Proton pump inhibitor (PPI)-based standard triple therapy, which comprises a PPI, amoxicillin, and clarithromycin, has been commonly recommended as the first-line treatment for Helicobacter pylori (HP) infections. However, the success rate of HP eradication has decreased worldwide due to increased antibiotic resistance, mainly against clarithromycin. Standard triple therapy is not recommended in regions with high resistance to clarithromycin (>15%) without testing for the susceptibility of HP.¹

Real-time polymerase chain reaction (RT-PCR) assay is known to be comparable to the culture method in terms of sensitivity and specificity for detecting clarithromycin resistance.² The RT-PCR assay detects point mutations in the 23S rRNA gene of HP, and the A2142G and A2143G point mutations have been reported to be found in clarithromycin-resistant HP strains.3,⁴ HP eradication with identification of clarithromycin resistance has shown high success rates.^5-8 Bismuth-based quadruple therapy (BQT), consisting of a PPI, bismuth subcitrate, metronidazole, and tetracycline, is one of therapeutic options for first-line treatment where local clarithromycin resistance rates are over 15% and for clarithromycin-resistant HP infections.^1,9-11 Current clinical guidelines recommend administering BQT for 10 to 14 days.^3,7,12 However, little evidence exists regarding the appropriate duration of BQT. Administering BQT for 7 to 14 days is recommended by the 2013 Korean HP treatment guidelines,¹³ and some physicians still use 7-day BQT as the first-line therapy for clarithromycin-resistant HP. There have been a few small prospective studies in Korea comparing eradication rates by BQT treatment duration with different results.^14-16 Seven-day BQT treatment has shown eradication success rates exceeding 90% in per-protocol (PP) analyses.^5,12,17,18 Reducing the duration of BQT may reduce healthcare costs and shorten the duration of adverse events. Thus, there is no definitive consensus on the treatment duration of first-line BQT therapy in confirmed clarithromycin-resistant HP infections. This prospective randomized trial investigated the effectiveness of 7- and 14-day BQT regimens as first-line therapy in HP infections identified as resistant to clarithromycin with the RT-PCR assay.

1. Study subjects

We prospectively enrolled 162 peptic ulcer disease subjects who had been confirmed as having clarithromycin-resistant HP infections with the RT-PCR assay using U-TOPTM HPy-ClaR Detection Kit (SeaSun Biomaterials, Daejeon, Korea) at Eunpyeong St. Mary’s Hospital, Catholic University, Seoul, the Republic of Korea, from March 2020 to April 2022 (Clinical Research Information Service: KCT0004414). HP infections were classified as clarithromycin-resistant based on the detection of the A2143G and A2142G point mutations using the RT-PCR assay from an endoscopic forceps biopsy specimen. HP treatment-naïve subjects between 19 and 75 years of age who had been confirmed as having clarithromycin-resistant HP infections were enrolled in the study. Subjects with serious concomitant illnesses, such as decompensated liver cirrhosis and chronic kidney disease, or with previous history of gastric surgery, were excluded from the study. Subjects who had taken medicine including antibiotics, bismuth, or nonsteroidal anti-inflammatory drugs during the previous 4 weeks were excluded. Pregnant and lactating women and subjects allergic to drugs were excluded.

2. BQT regimen and urea breath test

The BQT regimen used in this study comprised a double dose of ilaprazole (10 mg, two times a day), metronidazole (500 mg, three times a day), bismuth subcitrate (300 mg, four times a day), and tetracycline (500 mg, four times a day). Participants were allocated to groups with 7-day or 14-day treatment durations with simple randomization using a random number table in this open-label trial.

