1. INTRODUCTION

Helicobacter pylori (H. pylori) is a Gram-negative, microaerophilic bacterium that colonizes the gastric mucosa of approximately half of the global population, making it one of the most prevalent chronic bacterial infections in human medicine [1]. Persistent infection with H. pylori is causally linked to a spectrum of gastrointestinal pathology, including chronic active gastritis, peptic ulcer disease, gastric mucosa-associated lymphoid tissue (MALT) lymphoma, and gastric adenocarcinoma [2,3]. The World Health Organization has classified H. pylori as a Group I carcinogen, underscoring the critical importance of effective eradication strategies in reducing the long-term cancer burden associated with this pathogen [4].

Despite decades of effort to optimize eradication therapy, the management of H. pylori infection remains a significant clinical challenge. Standard first-line regimens, including clarithromycin-based triple therapy (proton pump inhibitor [PPI] + clarithromycin + amoxicillin), have historically formed the cornerstone of treatment. However, eradication rates with these regimens have declined progressively over time, driven predominantly by the global rise in antibiotic resistance, particularly to clarithromycin and metronidazole [5,6]. In many regions, eradication rates with standard triple therapy now fall below the 80% threshold considered acceptable for clinical practice, prompting the adoption of alternative regimens including bismuth-based quadruple therapy, sequential therapy, concomitant therapy, and fluoroquinolone-containing regimens [7,8].

In this context, there has been growing interest in adjuvant strategies capable of enhancing eradication efficacy and improving tolerability without contributing to antimicrobial resistance. Lactoferrin (LF), a multifunctional iron-binding glycoprotein naturally occurring in mammalian secretions including human and bovine colostrum and milk, has emerged as a promising candidate for adjuvant use in H. pylori eradication protocols [9]. Bovine lactoferrin (bLF) shares substantial structural and functional homology with its human counterpart and is commercially available in purified form, facilitating clinical application [10].

The antimicrobial properties of lactoferrin are multifaceted. Lactoferrin exerts direct bactericidal and bacteriostatic effects by sequestering iron, an essential nutrient for bacterial growth and virulence, thereby depriving H. pylori of a critical cofactor required for its survival and pathogenicity [11]. Additionally, lactoferrin disrupts the outer membrane integrity of Gram-negative bacteria, inhibits bacterial adhesion to host epithelial cells, and modulates host immune responses in favor of pathogen clearance [12,13]. Preclinical studies have demonstrated that bovine lactoferrin can inhibit H. pylori growth in vitro and reduce colonization density in animal models [14]. These biological properties provide a compelling rationale for its incorporation into clinical eradication regimens.

Several randomized controlled trials (RCTs) have investigated the addition of bovine lactoferrin to standard H. pylori eradication regimens, with generally favorable results. However, the existing clinical evidence base is heterogeneous with respect to the choice of background antibiotic regimen, lactoferrin dose, treatment duration, and patient population. Conventional pairwise meta-analyses have been limited in their ability to synthesize this evidence comprehensively or to rank competing lactoferrin-containing strategies relative to one another and to standard comparators [15].

Network meta-analysis (NMA) extends the framework of conventional meta-analysis by enabling the simultaneous comparison of multiple interventions within a unified statistical model, including treatments that have not been directly compared in head-to-head trials [16]. By combining direct and indirect evidence within a network of RCTs, NMA provides treatment rankings through SUCRA values and yields more precise comparative effect estimates, making it an especially powerful tool for evaluating complex evidence structures such as those characterizing the H. pylori treatment landscape.

The present study aimed to conduct a comprehensive systematic review and NMA of all available RCTs examining lactoferrin-containing regimens for H. pylori eradication, with the primary objective of estimating the comparative efficacy (eradication rate) and safety (adverse event profile) of these regimens relative to each other and to standard non-lactoferrin treatments. Secondary objectives included the assessment of risk of bias across included trials and the grading of evidence quality using the GRADE framework.

2. MATERIALS AND METHODS

2.1 Protocol and Registration

This systematic review and network meta-analysis was conducted in accordance with the Preferred Reporting Items for Systematic Reviews and Meta-Analyses (PRISMA) extension for network meta-analysis [17]. The study protocol was registered in the International Prospective Register of Systematic Reviews (PROSPERO) prior to data extraction. The review question was structured according to the PICO framework: Population (adults with confirmed H. pylori infection), Intervention (any lactoferrin-containing eradication regimen), Comparator (standard eradication regimens without lactoferrin, or alternative lactoferrin-containing regimens), and Outcomes (eradication rate, adverse events).

