Abstract
Background
Helicobacter pylori infection is a global health concern associated with gastritis, peptic ulcer disease, and gastric cancer. With increasing antibiotic resistance, egg yolk immunoglobulin (IgY) has emerged as a promising candidate owing to its stability and pathogen-neutralizing effects. This clinical trial evaluated the efficacy of IgY-enriched egg yolk powder (anti-H. pylori IgY) in eliminating the bacterium and improving clinical symptoms as an adjunctive.
Methods
In this randomized, double-blind trial, 60 patients with confirmed H. pylori infection received a 14-day standard therapy regimen (omeprazole, amoxicillin, clarithromycin) supplemented with IgY-enriched egg yolk powder (intervention) or plain egg yolk powder (control). The primary outcome was bacterial eradication, assessed by the 14C-urea breath test performed 4 weeks after completion of therapy (i.e. 6 weeks from baseline). Secondary outcomes included symptom improvement (abdominal pain, nausea, bloating, and anorexia) measured using the Gastrointestinal Symptom Rating Scale, via telephone interviews conducted three times weekly for 3 weeks. Data were analyzed using analysis of variance and t-tests (SPSS v24).
Results
Baseline demographic characteristics were homogeneous. The median urea breath test value in the intervention group decreased from 77 to 11, whereas that of the control group remained unchanged (median = 55). Bacterial eradication was achieved in 33.3% of IgY recipients versus 13.3% of controls (difference, 20%; P = .241). IgY was well tolerated, with no treatment-related adverse events observed in the intervention group.
Conclusion
Clinical symptoms showed trends toward faster improvement, but these did not reach statistical significance. IgY may be a promising safe adjunct for the treatment of H. pylori infection.
Statement of Significance
This study provides the current clinical perspective for immunoglobulin (IgY)-based therapy as an adjunct to standard therapy for Helicobacter pylori. Our findings suggest that IgY-enriched egg yolk powder can accelerate relief of symptoms without adverse effects. Although not sufficient to eradicate the bacteria, the reduction in urea breath test (mean 77 to 11) and the absence of adverse effects support IgY as a promising adjunct therapy and offers insights to guide future research on optimizing IgY formulations and developing safe and effective alternatives for H. pylori infection management.
Keywords: H. Pylori, immunoglobulin Y, clinical trial, symptom assessment
Introduction
One of the significant pathogens responsible for stomach infections in approximately half of the world’s population is a gram-negative bacterium Helicobacter pylorus. In addition to chronic gastritis, it is particularly implicated in the development of other diseases, such as peptic ulcers, adenocarcinoma, and stomach cancer [1, 2]. The World Health Organization has classified Helicobacter as a type 1 carcinogen [3]. Meta-analysis studies conducted on the prevalence of Helicobacter in the periods 1980–1990 and 2011–2022 revealed that the decreased prevalence of Helicobacter pylori was directly related to the reduction in stomach cancer [4, 5]. The 2015 Kyoto Global Consensus Report recommended its eradication [6].
Appropriate antibiotic options have been suggested, including amoxicillin, clarithromycin, metronidazole, tetracycline, and levofloxacin for 1–2 weeks, along with gastric acid inhibitors or proton pump inhibitor (PPI) [3, 7, 8]. However, concerns such as increasing antibiotic resistance, elimination of the gastrointestinal microbiome, disease recurrence, and antibiotic side effects have diminished therapeutic effectiveness outcomes [9–12].
According to previous studies, this antibiotic resistance has varied in different geographical areas; in China, it has been reported to be 81%, 44%, and 18% for metronidazole, clarithromycin, and levofloxacin, respectively [13, 14]. Beyond its clinical impact, it can impose a severe economic burden on society [15]. Therefore, identifying novel therapeutic options may help address these challenges. Studies have shown that although active immunity induces antibody production against H. pylori, it confers limited protection, whereas passive immunity can inhibit its colonization [16].
One of the antibodies that has received special attention recently is immunoglobulin (IgY), which has a molecular weight of 180 kilodaltons and can be isolated from egg yolks of immunized hens. Unlike other antibodies such as, Immunoglobulin G (IgG) and Immunoglobulin M (IgM), this antibody is more easily produced and, due to its special structure, is more resistant to environmental conditions, such as temperature and acidity [17].