The eradication of HP was assessed with a ¹³C-urea breath test (UBT) at least 4 weeks after the eradication regimen was completed. No PPIs, histamine 2 receptor blockers, or antibiotics were allowed until the UBT was done after completion of the BQT regimen. The subjects fasted for at least 4 hours before testing and breath samples before and 20 minutes after administration of 100 mg ¹³C-urea (UBIT^Ⓡtablet, Korea Otsuka Pharm., Seoul, Korea) were collected and measured by infrared spectral analyzer (POCone; Otsuka Electronics Co., Ltd., Hirakata, Japan). A cutoff value of UBT was 2.5‰.

3. Outcome measures and compliance

We compared the successful eradication rate between both groups. Compliance and adverse events were checked with a questionnaire during UBT. We investigated clinically significant adverse events in the study subjects. Adverse events were classified as mild, moderate, or severe, according to the degree of interruption to ordinary daily living. Mild adverse events were defined as those that did not cause any significant impact on subjects’ daily living and moderate adverse events as those that interrupted subjects’ daily living. Severe adverse events were defined as those that required discontinuation of the BQT regimen due to adverse events or hospitalization. Clinically significant adverse events were defined as moderate or severe adverse events for inclusion in the statistical analysis because mild adverse events were considered to have little clinical impact. Poor compliance was defined as taking less than 80% of BQT duration due to severe adverse events related to BQT or other causes.

4. Statistical analysis

The clinical significance criterion was set at 15% assuming an eradication rate of 90% based on the previous prospective studies,^14,16,19,20 and the number of subjects was calculated according to the formula below under the assumption that equivalence between the two groups was tested.

Assuming a significance level of 0.05, a power of 80%, and a dropout rate of 10%, the required number of subjects in each group was at least 81.

The HP eradication rate was analyzed using intention-to-treat (ITT) and PP analyses. Continuous variables were analyzed using the independent t-test, and categorical variables by the chi-square test or the Fisher exact test. Statistical significance was defined as a p-value less than 0.05. All statistical analyses were calculated using SPSS software version 23.0 for Windows (IBM Corp., Armonk, NY, USA).

5. Ethics statement

This study was approved by the Institutional Review Board of Eunpyeong St. Mary’s Hospital, Catholic University (IRB number: PC19MESI0089). Informed consent was obtained from all the study subjects. This study followed the ethical principles of the Declaration of Helsinki.

1. Characteristics of the study population

A total of 162 subjects were enrolled in this study, and 81 subjects were allocated to the 7- and 14-day groups, respectively. The ITT analysis included 81 subjects in the 7-day group and 78 subjects in the 14-day group. Three subjects in the 14-day group were excluded from the ITT analysis due to withdrawal of consent (n=2) and protocol violation (n=1). The PP analysis included 74 subjects in the 7-day group and 73 subjects in the 14-day group. Seven subjects in the 7-day group were excluded due to low compliance (n=2) and loss to follow-up (n=5), while five subjects in the 14-day group were excluded due to low compliance (n=5). A study flowchart is shown in Fig. 1. The baseline characteristics of the study subjects are shown in Table 1. No significant differences were found in age, sex, body mass index, alcohol intake, smoking, or underlying medical conditions. The baseline characteristics of all study subjects are shown in Table 1.

Figure 1. Study flowchart. ITT, intention-to-treat; PP, per-protocol analysis.

Table 1

Basic Characteristics.

Characteristic	7-day group	14-day group	p-value
No. of subjects	81	78
Age, yr	54.5±13.2	55.5±11.3	0.795
Male sex	29 (35.8)	26 (33.3)	0.747
Body mass index, kg/m2	24.0±3.2	23.8±3.4	0.777
Alcohol intake	7 (8.6)	6 (7.7)	0.827
Smoking	12 (14.8)	14 (18.0)	0.593
Diabetes mellitus	8 (9.9)	5 (6.4)	0.425
Hypertension	19 (23.5)	14 (18.0)	0.392

