Detection of Clarithromycin Resistance in Helicobacter pylori Using MmaxSure™ H. pylori & ClaR Assay

Abstract

Introduction

Clarithromycin resistance is a crucial factor in the eradication of Helicobacter pylori. This study aimed to evaluate the performance of MmaxSure™ H. pylori & ClaR Assay (MmaxSure™) in the diagnosis and detection of clarithromycin resistance in H. pylori.

Methods

Subjects who underwent esophagogastroduodenoscopy between April 2020 and October 2022 were enrolled. The diagnostic performances of MmaxSure™ and dual priming oligonucleotide (DPO)-based multiplex polymerase chain reaction (PCR) were compared with the rapid urease test and culture. Secondary gene sequencing analysis was performed in discordant cases of PCR tests.

Results

A total of 156 gastric biopsy samples were analyzed. In H. pylori detection, MmaxSure™ showed a 95.9% sensitivity (95% CI: 90.6–98.6), a 42.7% specificity (95% CI: 26.3–60.7), and a kappa value of 0.457. For the detection of A2143G mutation samples, MmaxSure™ showed a 91.2% sensitivity (95% CI: 76.3–98.1), a 93.4% specificity (95% CI: 87.5–97.1), and a kappa value of 0.804. There were a total of 10 discordant cases compared to gene sequencing in A2143G mutation detection for MmaxSure™.

Conclusion

In this study, MmaxSure™ showed comparable diagnostic performance to DPO-PCR in the detection of the H. pylori and A2143G mutation. Further research is needed to confirm the clinical effectiveness of the MmaxSure™ assay in H. pylori eradication.

Introduction

Helicobacter pylori is the main cause of gastrointestinal disorders, including peptic ulcer disease, and gastric cancer [1]. International guidelines recommend eradication in infected patients. A triple-therapy regimen composed of clarithromycin is traditionally used to treat H. pylori [2–4]. However, increasing resistance of H. pylori to clarithromycin has reduced the effectiveness of this regimen [5]. As a result, multiple guidelines recommend that triple therapy should be avoided in areas with clarithromycin resistance above 15% [2, 4, 6].

Resistance to clarithromycin is primarily caused by point mutations in H. pylori’s 23S ribosomal ribonucleic acid (rRNA) gene. These mutations prevent clarithromycin from binding to its ribosomal targets. An adenine-to-guanine transition of 23 rRNA at position 2143 (A2143G) and 2142 (A2142G), which represents 81.5% of clarithromycin resistance, is a point mutation most reported in H. pylori with clarithromycin resistance [7, 8]. Another possible cause of clarithromycin resistance is a thymine-to-cytosine transition of 23 rRNA at position 2182 with competing reports [9–11]. A tailored therapy through selective use of treatment with and without clarithromycin based on each patient’s H. pylori clarithromycin resistance is highly effective [12–15]. The polymerase chain reaction (PCR) method is used as one of the methodologies to identify clarithromycin resistance. Recent European guidelines recommend that PCR should be used whenever possible [4].

The Seeplex™ H. pylori-ClaR ACE Detection kit, developed by Seegene Inc. (Seoul, South Korea), utilizes dual priming oligonucleotide (DPO)-based multiplex PCR to diagnose H. pylori infection and detects two point mutations (A2143G and A2142G) associated with clarithromycin resistance [16]. Loop-mediated isothermal amplification (LAMP) is a fast, simple, and highly specific gene amplification [17]. The newly developed MmaxSure™ H. pylori & ClaR (MmaxSure™), developed by Mmonitor Inc. (Daegu, South Korea), reagent employs LAMP to detect H. pylori and the point mutations that cause resistance to clarithromycin, specifically A2143G and T2182C, in addition to H. pylori itself. This study aimed to evaluate and compare the diagnostic accuracy of MmaxSure™ and DPO-PCR for the detection of H. pylori and its point mutations related to clarithromycin resistance from gastric biopsy samples.

Materials and Methods

Patients

This study enrolled 156 patients who underwent upper gastrointestinal endoscopy at Incheon St. Mary’s Hospital between August 2020 and October 2022 for the diagnosis of H. pylori infection and who agreed to participate in the study. Biopsy samples from the antrum and body of the stomach were obtained from each patient. These samples were then analyzed using both DPO-PCR and MmaxSure™. Additional samples were also taken from each of the antrum and body to perform the rapid urease test (RUT) and culture. For cases with inconsistent results of the two PCR tests, a secondary gene sequencing analysis was performed selectively.

