Precision therapy guided by genotypic antibiotic resistance for Helicobacter pylori eradication: A prospective, randomized controlled trial

Abstract

BACKGROUND

Helicobacter pylori (H. pylori) is a Gram-negative bacillus classified as a Group I carcinogen by the World Health Organization. However, the efficacy of eradication therapies has declined in recent years, primarily due to the increasing prevalence of antibiotic resistance. The Maastricht VI/Florence Consensus Report highlights the importance of tailoring treatment strategies to local epidemiological data and individual antimicrobial susceptibility patterns.

AIM

To investigate the efficacy of precision-guided first-line therapy for H. pylori infection using genotypic antibiotic susceptibility testing (G-AST).

METHODS

This single-center randomized controlled trial enrolled 194 H. pylori-positive patients at a tertiary hospital in Qingdao, China (October 2022-August 2024). Participants were randomized to receive either a 14-day bismuth quadruple therapy (BQT: Amoxicillin, clarithromycin, esomeprazole, and bismuth) or a 14-day G-AST-guided regimen with tailored antibiotics (clarithromycin, levofloxacin, or tetracycline). Treatment efficacy and adverse events were compared between groups using intention-to-treat (ITT) and per-protocol (PP) analyses. Primary and secondary outcomes were analyzed with χ² tests.

RESULTS

Of 194 patients enrolled, 180 (92.8%) completed the study as planned. In the ITT analysis, the eradication rate was higher in the G-AST group than in the BQT group [92.8% (95%CI: 85.8-96.5) vs 79.4% (95%CI: 70.3-86.2), P = 0.007], with a risk difference of 13.4% (95%CI: 3.7-23.2). In the PP analysis, eradication rates were 97.8% (95%CI: 92.4-99.4) in the G-AST group and 84.1% (95%CI: 75.1-90.3) in the BQT group (P = 0.001), with a risk difference of 13.7% (95%CI: 5.5-23.0). Adverse event incidence did not differ significantly between groups (30.9% vs 28.9%, P = 0.754).

CONCLUSION

G-AST-guided therapy yielded higher eradication rates than empirical BQT in first-line H. pylori treatment without increasing adverse events, supporting the clinical utility of individualized, resistance-based therapy.

Key Words: Helicobacter pylori; Antibiotic resistance; Precision medicine; Eradication rate; First-line therapy

Core Tip: Antibiotic resistance in Helicobacter pylori (H. pylori) has reduced the efficacy of traditional eradication regimens. This randomized controlled trial in northern China used rapid genotypic antibiotic susceptibility testing (G-AST) to guide first-line therapy by tailoring regimens according to detected resistance mutations, including tetracycline for dual-resistant strains. G-AST-guided therapy significantly improved eradication rates compared with standard bismuth quadruple therapy without increasing adverse events. These findings demonstrate the feasibility and clinical benefit of molecular-guided personalized treatment, offering a promising strategy to overcome antibiotic resistance in H. pylori infection.

INTRODUCTION

With an adult global prevalence of approximately 43.9%, Helicobacter pylori (H. pylori) infection is a significant etiological factor in gastrointestinal disease[1]. H. pylori plays a crucial role in the pathogenesis of peptic ulcers and is closely associated with gastric cancer. Eradication substantially improves gastric health and reduces the risk of peptic ulcers, gastric malignancies, and mucosa-associated lymphoid tissue lymphoma.

In China, the standard approach for H. pylori eradication is bismuth quadruple therapy (BQT), which combines acid inhibitors and antibiotics for optimal results[2]. However, increasing antibiotic resistance, poor patient adherence, and low intragastric antibiotic concentrations have contributed to a gradual decline in the success rate of BQT[3]. Recent data from the European H. pylori Management Registration Center show that 14-day BQT achieves an 83% eradication rate[4]. In China, the average eradication rate for BQT is 81.3%[2]. The Maastricht VI/Florence Consensus Report places strong emphasis on antibiotic stewardship and recommends tailoring treatment strategies to local and individual antimicrobial resistance patterns[5].