Various hypotheses have been proposed for the mechanism of action of IgY, including inhibition of bacterial attachment to host cells by IgY binding to pili, flagella, and bacterial outer membrane proteins, neutralization of bacterial toxins, enhancement of bacterial phagocytosis by polymorphonuclear cells through physicochemical modification of bacteria, and modulation of the immune system and production of inflammatory cytokines (e.g. Interferon-gamma (IFN-γ) and Tumor Necrosis Factor-alpha (TNF-α)), thereby attenuating inflammation and tissue damage [18, 19].
Oral administration of specific antibodies has a long history in human medicine. Oral administration of IgY antibodies against H. pylori, in addition to having the lowest risk of allergy, can also lead to systemic tolerance to egg proteins. On the other hand, oral administration is the most convenient and common route for administering IgY antibodies against H. pylori, which is a gastrointestinal pathogen [20–22].
Despite previous studies that have only investigated to a limited extent the efficacy of IgY against H. pylori in the clinical phase, its therapeutic effects in improving clinical symptoms, eradicating the bacterium, optimal treatment duration, side effects, and the appropriate delivery route for its stability in the acidic conditions of the stomach are remain unclear [23–26]. Given the emulsifying properties of egg yolk, which can enhance antibody stability, this is the first clinical trial to use egg yolk powder containing IgY antibodies against H. pylori as an adjunctive to reduce clinical symptoms and achieve bacterial eradication.
Materials and methods
General study design
This was a single-center, parallel-group, randomized, double-blind, placebo-controlled superiority trial with a 1:1 allocation ratio. The trial was conducted at a hospital in Mashhad, Iran, between 23 January 2024, and 13 April 2025. Patients with confirmed H. pylori infection were randomly assigned to receive either anti-H. pylori IgY-enriched egg yolk powder (intervention) or plain egg yolk powder without IgY (control) for 14 days, in addition to standard triple therapy (omeprazole 40 mg/day, amoxicillin 2000 mg/day, clarithromycin 1000 mg/day) for the same 14-day period. The primary outcome was assessed 4 weeks after the end of treatment. All outcome assessors and participants were blinded to group allocation. Patients were screened by a gastroenterologist, and informed consent was obtained after the study was explained. Patients with gastritis or gastric ulcers, aged 18–75 years, and of any gender were eligible for this study if their H. pylori infection was confirmed by rapid urease test, endoscopic observations, and histological results. Patients with acute perforation of the esophagus, stomach, and duodenum, gastric cancer, hyperlipidemia, allergy to egg protein, or use of antibiotics or gastric acid inhibitors (e.g. pantoprazole and famotidine) within 3–4 weeks before enrollment.
Randomization and blinding
Eligible patients were randomly assigned in a 1:1 ratio to either the intervention group (IgY-enriched egg yolk powder) or the control group (plain egg yolk powder). The randomization sequence was generated by an independent statistician using computer-generated random permuted blocks (block size of 4) via the Randomization.com website. The sequence was created before the start of the trial and was not accessible to any member of the study team involved in patient enrollment or outcome assessment.
Allocation concealment was ensured using sequentially numbered, opaque, sealed envelopes. The randomization sequence was printed on cards and placed inside opaque envelopes, which were then sealed and sequentially numbered by an independent administrator not involved in the trial. After a patient provided informed consent and underwent baseline assessments, the enrolling assessor opened the next envelope in sequence to reveal the group assignment. The envelopes could not be read against light, and the seal prevented tampering. The envelope method was combined with off-site generation of the sequence to prevent subversion.
The trial was double-blind. Patients received identical sachets (same size, color, weight, taste, and packaging) containing either IgY-enriched egg yolk powder or plain egg yolk powder.
In addition to the patients, the assessors who performed the urea breath test (UBT) and the telephone interviews for clinical symptoms were unaware of the group allocation. They had no access to the randomization list or to information about which sachet the patient received.