H. pylori DNA Extraction

DNA extraction was performed using a QIAamp DNA Mini Kit (QIAGEN, Germany). Briefly, gastric biopsy tissue was collected and placed in a microcentrifuge tube containing ATL buffer (180 μL) and Proteinase K (20 μL). The tube was vortexed and incubated at 56°C until the tissue was completely lysed. Buffer AL (200 μL) was added to the tube and incubated at 70°C for 10 min. Then 240 μL of 100% ethanol was added to the tubes and mixed by vortexing for 15 s. Each tube was then loaded into a QIAamp spin column and centrifuged at 8,000 rpm for 1 min. The columns were washed with AW1 buffer (500 μL) and centrifuged at 8,000 rpm for 1 min. AW2 buffer (500 μL) was added to each column, and the column was centrifuged at 14,000 rpm for 3 min. AE buffer (200 μL) was added to each sample and incubated at room temperature for 1 min before centrifugation at 8,000 rpm to elute DNA. Eluted DNA was then used for molecular analysis [18].

To analyze nucleotide conservation in the region of 23S rRNA positions of 2142 and 2182, we amplified a segment of 300 bp using PCR primers (5′ – TGA​ATG​GCG​TAA​CGA​GAT​G – 3, 5′-CAG​GCC​AGT​TAG​CTA​ACA-3′). MmaxSure™ and DPO-PCR were used to detect H. pylori and its mutations according to the manufacturer’s recommendations. These tests were performed as previously published [16].

H. pylori Strains and Agar Dilution Method

The detailed methods of H. pylori culture and antibiotic resistance have been reported previously [19]. The Clinical and Laboratory Standards Institutes (CLSI) guideline was followed when performing the agar dilution method for H. pylori isolates (n = 44). Results were interpreted based on CLSI breakpoints for clarithromycin, with values assigned as follows: susceptible ≤0.25 μg/mL; intermediate = 0.5 μg/mL; and resistant >0.5 μg/mL [19].

Outcomes

The primary outcome was the diagnostic performance of MmaxSure™ and DPO-PCR for detecting H. pylori infection. The secondary outcome was the diagnostic performance of MmaxSure™ and DPO-PCR in detecting the point mutation A2143G. The reference standard for detecting HP was established using the results of the RUT and bacterial culture. In cases where PCR test results were inconsistent, direct gene sequencing was employed as the definitive reference standard. Additionally, we compared the concordance between genotype and phenotype specifically for point mutations.

Statistical Analysis

The sensitivity, specificity, positive predictive value (PPV), and negative predictive value (NPV) of each test were evaluated. Continuous variables were expressed as mean (±SD) or median (interquartile range) and compared using Student’s t test. Categorical variables were expressed as numbers (percentages) and analyzed using χ² and trend tests. The agreement between different PCR methods for each test was estimated using the Cohen’s kappa coefficient. SPSS software, version 24.0 (SPSS Inc., Chicago, IL, USA), was used for all statistical analyses.

Results

Patient Characteristics

The study cohort consisted of 156 patients ranging in age from 21 to 80 years with a median age of 61 years. Male subjects (n = 94) accounted for 60.3%. The primary indication for biopsy was atrophic gastritis, with intestinal metaplasia suspected in 76 (48.7%) patients. The predominant reason for endoscopic examination was screening for stomach (60.9%) (shown in Table 1).

Table 1.

Baseline characteristics of the study population

Characteristics	Total (n = 156)
Age, years	58.7±11.9
Male	94 (60.3)
Comorbidity
Hypertension	60 (38.5)
Diabetes mellitus	30 (19.2)
Dyslipidemia	25 (16.0)
Current smoking	12 (7.7)
Alcohol	50 (32.1)
Endoscopy indication
Symptoma	24 (15.4)
Screening	25 (16.0)
Alarm featureb	12 (7.7)
Surveillance	95 (60.9)
Endoscopic biopsy indication
Atrophic gastritis	151 (96.8)
Closed type	61 (39.1)
Open type	90 (57.7)
Intestinal metaplasia	76 (48.7)
Peptic ulcer	16 (10.3)
Neoplasm of stomach	67 (42.9)

Data are presented as the number of patients (%) or mean ± SD.

BMI, body mass index; EGD, esophagogastroduodenoscopy.

^aSymptoms include dyspepsia, reflux symptom, and abdominal pain.

^bAlarm features include anemia, dysphagia, weight loss, GI bleeding, vomiting, and bowel habit change.