Currently, H. pylori antimicrobial susceptibility is assessed with two primary approaches: Phenotypic testing, which relies on H. pylori culture, and genotypic antibiotic susceptibility testing (G-AST). Phenotypic drug resistance testing via H. pylori culture is complex and costly, which limits its widespread clinical application. G-AST has emerged as a promising strategy for personalized H. pylori treatment, offering advantages in efficiency, speed, and diagnostic accuracy[6]. It has demonstrated clinical value across diverse infectious diseases by rapidly detecting resistance mutations to guide therapy. It has been applied in the treatment of tuberculosis and bloodstream infections to optimize treatment and reduce antibiotic misuse[7,8]. This broad applicability supports the use of G-AST for targeted H. pylori eradication. Moreover, there is strong concordance between genotypic and phenotypic resistance to clarithromycin and levofloxacin, indicating that these genetic markers can serve as reliable and practical targets for directing personalized treatment strategies in H. pylori infections[9].

Polymerase chain reaction (PCR) testing was performed on gastric mucosal samples from treatment-naive patients, guiding individualized therapy based on G-AST for levofloxacin and clarithromycin. Previous studies have demonstrated that this PCR method yields results comparable to those of gastric biopsy analysis and Sanger sequencing, while maintaining practicality and ease of use[10]. This study aimed to investigate the efficacy of precision-guided first-line therapy for H. pylori infection using the G-AST method.

MATERIALS AND METHODS

Study design and patients

This single-center prospective randomized controlled trial was conducted at a tertiary hospital in Qingdao, China, between October 2022 and August 2024. Participants aged 18-70 years who tested positive for H. pylori infection by the ¹³C/¹⁴C urea breath test (UBT) and consented to gastroscopy with biopsy for histopathological evaluation were enrolled. Primary exclusion criteria were active gastrointestinal bleeding; prior upper gastrointestinal surgery; pregnancy or lactation without willingness to use contraception during the study; prior unsuccessful H. pylori eradication therapy; severe comorbidities potentially affecting evaluation (e.g., advanced hepatic, pulmonary, cardiac, or renal disease; malignancy), and known allergy to study medications.

Ethical issues

Approval was obtained from the Ethics Committee of the Affiliated Hospital of Qingdao University (Approval No. QYFYKYLL 923711921). All trial procedures complied with Clinical Trial Standards/Code guidelines. Participants provided informed consent before enrollment. The study is registered with the China Clinical Trial Registry (ChiCTR 2500095247).

Randomization and blinding

Eligible participants were randomized 1:1 to the G-AST or BQT group using a computer-generated random sequence. Allocation was concealed with opaque sealed envelopes labeled with unique subject identifiers. The trial used an open-label design, and all participants and healthcare providers were aware of the assigned treatment after randomization.

Endoscopy and H. pylori antibiotic susceptibility testing

All patients underwent gastroscopy at the hospital. Two gastric mucosal biopsy specimens, approximately 0.1 cm in diameter, were obtained from the antrum 1-2 cm proximal to the pylorus. For patients in the G-AST group, an H. pylori drug resistance gene nucleic acid detection kit (Qingdao Jianma Gene Detection Company) was used for PCR amplification of 23S rRNA and gyrA gene fragments extracted from the specimens. A TaqMan-MGB probe-based multiplex real-time PCR system detected the C261A/G mutation in gyrA and the A2142G/C mutation in 23S rRNA, mutations linked to resistance to levofloxacin and clarithromycin, respectively.

Therapy regimens

The BQT group received a twice-daily regimen of esomeprazole, bismuth potassium citrate, amoxicillin, and clarithromycin for 14 days. The G-AST group received regimens tailored to G-AST results. The detailed procedure is shown in Figure 1. Because levofloxacin-based quadruple therapy is infrequently used as a primary strategy for H. pylori eradication[11], the specific regimens for the trial group are presented in Table 1.