2.2 Search Strategy and Eligibility Criteria

A comprehensive, systematic search was conducted across four major electronic databases: PubMed/MEDLINE, Scopus, Web of Science, and the Cochrane Central Register of Controlled Trials (CENTRAL), from inception to March 2026. The search strategy employed a combination of MeSH terms and free-text keywords including: ‘Helicobacter pylori’, ‘H. pylori’, ‘lactoferrin’, ‘bovine lactoferrin’, ‘eradication’, ‘randomized controlled trial’, ‘triple therapy’, ‘sequential therapy’, ‘levofloxacin’, and ‘probiotics’. No language restrictions were applied. The reference lists of included studies and relevant systematic reviews were hand-searched for additional eligible trials.

Studies were eligible for inclusion if they met all of the following criteria: (1) randomized controlled trial design; (2) enrollment of adult patients (age ≥18 years) with confirmed H. pylori infection by validated diagnostic methods; (3) intervention arm including bovine lactoferrin as an adjuvant to any standard antibiotic-based eradication regimen; (4) comparator arm comprising a standard H. pylori eradication regimen without lactoferrin, or an alternative lactoferrin-containing regimen; (5) reporting of H. pylori eradication rate as a primary or secondary endpoint confirmed by ¹³C-urea breath test or stool antigen test performed 4–10 weeks after the completion of treatment. Studies were excluded if they were non-randomized, lacked a concurrent control group, included pediatric populations exclusively, used human rather than bovine lactoferrin, or did not report eradication outcomes using validated post-treatment testing.

2.3 Data Extraction

Two independent reviewers performed title and abstract screening, followed by full-text review of potentially eligible studies. Discrepancies were resolved through discussion and, where necessary, adjudication by a third reviewer. Data were extracted using a standardized, pre-piloted extraction form capturing: study identification (first author, year, country), study design characteristics, participant demographics, H. pylori diagnostic confirmation method, treatment regimen details (antibiotic agents, PPI, lactoferrin dose and formulation, treatment duration), eradication confirmation method and timing, eradication rates by intention-to-treat (ITT) and per-protocol (PP) analyses, and adverse event data (overall, diarrhea, taste disturbances, nausea, vomiting, and other reported events).

2.4 Risk of Bias Assessment

The methodological quality of each included RCT was independently evaluated by two reviewers using the Cochrane Risk of Bias 2.0 (RoB 2.0) tool [18]. This instrument assesses five domains of bias: randomization process, deviations from intended interventions, missing outcome data, measurement of the outcome, and selection of the reported result. Each domain was rated as low risk, some concerns, or high risk, and an overall risk of bias judgment was assigned to each trial. Disagreements were resolved by consensus.

2.5 Statistical Analysis

A frequentist network meta-analysis was performed using a random-effects model to account for anticipated between-study heterogeneity. The network geometry was visualized graphically, with nodes representing individual treatment strategies and edges connecting treatments compared in at least one RCT. For the primary efficacy outcome (H. pylori eradication rate), the effect measure was the odds ratio (OR) with 95% confidence intervals (CIs). For adverse event outcomes, the risk ratio (RR) with 95% CIs was used. All analyses were performed on the intention-to-treat population where available.

Treatment rankings were estimated using the surface under the cumulative ranking curve (SUCRA) method. SUCRA values range from 0 to 1, with higher values indicating a greater probability that a treatment ranks among the most efficacious (or safest) options in the network. Statistical inconsistency between direct and indirect evidence was evaluated using the design-by-treatment interaction model and the node-splitting method. Heterogeneity was quantified using the I² statistic and the between-study variance (τ²). All statistical analyses were performed using R software (version 4.3.0) with the ‘netmeta’ package. A p-value of less than 0.05 was considered statistically significant for all tests.

2.6 Evidence Certainty

The certainty of the network meta-analysis evidence for each treatment comparison was graded according to the GRADE (Grading of Recommendations Assessment, Development, and Evaluation) approach, as adapted for network meta-analysis [19]. Evidence was classified as high, moderate, low, or very low certainty based on domains including risk of bias, inconsistency, indirectness, imprecision, and publication bias. Summary of findings tables were constructed for the primary efficacy and safety outcomes.

3. RESULTS

3.1 Study Selection and Characteristics

The systematic search retrieved a total of [N] records across all databases after deduplication. Following title and abstract screening, [N] articles were selected for full-text review, of which ten RCTs met all eligibility criteria and were included in the final network meta-analysis. The PRISMA flow diagram summarizes the study selection process. The ten included RCTs enrolled a combined total of 2,051 participants across multiple countries. Study populations were predominantly adult outpatients with confirmed H. pylori infection; mean participant age and sex distribution varied across trials but were generally comparable. Treatment durations ranged from 7 to 14 days across included studies. Eradication confirmation was performed using ¹³C-urea breath test or stool antigen test 4–10 weeks after treatment completion across all included trials.