Diagnostic Performance for H. pylori

Culture (44 positives over 92 subjects) or RUT (118 positives over 156 subjects) identified 121 H. pylori infections among the 156 subjects. DPO-PCR and MmaxSure™ detected 135 and 136 H. pylori infections, respectively (shown in Table 2). DPO-PCR had a sensitivity of 98.4%, a specificity of 54.3%, a PPV of 88.2%, an NPV of 90.5%, an overall accuracy of 88.5%, and a kappa coefficient of 0.614. MmaxSure™ had a sensitivity of 95.9%, a specificity of 42.9%, a PPV of 85.3%, an NPV of 75.0%, an overall accuracy of 84.0%, and a kappa coefficient of 0.457. MmaxSure™ demonstrated comparable sensitivity, NPV, and diagnostic accuracy for H. pylori detection to DPO-PCR. In addition, we expanded our analysis of the H. pylori detection performance of MmaxSure™ by including DPO-PCR as an additional reference for H. pylori infection in addition to RUT and culture results. MmaxSure™ showed a sensitivity of 96.4%, a specificity of 79.0%, a PPV of 97.1%, a NPV of 75.0%, an overall accuracy rate of 94.2%, and a kappa coefficient of 0.736 (shown in online suppl. Table 1; for all online suppl. material, see https://doi.org/10.1159/000538655).

Table 2.

Performance of the MmaxSure™ H.pylori & ClaR and DPO-PCR in H. pylori detection compared with RUT and culture

	N (%) of results by the reference standard				Analytical performance, %a
	TP	TN	FP	FN	sensitivity	specificity	PPV	NPV	accuracy	kappa (κ)b
MmaxSure™	116 (74.4)	15 (9.6)	20 (12.8)	5 (3.2)	95.9 (90.6–98.6)	42.7 (26.3–60.7)	85.3 (78.2–90.8)	75.0 (50.9–91.3)	84.0 (77.3–89.4)	0.457 (0.262–0.652)
DPO-PCR	119 (76.3)	19 (12.2)	16 (10.3)	2 (1.3)	98.4 (94.2–99.8)	54.3 (36.7–71.2)	88.2 (81.5–93.1)	90.5 (69.6–98.8)	88.5 (82.4–93.0)	0.614 (0.446–0.781)

MmaxSure™, MmaxSure™ H. pylori & ClaR; DPO-PCR, dual priming oligonucleotide-based multiplex polymerase chain reaction; RUT, rapid urease test; TP, true positive; TN, true negative; FP, false positive; FN, false negative; PPV, positive predictive value; NPV, negative predictive value.

^aValues in parentheses are 95% confidence intervals.

^bCohen’s kappa coefficient, kappa values representing levels of agreement are categorized as follows: >0.90, almost perfect; 0.80–0.90, strong; 0.60–0.79, moderate; 0.40–0.59, week; 0.21–0.39, minimal; (0–0.20), none.

Diagnostic Performance for Clarithromycin Resistance Detection

Among the 156 subjects, DPO-PCR demonstrated a sensitivity of 88.2%, a specificity of 91.0%, a PPV of 73.2%, an NPV of 96.5%, an overall accuracy of 90.4%, and a kappa coefficient of 0.737 for the detection of the A2143G mutation. MmaxSure™ had a sensitivity of 91.2%, a specificity of 93.4%, a PPV of 79.5%, an NPV of 97.4%, an overall accuracy of 93.0%, and a kappa coefficient of 0.804. MmaxSure™ demonstrated comparable accuracy to DPO-PCR for detection of the A2143G mutation (shown in Table 3). Regarding the detection of the A2142G mutation, only 5 cases were identified as mutated by DPO-PCR. Conversely, MmaxSure™ revealed that 106 cases exhibited the T2182C mutation.

Table 3.

Performance of the MmaxSure™ H.pylori & ClaR and DPO-PCR in A2143G mutation

	N (%) of results by the reference standard				Analytical performance, %a
	TP	TN	FP	FN	sensitivity	specificity	PPV	NPV	accuracy	kappa (κ)b
MmaxSure™	31 (19.9)	114 (73.1)	8 (5.1)	3 (1.9)	91.2 (76.3–98.1)	93.4 (87.5–97.1)	79.5 (63.5–90.7)	97.4 (92.7–99.5)	93.0 (87.7–96.4)	0.804 (0.692–0.916)
DPO-PCR	30 (19.2)	111 (71.2)	11 (7.1)	4 (2.6)	88.2 (72.6–96.7)	91.0 (84.4–95.4)	73.2 (57.1–85.8)	96.5 (91.3–99.0)	90.4 (84.6–94.5)	0.737 (0.611–0.864)

MmaxSure™, MmaxSure™ H. pylori & ClaR; DPO-PCR, dual priming oligonucleotide-based multiplex polymerase chain reaction; TP, true positive; TN, true negative; FP, false positive; FN, false negative; PPV, positive predictive value; NPV, negative predictive value.