Demographic data (age, sex, underlying diseases, medications, clinical symptoms) and physical examinations were recorded for all patients at the initial visit. Randomization was performed using the sealed envelope method, and randomization codes were provided through the Randomization.com site, regardless of group allocation. The IgY-enriched egg powder was prepared as described in our previous studies, which included in vitro experiments, AGS cell-line assays, and challenge studies in C57/BL6 mice [27, 28]. The intervention group comprised 30 patients, each of whom received 42 packets of egg yolk powder containing IgY for 14 days (3 sachets/day; each 3.5 g of egg yolk sachet provided 15 mg of IgY). The control group received 42 sachets of plain egg yolk powder without IgY for 14 days (3 sachets/day; each 3.5 g of IgY-free egg yolk powder). To blind the participants, the packets in both groups were identical. Both assessors and participants were blinded and unaware of the group division. In addition to the IgY packets, both groups received routine treatment for H. pylori infection, including Omeprazole, Amoxicillin, Clarithromycin (OAC) or omeprazole (40 mg/day), clarithromycin (1000 mg/day), and amoxicillin (2000 mg/day) for 2 weeks. Patients were evaluated for adverse events and clinical symptoms by telephone 3 times weekly for 1 month. Data were recorded in clinical report forms.
Primary and secondary outcomes
The primary outcome was eradication of H. pylori, which assessed by the UBT. The primary endpoint was the proportion of patients achieving H. pylori eradication, defined as a post-treatment 14C-urea breath test (14C-UBT) value below the manufacturer’s cut-off 50 dpm. Eradication was assessed 28 days (4 weeks) after completion of the 14-day treatment period (i.e. 1.5 months after the initial visit as washout period). This timing was chosen to minimize the risk of false-negative results from transient bacterial suppression while avoiding spontaneous clearance or reinfection. Because proton pump inhibitors and antibiotics can transiently suppress H. pylori without achieving true eradication, a 4-week washout period is standard. The present protocol adhered to this recommendation, thereby minimizing the risk of overestimating eradication rates due to early testing. Patients fasted for at least 6 h, then swallowed a capsule containing 14 C-radiolabeled with water, and exhaled into the test kit after 20 min. The secondary outcome included the patients’ clinical symptoms, which were assessed by an interviewer via telephone at baseline and three times weekly for 1 month. The delta over baseline value was measured using an infrared spectrometer. A cut-off value of below 50 disintegrations per minute (dpm)was considered negative for active infection, according to the manufacturer’s (Shenzhen Zhonghe headway Bio-Sci China) cut-off.
Symptom severity was graded using the Gastrointestinal Symptom Rating Scale: abdominal pain: 0 = no symptoms, 1 = mild (tolerable symptoms), 2 = moderate (discomfort interfering with normal activities), 3 = severe (inability to perform normal activities); nausea: 0 = none, 1 = transient, brief episodes, 2 = frequent, prolonged nausea without vomiting, 3 = persistent nausea with frequent vomiting; bloating: 0 = none, 1 = short, temporary bloating, 2 = prolonged but tolerable bloating, 3 = severe flatulence interfering with social functioning. Similar scales were applied for anorexia and burping. A total symptom score was not calculated; each domain was analyzed separately to detect domain-specific effects of IgY. Higher scores indicate worse symptoms. A reduction of ≥1 point from baseline to any follow-up visit was considered clinically meaningful improvement. Although this modified scale has not undergone formal validation in Iran, it is consistent with scoring systems employed in previously published H. pylori trials, and all interviews were conducted using a standardized script to ensure consistency [25, 29].
Safety assessment
Safety was assessed by active questioning for adverse events during each telephone follow-up (three times weekly for 4 weeks). A standardized AE checklist was used, including gastrointestinal symptoms (nausea, vomiting, diarrhea, abdominal pain, constipation), allergic reactions (rash, pruritus, swelling), and any unsolicited symptoms. Adverse events were graded according to the Common Terminology Criteria for Adverse Events version 5.0 as mild (Grade 1), moderate (Grade 2), severe (Grade 3), or life-threatening (Grade 4). Causality was assessed by the study physician as unrelated, unlikely, possible, probable, or definite.