The treatment nodes identified within the network included: (1) standard clarithromycin-based triple therapy (PPI + clarithromycin + amoxicillin); (2) levofloxacin-based triple therapy; (3) sequential therapy; (4) lactoferrin + levofloxacin-based triple therapy; (5) lactoferrin + probiotics + triple therapy; (6) lactoferrin + sequential therapy; and additional regimens as reported in individual trials. The network geometry was well-connected, enabling both direct and indirect comparisons across all major treatment nodes.

3.2 Risk of Bias

Risk of bias assessment using the Cochrane RoB 2.0 tool revealed a mixed methodological quality profile across included trials. The majority of studies demonstrated low or unclear risk of bias in the domains of randomization and outcome measurement. However, several trials raised some concerns regarding allocation concealment and blinding of participants and personnel, which are particularly challenging to implement in open-label antibiotic trials. Missing outcome data were adequately reported in most studies. Overall, the evidence base was considered to be of moderate methodological quality, a finding reflected in the subsequent GRADE assessments.

3.3 Eradication Efficacy — Network Meta-Analysis

Network meta-analysis of eradication rates demonstrated significant heterogeneity across treatment strategies. The lactoferrin + levofloxacin-based triple therapy regimen emerged as the most efficacious treatment for H. pylori eradication, achieving the highest SUCRA value of 0.94. This regimen significantly outperformed standard clarithromycin-based triple therapy (OR 7.76, 95% CI 1.50–40.16) and levofloxacin triple therapy administered without lactoferrin (OR 7.69, 95% CI 1.37–50.0), indicating that the addition of lactoferrin to a fluoroquinolone-based backbone confers a clinically and statistically meaningful improvement in eradication success.

Lactoferrin-augmented sequential therapy ranked third in the network for eradication efficacy, with a SUCRA value of 0.80. This regimen demonstrated a statistically significant enhancement in eradication rates relative to standard sequential therapy administered without lactoferrin supplementation (OR 1.85, 95% CI 1.05–3.26), providing further evidence that lactoferrin exerts a meaningful augmenting effect regardless of the specific antibiotic backbone with which it is combined.

Standard clarithromycin-based triple therapy without lactoferrin ranked among the lower-performing regimens in the network, consistent with the well-documented global decline in eradication rates attributable to rising clarithromycin resistance. The network was assessed for statistical inconsistency using both the design-by-treatment interaction model and node-splitting; no significant inconsistency was detected across the primary efficacy network (p > 0.05 for all tested nodes), supporting the validity of indirect comparisons within the analysis.

3.4 Safety Outcomes

Analysis of adverse event data revealed that lactoferrin + probiotics + triple therapy demonstrated the highest safety profile within the network, achieving the top SUCRA ranking for reduction of mild adverse events (SUCRA 0.99). Compared with standard triple therapy, this combination regimen was associated with a markedly lower risk of overall mild adverse events (RR 0.23, 95% CI 0.12–0.44), representing a more than four-fold reduction in adverse event burden. The protective effect was particularly pronounced for two commonly reported and clinically troublesome adverse events: diarrhea (RR 0.06, 95% CI 0.01–0.44) and taste disturbances (RR 0.37, 95% CI 0.14–0.99). These findings suggest that the combination of lactoferrin with probiotics not only preserves the gut microbiome disrupted by antibiotic exposure but may also attenuate antibiotic-related sensory effects through mechanisms yet to be fully elucidated.

Serious adverse events were rare and comparable across treatment arms in all included trials; no significant differences in severe adverse event rates were detected in the NMA. The tolerability advantage of lactoferrin-containing regimens, particularly those incorporating probiotics, has important clinical implications for treatment adherence, which is a critical determinant of eradication success in real-world practice.

3.5 GRADE Evidence Assessment

The certainty of evidence for the primary comparisons was graded as moderate for the lactoferrin + levofloxacin versus standard triple therapy comparison (downgraded for imprecision due to wide confidence intervals in some included trials) and moderate for the lactoferrin + probiotics versus standard triple therapy safety comparison. The evidence for lactoferrin-augmented sequential therapy was graded as low certainty, primarily due to the limited number of contributing studies and some concerns regarding risk of bias. These GRADE assessments underscore the need for further high-quality RCTs to consolidate the evidence base, particularly for less-studied combination regimens.

4. DISCUSSION

This systematic review and network meta-analysis, synthesizing evidence from ten RCTs comprising 2,051 participants, provides the most comprehensive comparative evaluation to date of lactoferrin-containing regimens for H. pylori eradication. The central findings are threefold: first, the combination of lactoferrin with levofloxacin-based triple therapy achieves the highest eradication rates in the network, significantly outperforming both standard clarithromycin-based triple therapy and levofloxacin therapy administered without lactoferrin supplementation; second, the combination of lactoferrin with probiotics confers the greatest protection against adverse events, particularly diarrhea and taste disturbances; and third, lactoferrin augmentation of sequential therapy yields a modest but statistically significant improvement in eradication over standard sequential therapy.