^aValues in parentheses are 95% confidence intervals.

^bCohen’s kappa coefficient, kappa values representing levels of agreement are categorized as follows: >0.90, almost perfect; 0.80–0.90, strong; 0.60–0.79, moderate; 0.40–0.59, week; 0.21–0.39, minimal; (0–0.20), none.

Discordance between the PCR Methods

In detection of H. pylori, a disparity was observed in 6 cases between results from MmaxSure™ and DPO-PCR. Among the 6, only one result of DPO-PCR was concordant with secondary gene sequencing. Additionally, a notable discordance was observed in 24 cases in the 2143G mutation from the two PCR results. In secondary gene sequencing, 14 DPO-PCR results from DPO-PCR and 10 MmaxSure™ results were valid (shown in Table 4).

Table 4.

Performance of MmaxSure™ H pylori and ClaR reagent, DPO-PCR, and gene sequencing methods for detection of A2143G mutation of H. pylori in discordant cases

DPO-PCR	MmaxSure™	Gene sequencing	Number of specimens
Mutated	Wild	Wild	10
Mutated	Wild	Mutated	3
Wild	Mutated	Wild	7
Wild	Mutated	Mutated	4

MmaxSure™, MmaxSure™ H. pylori & ClaR; DPO-PCR, dual priming oligonucleotide-based multiplex polymerase chain reaction.

Concordance between the Genotype and Phenotype

In this study, out of 156 samples collected for culture testing, 64 were excluded due to inadequate quality and volume, leaving 92 samples for analysis. Of these, 44 were successfully cultured and were then tested for antibiotic susceptibility. However, 48 cultures were found to be nonviable (shown in online suppl. Table 2).

Among the 44 viable isolates, 11 showed resistance to clarithromycin. Specifically, 10 of the 11 isolates that harbored the A2143G mutation, as identified by DPO-PCR, were resistant to clarithromycin. Additionally, 1 out of 3 isolates with the A2142G mutation also exhibited resistance to clarithromycin. In total, 11 out of 14 strains with any mutation detected by DPO-PCR were clarithromycin-resistant. On the contrary, DPO-PCR found no mutations in the 30 strains, and culture tests confirmed that 29 of these were susceptible to clarithromycin.

Using the MmaxSure™ method, 10 out of 15 strains with the A2143G mutation and 9 out of 37 strains with the T2182C mutation were found to be clarithromycin-resistant. Twelve strains showed simultaneous mutations of the A2143G and T2182C. Furthermore, 11 out of 40 strains with any detected mutation showed resistance to clarithromycin. Notably, 1 of the 4 strains without any detected mutations was also found to be resistant to clarithromycin.

Discussion

Antibiotic susceptibility is a critical factor in the eradication of H. pylori, making detection of resistance essential for successful treatment. The gold standard for detection of antibiotic resistance is a culture-based method. However, these techniques take at least 20–72 h, have low output, and are limited in accessibility. Consequently, culture methods are gradually being replaced by molecular biology methods that overcome these limitations, such as PCR testing [20]. Recent PCR techniques offer faster, simpler, and more cost-effective solutions for the detection of antibiotic resistance [21, 22]. Additionally, PCR methods can identify H. pylori infection in specimens that are negative for conventional staining or culture tests. Recent European guidelines advocate for clarithromycin susceptibility testing, either by molecular techniques or culture, before prescribing clarithromycin-containing therapy [4]. In the absence of information on clarithromycin resistance, the guidelines recommend a quadruple treatment. However, the increase in the number of antibiotics can disrupt the gut microbiota and contribute to the development of resistance in other bacterial species [23]. PCR methods allow clinicians to optimize triple therapy in 60–90% of patients, leading to more effective and targeted treatment strategies [4].

In South Korea, DPO-PCR is one of the methods used to assess resistance to clarithromycin. Seeplex H. pylori-ClaR ACE Detection is a DPO-PCR-based test for detecting H. pylori, A2143G, and A2142G mutations. DPO-PCR was consistent with the E test for sensitivity to clarithromycin in 95.3% of cases [24]. Eradication rates for clarithromycin-susceptible individuals determined by DPO-PCR ranged from 89.2% to 97.4%. However, for individuals with clarithromycin-resistant strains identified by DPO-PCR, bismuth quadruple therapy was more effective, resulting in eradication rates of 91.7–93.5% in individuals with clarithromycin resistance identified by DPO-PCR [14, 25, 26].