Figure 1 Flowchart of therapeutic decision-making in the genotypic antibiotic susceptibility testing group based on resistance mutations detected by real-time polymerase chain reaction. Treatment was tailored as clarithromycin-containing bismuth quadruple therapy (BQT), levofloxacin-containing BQT, or tetracycline-containing BQT according to clarithromycin and levofloxacin resistance. H. pylori: Helicobacter pylori; PCR: Polymerase chain reaction; Cla-BQT: Clarithromycin-containing bismuth quadruple therapy; Lev-BQT: Levofloxacin-containing bismuth quadruple therapy; Tet-BQT: Tetracycline-containing bismuth quadruple therapy.

Table 1 Drug selection for the genotypic antibiotic susceptibility testing group.

Regimen	Clarithromycin	Levofloxacin	Drugs
			Clarithromycin	Levofloxacin	Tetracycline
Cla-BQT	S	-	500 mg, bid	-	-
Lev-BQT	R	S	-	500 mg, qd	-
Tet-BQT	R	R	-	-	500 mg, tid

All regimens were administered for 14 days and included esomeprazole 20 mg twice daily, bismuth potassium citrate 220 mg twice daily, and amoxicillin 1000 mg twice daily. R: Resistant; S: Susceptible; “-” indicates this drug is not chosen; bid: Twice daily; qd: Once daily; tid: Three times daily; Cla-BQT: Clarithromycin-containing bismuth quadruple therapy; Lev-BQT: Levofloxacin-containing bismuth quadruple therapy; Tet-BQT: Tetracycline-containing bismuth quadruple therapy.

Assessment

Investigators completed a case report form and recorded baseline demographic data for each patient. After treatment initiation, investigators conducted telephone follow-ups to monitor adherence and document any adverse events that occurred during therapy.

After completing eradication therapy, participants underwent a UBT (¹³C or ¹⁴C) 5 weeks later to assess efficacy (proton pump inhibitors and H₂ receptor antagonists were prohibited for 2 weeks before the test; antibiotics or probiotics were not permitted within 4 weeks). A delta over baseline (DOB) value < 4‰ in the ¹³C-UBT was considered negative (successful eradication); a DOB ≥ 4‰ was considered positive (eradication failure).

Statistical analysis

Sample size estimation was based on eradication rates reported in earlier studies[11], with expected rates of 80% in the control group and 94% in the trial group. With an 80% power (1 - β), a two-sided α level of 0.05, and calculations performed using PASS 2021, the minimum required sample size was determined. Allowing for an anticipated 10% dropout, the target enrollment was 194 participants (n = 97 per group).

Demographic characteristics and the incidence of adverse events were recorded. Eradication rates were evaluated by intention-to-treat (ITT) and per-protocol (PP) analyses. ITT included all randomized patients; PP included only those who completed treatment with ≥ 80% adherence and without loss to follow-up. Continuous variables with a normal distribution were expressed as mean ± SD; Categorical variables were presented as frequencies and percentages. For continuous variables, a t-test was performed if data were normally distributed; otherwise, a nonparametric test was used. For group comparisons, the χ² test or Fisher exact test was applied. Statistical significance was defined as a two-tailed P < 0.05. All analyses were performed with SPSS 26.0.

RESULTS

Participants’ characteristics

Between October 2022 and August 2024, 194 patients were enrolled; 180 completed all required study procedures (Figure 2). Baseline clinical characteristics did not differ significantly between groups (P > 0.05; Table 2).

Figure 2 Flowchart of the recruitment of study subjects. H. pylori: Helicobacter pylori; ITT: Intention-to-treat analysis; PP: Per-protocol analysis; G-AST: Genotypic antibiotic susceptibility testing; BQT: Bismuth quadruple therapy.

Table 2 Clinical and demographic characteristics of the studied groups, n (%).