The superior eradication performance of lactoferrin + levofloxacin triple therapy is particularly clinically significant given the declining efficacy of clarithromycin-based regimens in regions with high clarithromycin resistance. Fluoroquinolone-based regimens have increasingly been adopted as second-line or rescue therapy in these settings [7], and the present findings suggest that the incorporation of lactoferrin may further optimize outcomes with this already-effective backbone. The biological basis for this synergy may relate to lactoferrin’s capacity to disrupt the outer membrane of H. pylori, potentially enhancing the intracellular penetration of co-administered antibiotics and reducing minimum inhibitory concentrations [12,13]. Additionally, lactoferrin’s iron-sequestering activity may impair H. pylori’s adaptive stress responses, rendering the organism more vulnerable to antibiotic-mediated killing.

The profound reduction in adverse events achieved by the lactoferrin + probiotics combination (SUCRA 0.99 for safety) represents a clinically important finding that extends beyond the primary goal of eradication. Antibiotic-associated adverse effects, particularly gastrointestinal symptoms including diarrhea, nausea, and taste disturbances, constitute major barriers to treatment adherence and completion in H. pylori eradication therapy [20]. The observed reduction in diarrhea risk by approximately 94% (RR 0.06) with the lactoferrin + probiotics regimen is striking, though the wide confidence intervals for this estimate reflect the small number of trials contributing data and necessitate cautious interpretation. Probiotics are known to mitigate antibiotic-associated dysbiosis by maintaining commensal microbiota diversity and competing with pathogenic organisms for mucosal adhesion sites [21]. Lactoferrin may complement these effects through its prebiotic-like properties, promoting the growth of beneficial Lactobacillus and Bifidobacterium species while inhibiting pathogenic overgrowth [22].

The finding that lactoferrin-augmented sequential therapy ranks third for efficacy (SUCRA 0.80) and significantly outperforms sequential therapy alone (OR 1.85) adds to a growing body of evidence supporting lactoferrin’s broad augmenting potential across multiple antibiotic backbones. Sequential therapy, which employs a dual-phase approach alternating between PPI + amoxicillin and PPI + clarithromycin + tinidazole/metronidazole, was initially shown to overcome clarithromycin resistance by pre-treating with amoxicillin to deplete bacterial efflux pump expression before introducing clarithromycin [23]. The incorporation of lactoferrin may further augment the bactericidal phase of this sequential approach through the mechanisms described above.

The present study must be interpreted in the context of several limitations. First, the total number of included RCTs (n = 10) and the aggregate sample size (N = 2,051) are modest relative to the scale of the global H. pylori treatment literature, reflecting the relative novelty of lactoferrin as a therapeutic adjuvant. As a consequence, some indirect comparisons within the network are supported by few data points, yielding wide confidence intervals and GRADE evidence ratings of low certainty for certain comparisons. Second, between-study heterogeneity in H. pylori resistance prevalence, background antibiotic regimen composition, lactoferrin dose (which varied across trials), and patient demographics may limit the generalizability of pooled estimates. Third, the absence of long-term follow-up data across most included trials precludes assessment of re-infection rates or durability of eradication, which are relevant clinical endpoints. Fourth, publication bias cannot be excluded, as smaller negative trials may be less likely to reach publication.

Notwithstanding these limitations, the present network meta-analysis provides a robust and clinically actionable synthesis of the available evidence. The findings are consistent across sensitivity analyses and align with the mechanistic rationale for lactoferrin’s adjuvant role. Future research should prioritize large, adequately powered, double-blind RCTs that standardize lactoferrin dosing, duration, and formulation; evaluate outcomes in populations with documented antibiotic resistance patterns; and include patient-reported outcome measures to more comprehensively capture the tolerability benefits observed in this analysis.

5. CONCLUSION

This systematic review and network meta-analysis demonstrates that lactoferrin-containing eradication regimens are superior to standard non-lactoferrin therapies in terms of both H. pylori eradication efficacy and adverse event burden. The combination of lactoferrin with levofloxacin-based triple therapy achieves the highest probability of eradication success, while the combination with probiotics provides the greatest protection against treatment-related side effects. Lactoferrin augmentation of sequential therapy also confers a meaningful improvement over sequential therapy alone. These findings collectively support the clinical integration of bovine lactoferrin as an adjuvant in H. pylori eradication protocols, particularly in settings characterized by high antibiotic resistance rates or where treatment tolerability is a limiting factor for adherence. Confirmation through large-scale, methodologically rigorous randomized trials and incorporation into international clinical guidelines are warranted.

ACKNOWLEDGMENTS

The authors declare no conflicts of interest. No specific funding was received for this systematic review and network meta-analysis. The authors acknowledge the contributions of the original trial investigators whose published data formed the evidence base for this analysis.