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

2. Eradication rate according to the treatment duration

The overall eradication rates in the ITT and PP analyses were 83.0% (95% confidence interval [CI], 77.2% to 88.9%; 132/159) and 89.8% (95% CI, 84.9% to 94.7%; 132/147), respectively. The eradication rates in the ITT analysis were 79.0% (95% CI, 70.1% to 87.9%; 64/81) in the 7-day group and 87.2% (95% CI, 79.8% to 94.6%; 68/78) in the 14-day group (p=0.170). The eradication rates in the PP analysis were 86.5% (95% CI, 78.7% to 94.3%; 64/74) in the 7-day group and 93.2% (95% CI, 87.4% to 99.0%; 68/73) in the 14-day group (p=0.182). The eradication rate was not significantly different in either the ITT analysis or the PP analysis (Fig. 2). Among the five subjects in the 14-day group who were excluded from the analysis due to low compliance, four subjects took the BQT regimen for 7 days and the UBT results showed successful HP eradication. One subject took the BQT regimen for 11 days, and the UBT result showed persistent HP infection. Considering the 7-day group and the four subjects mentioned above from the 14-day group, the eradication rates of the 7-day BQT regimen could be estimated at 80.0% (95% CI, 71.5% to 88.5%; 68/85) in the ITT analysis and 87.2% (95% CI, 79.8% to 94.6%; 68/78) in the PP analysis.

Figure 2. Eradication rates of the two study groups. ITT, intention-to-treat; PP, per-protocol analysis.

3. Eradication rate according to the type of mutation

The A2143G mutation was the most frequent point mutation (94.3%). The A2142G mutation and both mutations (A2143G and A2142G) were found in 2.5% and 3.1% of subjects, respectively. The eradication rate of the A2142G mutation in the 7-day group was 100% (1/1) in the ITT and PP analyses. The eradication rate of the A2143G mutation in the 7-day group was 77.9% (60/77) in the ITT analysis and 85.7% (60/70) in the PP analysis. The eradication rate for subjects with both mutations in the 7-day group was 100% (3/3) in the ITT and PP analyses. There was no statistically significant difference in eradication rates among different mutations in the 7-day group (Table 2). The eradication rate of the A2142G mutation in the 14-day group was 100% (2/2) in the ITT and PP analyses. The eradication rate of the A2143G mutation in the 14-day group was 86.3% (63/73) in the ITT analysis and 92.3% (63/68) in the PP analysis. The eradication rate for subjects with both mutations in the 14-day group was 100% (2/2) in the ITT and PP analyses. No statistically significant difference was found in the eradication rates among different mutations in the 14-day group (Table 2). No clinical factors showed a statistically significant association with the eradication rate in the multivariate analysis (Table 3).

Table 2

Eradication Rates According to Type of Mutations.

Group	Analysis	A2142G	A2143G	Both	p-value
7-day group	ITT	1/1 (100)	60/77 (77.9)	3/3 (100)	1.000
	PP	1/1 (100)	60/70 (85.7)	3/3 (100)	1.000
14-day group	ITT	3/3 (100)	63/73 (86.3)	2/2 (100)	1.000
	PP	3/3 (100)	63/68 (92.3)	2/2 (100)	1.000

Data are presented as number (%)..

ITT, intention-to-treat; PP, per-protocol..

Table 3

Clinical Factors-Associated with Helicobacter pylori Eradication.

Variable	Multivariate analysis (n=147)*
	Odds ratio (95% CI)	p-value
Age	0.975 (0.929–1.023)	0.297
Sex
Male	Reference
Female	0.764 (0180–3.255)	0.716
Alcohol intake
Non-drinker	Reference
Drinker	1.567 (0.175–14.042)	0.688
Smoking state
Non-smoker	Reference
Smoker	0.416 (0.079–2.195)	0.301
Hypertension
No	Reference
Yes	6.064 (0.716–51.391)	0.098
Diabetes mellitus
No	Reference
Yes	0.598 (0.074–4.8843)	0.630
Treatment duration
7-day	Reference
14-day	2.745 (0.84–8.971)	0.095

CI, confidence interval..