Baseline characteristics	BQT group (n = 97)	G-AST group (n = 97)	P value	χ²/t/z
Age (years), mean ± SD	52.81 ± 10.69	50.20 ± 9.45	0.072	1.808
Gender (male)	46 (47.4)	48 (49.5)	0.083	0.774
Height (m), mean ± SD	1.67 ± 0.08	1.68 ± 0.09	0.230	-1.2
Weight (kg), mean ± SD	66.14 ± 10.56	68.24 ± 11.62	0.189	-1.318
BMI, mean ± SD	23.73 ± 2.89	24.08 ± 3.24	0.432	-0.787
Smoking	20 (20.6)	14 (14.4)	0.257	1.284
Alcohol consumption	22 (22.7)	22 (22.7)	1.0	0
Hypertension	23 (23.7)	19 (19.6)	0.486	0.486
Diabetes	22 (22.7)	13 (13.4)	0.093	2.824
Family history of gastric cancer	11 (11.3)	13 (13.4)	0.663	0.190
Bile reflux	10 (10.3)	15 (15.5)	0.284	1.148
Atrophic gastritis	63 (64.9)	52 (53.6)	0.108	2.584

BMI: Body mass index; G-AST: Genotypic antibiotic susceptibility testing; BQT: Bismuth quadruple therapy.

Eradication rates of H. pylori

The G-AST group achieved eradication rates of 92.8% (90/97) in the ITT analysis and 97.8% (90/92) in the PP analysis. Eradication rates in the G-AST group were significantly higher than those in the BQT group in both analyses (both P < 0.05; Table 3).

Table 3 Comparison of Helicobacter pylori eradication rates between the control and trial groups, n (%).

	BQT group	G-AST group	Difference in eradication rate (%)	P value	χ²
ITT	77 (79.4)	90 (92.8)	13.4	0.007	7.271
95%CI	70.3-86.2	85.8-96.5	3.7-23.2
PP	74 (84.1)	90 (97.8)	13.7	0.001	10.477
95%CI	75.1-90.3	92.4-99.4	5.5-23.0

Bismuth quadruple therapy group received esomeprazole 20 mg twice daily + bismuth potassium citrate 220 mg twice daily + amoxicillin 1000 mg twice daily + clarithromycin 500 mg twice daily; genotypic antibiotic susceptibility testing group received esomeprazole 20 mg twice daily + bismuth potassium citrate 220 mg twice daily + amoxicillin 1000 mg twice daily + clarithromycin 500 mg twice daily/Levofloxacin 500 mg once daily/tetracycline 500 mg three times daily. G-AST: Genotypic antibiotic susceptibility testing; ITT: Intention-to-treat analysis; PP: Per-protocol analysis; BQT: Bismuth quadruple therapy.

In the G-AST group, 60 patients received clarithromycin-containing BQT (Cla-BQT), 19 received levofloxacin-containing BQT (Lev-BQT), and 18 received tetracycline-containing BQT (Tet-BQT). H. pylori eradication rates in these subgroups were 91.7%, 94.7%, and 94.4% (ITT), and 98.2%, 94.7%, and 100% (PP), respectively. Eradication rates did not differ significantly among the three subgroups (ITT: P = 0.863; PP: P = 0.530; Table 4).

Table 4 Subgroup analysis of the trial group.

G-AST group	Eradication rate, n/N [% (95%CI)]
	ITT	PP
Overall eradication rates	90/97 [92.8 (85.8-96.5)]	90/92 [97.8 (92.4-99.4)]
Cla-BQT	55/60 [91.7 (81.9-96.4)]	55/56 [98.2 (90.6-99.7)]
Lev-BQT	18/19 [94.7 (75.4-99.1)]	18/19 [94.7 (75.4-99.1)]
Tet-BQT	17/18 [94.4 (74.2-99.0)]	17/17 [100 (81.6-100.0)]
P value	0.863	0.530
χ²	0.294	1.270

Genotypic antibiotic susceptibility testing group received esomeprazole 20 mg twice daily + bismuth potassium citrate 220 mg twice daily + amoxicillin 1000 mg twice daily + clarithromycin 500 mg twice daily/Levofloxacin 500 mg once daily/tetracycline 500 mg three times daily. G-AST: Genotypic antibiotic susceptibility testing; ITT: Intention-to-treat analysis; PP: Per-protocol analysis; Cla-BQT: Clarithromycin-containing bismuth quadruple therapy; Lev-BQT: Levofloxacin-containing bismuth quadruple therapy; Tet-BQT: Tetracycline-containing bismuth quadruple therapy.