MmaxSure™ is a PCR reagent that utilizes the LAMP technique. The test results are easily confirmed by a color change (shown in Figure 1). Compared to other PCR methods such as DPO-PCR and quantitative RT-PCR, MmaxSure™ does not require additional procedures, such as Western blot or equipment beyond standard PCR tools. This simpler process results in lower price and a faster test time than DPO-PCR and RT-PCR.

Fig. 1.

Example of the MmaxSure™ H. pylori & ClaR result. Blue: positive (detected), purple: negative (not detected). When the specific target is amplified, the color of the solution shows a blue color.

In a European study, point mutations identified through whole-genome sequencing demonstrated a strong correlation with the clarithromycin resistance phenotype (congruence at 99%) [27]. In South Korea, the A2143G mutation exhibited 85.7% congruence with the phenotype (6 out of 7 strains) [16], while the A2142G mutation displayed 100% congruence with the phenotype (20 out of 20 strains) [28]. These point mutations were associated with failure in eradication when treated with a clarithromycin-based regimen [28–30].

In this study, we compared the diagnostic performance of MmaxSure™ and DPO-PCR. MmaxSure™ showed comparable diagnostic performance for H. pylori detection and A2143G mutation detection. Our results suggest that MmaxSure™ can be considered for the diagnosis of H. pylori and A2143G mutations. We also found a diminished association of the T2182C mutations with clarithromycin resistance. Future studies are required to investigate the role of these two mutations in clarithromycin resistance.

This study has several limitations. The first limitation was a relatively restricted sample size employed for this study. Second, T2182C is relatively less clinically significant than A2143G. Most cases of clarithromycin resistance in South Korea are due to the A2143G mutation [12]. The relationship between T2182C and clarithromycin resistance is unclear with conflicting results [9, 31–33]. However, since A2143G can explain most of clarithromycin resistance, the identification of the A2143G variant alone may have clinical value [32]. In addition, we did not perform gene sequencing for the entire sample, which might have limited our ability to detect false positives or negatives. However, previous studies have reported that DPO-PCR produces few false positives and a small number of false negatives for both H. pylori and A2143G detection [24]. In this study, MmaxSure™ showed almost similar results to DPO-PCR for H. pylori detection. Thus, we can expect comparable characteristics. Furthermore, although this study used culture or RUT as a reference standard, previous studies have reported cases of PCR detection even when these traditional methods were negative [24]. As a result, the assessment of PCR performance tested in this study may not be accurate. The final constraint was that we did not assess other mutations such as A2115G, A2142T, and G2141A implicated in clarithromycin resistance, which have been reported to impact susceptibility to clarithromycin [34]. However, it is noteworthy that previous research has primarily associated clarithromycin resistance with point mutations, particularly A2143G (69.8%), A2142G (11.7%), and A2142C (2.6%), which can inhibit the establishment of macrolide binding sites [8].

Conclusion

In conclusion, MmaxSure™ demonstrated diagnostic performance comparable to DPO-PCR. Further clinical research is needed to confirm whether the improved diagnostic performance of MmaxSure™ could translate into more effective eradication therapy.

Statement of Ethics

All participants provided written informed consent. This study was approved by the Institutional Review Board (IRB) of Incheon St. Mary’s Hospital, Incheon, South Korea. (OC20OISI0065).

Conflict of Interest Statement

The authors declare that they have no competing interests.

Funding Sources

This work has received funding from Hanmi Science Co., Ltd. The funding body had no role in study design, data collection, analysis, interpretation of data, or writing the manuscript.

Author Contributions

Ilsoo Kim and Yu Ri Shin contributed equally to this work as co-authors. Joon Sung Kim designed the research study. Yu Ri Shin, Lee-So Maeng, and Jung Min Kim contributed to the design of the study. All the authors performed the research and collected and analyzed the data. Ilsoo Kim and Yu Ri Shin wrote the manuscript and performed data visualization. Byung-Wook Kim contributed to the revision of the manuscript. All the authors approved the final version of the manuscript including the authorship list.

Funding Statement

This work has received funding from Hanmi Science Co., Ltd. The funding body had no role in study design, data collection, analysis, interpretation of data, or writing the manuscript.

Data Availability Statement

The datasets generated during and/or analyzed during the current study are not publicly available due to the restriction from the Institutional Review Board for this specific study but are available from the corresponding author on reasonable request.