*Per-protocol analysis..

4. Adverse event rate according to the therapeutic duration

Clinically significant adverse events occurred in 18.2% (95% CI, 12.2% to 24.2%; 29/159) of the study subjects during HP eradication. There was no statistically significant difference in the rates of individual adverse events and all adverse events between the 7-day group and the 14-day group (Table 4). Severe adverse events leading to low compliance occurred in 4.4% (95% CI, 1.2% to 7.6%; 7/159) of the study subjects. Nausea and vomiting were the main causes of low compliance. No statistically significant difference in the severe adverse event rate was found between the two groups (7-day group 2.4% [95% CI, –0.9% to 5.8%; 2/81] vs 14-day group 6.4% [95% CI, 1.0% to 11.8%; 5/78], p=0.226).

Table 4

The Adverse Event Profile of the Study Subjects.

Variable	7-day group	14-day group	p-value
No. of subjects	81	78
Bloating	0	1 (1.3)	0.128
Epigastric soreness	1 (1.3)	1 (1.3)	1.000
Anorexia	3 (4.0)	1 (1.3)	0.364
Dysgeusia	1 (1.3)	2 (2.6)	1.000
Nausea	4 (5.3)	7 (9.0)	0.371
Vomiting	3 (4.0)	4 (5.1)	1.000
Abdominal pain	3 (4.0)	0	0.118
Headache	7 (9.2)	2 (2.6)	0.096
Dyspepsia	3 (4.0)	3 (3.9)	1.000
Diarrhea	5 (6.6)	1 (1.3)	0.114
Reflux	1 (1.3)	2 (2.6)	1.000
Pruritus or skin rash	0	0	-
Sense of weakness or tiredness	4 (5.3)	1 (1.3)	0.207
Xerostomia	0	2 (2.6)	0.497
Dizziness	2 (2.6)	3 (3.9)	1.000
Somnolence	1 (1.3)	2 (2.6)	1.000
All adverse events	18 (23.7)	11 (14.1)	0.128

Data are presented as number (%)..

This study showed that the eradication rate of the 7-day BQT regimen was not significantly different from that of the 14-day BQT regimen as first-line therapy for HP infections identified as resistant to clarithromycin using the RT-PCR assay. The compliance and clinically significant adverse event rates of both study groups were not significantly different.

The eradication rate of PPI-based standard triple therapy has declined and the prevalence of HP antibiotic resistance has been increasing in most parts of the world.²¹ The clarithromycin resistance rate strongly influences the eradication rate of PPI-based standard triple therapy. HP culture testing for clarithromycin susceptibility is the best method for selecting an HP eradication regimen.²² However, the slow growth of HP and demanding culture conditions make applying culture-based results to clinical practice difficult. Recent Korean guidelines strongly recommend the PCR assay for clarithromycin resistance when 7-day standard triple therapy is considered as a first-line treatment.¹⁰ The Maastricht guideline has recommended clarithromycin resistance testing before prescribing clarithromycin triple therapy when clarithromycin resistance is common.²³ Recent guidelines for HP treatment recommend a 10- to 14-day BQT regimen.^1,9,10 Some physicians use 7-day BQT as the first-line therapy for clarithromycin-resistant HP because the 2013 Korean HP treatment guidelines recommend BQT for 7 to 14 days.¹³ Previous retrospective studies of the first-line treatment of HP infections confirmed to be clarithromycin-resistant by the PCR assay showed no significant differences between 7- and 14-day BQT treatment.^18,20,24 The eradication rates of 7- and 14-day BQT treatment in those studies were 91.1% to 95.6% and 90.3% to 100% in PP analysis. Our prospective randomized study showed that the eradication rate of 14-day BQT treatment was higher than that of 7-day treatment, but the difference was not statistically significant. This finding is consistent with previous retrospective studies and suggests that first-line 7-day BQT treatment for confirmed clarithromycin-resistant HP infections may be considered as an alternative option to 14-day BQT treatment. Nonetheless, further larger-scale studies are required to clarify the selection of treatment duration of first-line BQT treatment for confirmed clarithromycin-resistant HP infections.