Resistance rates in the G-AST group

In the G-AST group, 60 H. pylori strains were susceptible to clarithromycin (61.9%), and 37 were resistant (38.1%). The levofloxacin resistance rate was 40.2%, and dual resistance to clarithromycin and levofloxacin was 17.5% (Table 5).

Table 5 Helicobacter pylori resistance rate, n (%).

Antibiotics	Susceptible	Resistant
Clarithromycin	60 (61.9)	37 (38.1)
Levofloxacin	58 (59.8)	39 (40.2)
Clarithromycin and levofloxacin	38 (39.2)	17 (17.5)

Safety and compliance

Fifty-eight patients reported adverse events: 28.9% (28/97) in the BQT group and 30.9% (30/97) in the G-AST group. All adverse symptoms resolved spontaneously after treatment completion. No significant difference in adverse events was observed between the groups (P = 0.754). Adverse event incidence in the Cla-BQT, Lev-BQT, and Tet-BQT subgroups was 30.0% (18/60), 31.6% (6/19), and 33.3% (6/18), respectively, with no significant differences (P = 0.890). The most common adverse events were bitter taste (16/194) and nausea (9/194) (Table 6).

Table 6 Adverse events and patient compliance, n (%).

Adverse events	BQT group (n = 97)	G-AST group (n = 97)	P value
Bitter taste	9 (9.3)	7 (7.2)
Nausea	5 (5.2)	4 (4.1)
Abdominal pain	3 (3.1)	4 (4.1)
Diarrhea	4 (4.1)	3 (3.1)
Abdominal discomfort	2 (2.1)	2 (2.1)
Bloating	1 (1.0)	3 (3.1)
Heartburn	1 (1.0)	2 (2.1)
Skin rash	1 (1.0)	2 (2.1)
Dizziness	2 (2.1)	3 (3.1)
Total	28/97 (28.9)	30/97 (30.9)	0.754

Bismuth quadruple therapy group received esomeprazole 20 mg twice daily + bismuth potassium citrate 220 mg twice daily + amoxicillin 1000 mg twice daily + clarithromycin 500 mg twice daily; genotypic antibiotic susceptibility testing group received esomeprazole 20 mg twice daily + bismuth potassium citrate 220 mg twice daily + amoxicillin 1000 mg twice daily + clarithromycin 500 mg twice daily/Levofloxacin 500 mg once daily/ tetracycline 500 mg three times daily. G-AST: Genotypic antibiotic susceptibility testing; BQT: Bismuth quadruple therapy.

DISCUSSION

Antibiotic resistance has gradually increased, and the effectiveness of eradicating H. pylori infection has steadily declined[12]. A recent systematic review of antibiotic resistance in the Asia-Pacific region from 1990 to 2023 reported primary resistance rates of 30% for clarithromycin and 35% for levofloxacin[13]. Amoxicillin and tetracycline showed substantially lower resistance rates of 2.41% and 2.53%, respectively[14].

The A2142G/C mutation in 23S rRNA and the C261A/G mutation in gyrA are associated with resistance to clarithromycin and levofloxacin, respectively[15,16]. Genotypic resistance correlates strongly with phenotypic resistance to these agents in H. pylori strains[17], and a meta-analysis confirmed the high sensitivity and specificity of genotypic detection for clarithromycin and fluoroquinolone resistance[18]. Moreover, genotypic testing outperforms phenotypic methods in predicting treatment outcomes[19] and is increasingly favored when performed on gastric mucosal samples to guide eradication therapy[20].

