. The Maastricht VI/Florence consensus report recommends testing for clarithromycin resistance, using either culture or molecular methods, before starting clarithromycin-based therapy. It also recommends using quadruple therapy over clarithromycin-containing triple therapy when the clarithromycin resistance status is unknown [3]. However, using more antibiotics may disrupt the gut microbiota and lead to the development of new types of bacterial resistance [12]. Thus, clinicians can use modern PCR methods to optimize clarithromycin-based triple therapy.
The Allplex method uses a multiplex quantitative real-time reverse transcription (RT-PCR) procedure that employs multiple temperature detection techniques [13]. Ezplex is a diagnostic kit that employs a real-time PCR assay. Both PCR methods can detect H. pylori infections and the A2143G and A2142G mutations that are linked to clarithromycin resistance. In this study, both methods showed high accuracy and inter-test agreement with RUT and culture techniques, for detecting H. pylori. A European study found a strong correlation between specific point mutations and clarithromycin resistance [14]. Korean studies revealed that the A2143G mutation matched the resistance phenotype in 85.7% of cases, whereas the A2142G mutation match was 100% [11,15]. Both A2143G and A2142G mutations are associated with the failure of H. pylori eradication when using clarithromycin-based treatments [16].
In this study, we assessed the diagnostic effectiveness of the Allplex and Ezplex methods for detecting both H. pylori infection and clarithromycin resistance. Allplex showed 85% concordance, whereas Ezplex demonstrated 87% concordance with gene sequencing results for identifying clarithromycin resistance. These findings suggest that both PCR methods are clinically reliable for detecting the point mutations associated with clarithromycin resistance.
This study has several limitations. First, the number of enrolled patients was small. Owing to the limited number of clarithromycin-resistant patients, the diagnostic performance of the tests in this group may not have been accurately assessed. Nevertheless, this study is the first to evaluate the diagnostic performance of currently available RT-PCR tests with gene sequencing. Second, UBT was excluded as a diagnostic test. However, because the diagnostic use of UBT is not covered by insurance in Korea, it is not commonly used in clinical practice. Therefore, we believe the results of this study accurately reflect real-world clinical practice. Third, the presence of the A2142C mutation was not reported. Moreover, other mutations associated with clarithromycin resistance, such as A2115G, A2142T, G2141A, and T2182C, were also not considered [17,18]. There have been no large-scale investigations of point mutations associated with clarithromycin resistance in Korea. Currently, most RT-PCR kits used in clinical settings, in Korea, detect only A2143G and A2142G mutations. Therefore, this study focused on these two mutations, and investigated the A2142C mutation, which is commonly found in Western populations. Further research is needed to examine the distribution of point mutations related to clarithromycin resistance in Korea and whether this distribution varies regionally. Finally, the causes of the discrepancies between the RT-PCR and gene sequencing results were not determined. However, the few discrepancies observed when gene sequencing was used as the gold standard highlight the strong diagnostic performance of both PCR methods. Further studies are required to investigate the reasons for these discrepancies.
In conclusion, Allplex demonstrated comparable diagnostic performance for detecting H. pylori infection when compared with RUT, culture, and Ezplex. The Allplex and Ezplex diagnostic methods showed high accuracy and consistency with gene sequencing for detecting clarithromycin resistance. The high eradication rates achieved with the Ezplex results-based tailored therapy further confirmed its clinical utility detecting H. pylori and gene mutations associated with clarithromycin resistance.
Notes
Availability of Data and Material
The datasets generated or analyzed during the study are available from the corresponding author on reasonable request.
Conflicts of Interest
The authors have no financial conflicts of interest.
Funding Statement
This research was funded by Seegene, Inc., Korea. Although funding was provided by Seegene, the Sponsor played no role in the design or analysis of the experiment nor did they influence the decision to publish the results.
Acknowledgements
None
Authors’ Contribution
Conceptualization: Joon Sung Kim, Tae-Woo Kim, Won Seok Lee. Data curation: Tae-Woo Kim, Won Seok Lee. Formal analysis: Tae-Woo Kim, Won Seok Lee. Funding acquisition: Joon Sung Kim. Investigation: Tae-Woo Kim, Won Seok Lee, Dong Jin Yoon, Ilsoo Kim. Methodology: Tae-Woo Kim, Won Seok Lee. Project administration: Joon Sung Kim. Resources: Joon Sung Kim. Software: Joon Sung Kim, Tae-Woo Kim, Won Seok Lee. Supervision: Joon Sung Kim. Validation: Joon Sung Kim, Tae-Woo Kim, Won Seok Lee. Visualization: Tae-Woo Kim, Won Seok Lee. Writing—original draft: Tae-Woo Kim, Won Seok Lee. Writing—review & editing: Joon Sung Kim. Approval of final manuscript: all authors.