This study showed that the A2143G mutation was the dominant mutation conferring clarithromycin resistance in this study, coinciding with the known distribution of clarithromycin resistance mutations.^7,20,24,25 Previous retrospective studies showed that eradication rates in A2142G mutation and both (A2142G and A2143G) mutation were 90.6% to 97.5% and 90.0% to 100% without significant differences in the PP analysis.^18,20,24 Our finding was consistent with those studies. Although eradication rates in A2142G mutation were 100% in both 7- and 14-day BQT treatment in PP analysis of our study. Previous retrospective studies showed similar eradication results with 80.0 to 100% in both 7- and 14-day BQT treatment in the PP analysis.^18,20,24 Although the number of A2142G mutation was small and a larger number of A2142G mutation data will be required, the results of our study and previous studies suggest that the type of 23S rRNA point mutation conferring clarithromycin resistance may not influence the eradication rate of BQT or outcomes according to treatment duration.

Adverse events during HP eradication treatment influence treatment compliance. The adverse event rate of BQT has been reported to be significantly higher than those of other eradication treatments.¹⁰ One study reported that 14-day BQT treatment showed a higher adverse event rate than 7-day BQT for second-line HP eradication,¹⁵ but another study showed no significant difference.¹⁶ Our retrospective study on first-line HP eradication of confirmed clarithromycin-resistant HP infections showed no significant difference in the adverse event rate between 7- and 14-day BQT treatment regimens.²⁰ Clinically significant adverse events occurred in 18.2% of the study subjects, and the adverse event rate was not significantly different between the 7-day group and the 14-day group in this study. The treatment duration may not be related to the occurrence or severity of clinically significant adverse events. Clinically, if there would be no major adverse events after taking a 7-day BQT therapy, it may be recommended to take BQT up to 10–14 days.

Our study has the following limitations. First, this study was conducted at a single center in Korea. Thus, the findings may not be generalized because regional differences in metronidazole resistance rates of HP in Korea has been reported.²⁶ Second, an RT-PCR assay is limited to the detection of A2142G and A2143G point mutations. Other point mutations including A2142C or A2144G were not included in this study. Third, total duration of adverse events during BQT treatment was not investigated. Although the adverse event rate was not significantly different in this study, there is a possibility that the total duration of adverse events during BQT treatment may be different.

Our study has the following strengths. We performed first-line BQT treatment of treatment-naïve subjects with clarithromycin-resistant HP infections confirmed using the RT-PCR method, and we conducted a prospective randomized comparative trial of 7-day or 14-day treatment regimens with a detailed adverse event profile.

In conclusion, both 7- and 14-day BQT regimens showed effectiveness and safety for first-line therapy in HP infections identified as resistant to clarithromycin with the RT-PCR assay. Seven-day BQT treatment duration may be sufficient in first-line therapy in clarithromycin-resistant HP infections. More large-scale trials and meta-analyses would be required to clarify the optimal duration of first-line BQT therapy in clarithromycin-resistant HP infections.

This work was supported by the Research Institute of Medical Science, Eunpyeong St. Mary’s Hospital, the Catholic University of Korea and by Il-Yang Pharm. Co., Ltd.

This work was supported by Il-Yang Pharm. Co., Ltd. Except for that, no potential conflict of interest relevant to this article was reported.

Study concept and design: J.H.O. Data acquisition; Data analysis and interpretation: C.H.L. Drafting of the manuscript: C.H.L. Critical revision of the manuscript for important intellectual content: C.H.L., J.H.O. Statistical analysis: C.H.L. Obtained funding: C.H.L. Administrative, technical, or material support; study supervision: J.H.O. Approval of final manuscript: all authors.