According to the Maastricht VI/Florence consensus, the goal of antimicrobial therapy is to reliably cure H. pylori infection in most patients (e.g., ≥ 90%)[5]. Our findings demonstrate that G-AST-guided therapy, tailored to clarithromycin and levofloxacin resistance, meets and exceeds this efficacy threshold. All three G-AST-based regimens—Cla-BQT, Lev-BQT, and Tet-BQT—achieved ITT and PP eradication rates above 90%. These outcomes highlight the clinical effectiveness of individualized therapy guided by G-AST.

The resistance rates to clarithromycin and levofloxacin were both approximately 40%, with a dual resistance rate of 17.5%, consistent with recent Chinese literature[14], reaffirming the relevance of G-AST in settings with substantial antimicrobial resistance. Patients receiving G-AST-guided Cla-BQT or Lev-BQT achieved eradication rates markedly higher than previously reported empirical rates for the same regimens (75.2% and 68.0%, respectively)[21]. Patients with dual resistance were successfully treated with Tet-BQT, reaching 100% eradication in the PP analysis. Although this result likely reflects the small sample size, it is concordant with the low tetracycline resistance reported in Chinese H. pylori strains[14]. Widespread empirical tetracycline use remains challenging due to limited availability, concerns about adverse effects, and dosing complexity[22,23]. Within a G-AST framework, however, tetracycline can be deployed selectively and effectively rather than as a universal empirical option. Overall, the success of G-AST-guided therapy across all subgroups highlights its practical value for optimizing eradication outcomes while advancing antibiotic stewardship. This precision-based strategy is particularly suitable for regions with moderate to high resistance rates where empirical regimens often underperform, and it provides a feasible pathway for integrating molecular diagnostics into routine care.

Adverse event rates were comparable between the G-AST and BQT groups, with no significant differences among G-AST subgroups. The most common adverse reactions were a bitter taste and nausea, likely related to the effects of bismuth potassium citrate or antibiotics. Structured follow-up calls, both during and after therapy, facilitated systematic assessment of adverse events. The adverse reaction rate in the Lev-BQT subgroup was 31.6%. No serious adverse events (e.g., tendinitis, tendon rupture, neuropathy) occurred with levofloxacin, which may be related to the 14-day course, standard dosing (500 mg once daily), and exclusion of high-risk individuals. The predominant side effects were mild gastrointestinal symptoms that resolved spontaneously. These findings align with prior studies[24,25] that support the safety of levofloxacin-based regimens for H. pylori eradication when used appropriately and monitored.

Clinically, G-AST-guided therapy significantly improves eradication outcomes without increasing adverse events, providing a practical and effective approach to personalized treatment. Although PCR-based genotypic testing and gastric biopsy entail higher upfront costs than empirical therapy, long-term benefits—including reduced treatment failures, fewer retreatments, and mitigation of antibiotic resistance—justify the investment. The broader applicability depends on local resistance patterns and the healthcare infrastructure. G-AST is particularly advantageous in areas with moderate to high clarithromycin and levofloxacin resistance, where empirical regimens are less effective. In low-resistance settings, adoption should be guided by local cost-effectiveness assessments and diagnostic capacity.

This study has several limitations. First, the study was conducted at a single tertiary center in Northern China, which may limit its generalizability to regions with different resistance profiles or healthcare systems. Second, all participants were receiving initial H. pylori therapy. Further studies are needed to evaluate the effectiveness of personalized eradication strategies using G-AST in treatment-resistant H. pylori infection, where resistance is more prevalent and therapeutic options are more constrained.

CONCLUSION

G-AST-guided precision therapy achieved higher first-line H. pylori eradication rates than empirical BQT (92.8% vs 79.4%, ITT), without increasing adverse events. These findings highlight the clinical utility of individualized, resistance-based therapy and support the integration of G-AST into routine clinical practice to improve treatment outcomes.

ACKNOWLEDGEMENTS

We sincerely thank all personnel who supported patient recruitment and sample collection, as well as the laboratory staff for their PCR technical assistance.