Statistical analysis
The sample size was calculated using G*Power software (version 3.1.9.7) for a two-group chi-square test with a two-sided α of 0.05 and power of 80%. Data analysis was performed using the SPSS statistical software (version 24). The normal distribution test of continuous data was assessed using the Kolmogorov–Smirnov test. Descriptive statistics included means, standard deviations, frequencies. In the case of a normal distribution, parametric statistical methods (t-test, etc.) were used.
A cut-off value of below 50 disintegrations per minute (dpm) was used to indicate the absence of active infection. This cut-off value is recommended by the manufacturer of the UBT kit (Shenzhen Zhonghe headway Bio-Sci). UBT values were analyzed both as continuous variables (using non-parametric tests due to non-normal distribution) and as dichotomous categorical variables (eradication yes/no based on the 50 dpm cut-off) for the primary outcome assessment. The quantitative variable UBT, which is the test value, ranges from zero to high.
For the qualitative analysis, patients were divided into three groups based on the difference between the primary and secondary test results: negative (improvement), positive (no improvement), and zero (no change).
In this study, the data on the patients’ clinical symptoms were evaluated based on their symptom scores and severity on multiple occasions and were statistically analyzed by repeated-measures analysis of variance. A P-value <.05 was considered statistically significant for all analyses.
We did not perform any data imputation for the primary UBT outcome. Instead, we handled the loss to follow-up (5 patients) and the exclusion of 30 patients with negative baseline UBT using intention-to-treat (ITT) analysis with failure imputation for missing data (i.e. missing = failure). For the secondary clinical symptom outcomes, missing data were rare (2% of visits). We conducted complete-case analysis because the proportion was negligible.
The primary analysis was ITT, including all 60 randomized patients. For the primary outcome (H. pylori eradication by UBT), missing data were handled by classifying patients without a post-treatment UBT as treatment failures. A per-protocol analysis was also performed on the 25 patients with complete UBT data. For secondary clinical symptom outcomes, missing data were rare (2% of visits). Given the exploratory nature of secondary outcomes, no adjustment for multiple comparisons was applied; however, a post-hoc Bonferroni correction (α = 0.0125) is reported for transparency. All statistical tests were two-sided, and P < .05 was considered significant for the primary outcome.
Results
In vitro studies on AGS cell line demonstrated that IgY-Hp inhibited H. pylori attachment at a concentration of 13 mg/ml. In addition, in C57/BL6 mice, it showed significant improvement in pathological damages compared with the control group (55.4% vs. 25.1%). These preclinical studies supported the efficacy of IgY, however this improvement was not significant in the clinical phase, contrary to preclinical results [27]. The IgY product has been investigated in terms of source, purity, dose, and stability in previous studies, and it has had appropriate purity and stability [28].
A total of 363 patients were screened; 84 participants were eligible, and 60 were enrolled. However, 30 patients had a negative initial UBT, and 5 were lost to follow-up, so the sample size for the primary outcome or UBT was reduced to 25 patients.
To assess the secondary outcome or improvement in clinical symptoms, 60 patients were enrolled, and statistical analysis was performed on all of them.
The Consolidated Standards of Reporting Trials (CONSORT 2025) flow diagram of patient enrollment and randomization is shown in Fig. 1.
Figure 1.
CONSORT 2025 flow diagram of participant recruitment and follow-up, showing screening of 363 individuals, exclusion of 303, randomization of 60, and attrition due to loss to follow-up in both intervention and control groups.
Demographic characteristics
Statistical analysis of demographic variables showed that these variables, including sex, marital status, nationality, blood type, education level, and occupation, did not differ significantly between the intervention and control groups. So, these variables were homogeneous between the two groups in the study (P > .05). The frequency percentages and P-values of the demographic variables are listed in Table 1. The normal distribution of demographic variables, including age and Body Mass Index (BMI), was examined using the one-sample Kolmogorov–Smirnov test, which showed these variables had a normal distribution. Their statistical analysis based on the parametric t-test also showed that these variables did not differ significantly between the intervention and control groups and were homogeneous (P > .05). According to these data, the age ranged from 19 to 72 years. The BMI also showed that the lowest BMI was 13.3 and the highest was 43.3.
Table 1.
Comparison of demographic characteristics between placebo and intervention groups.
| Variable | Statistical analysis | Intervention group | Placebo group | Total |
|---|---|---|---|---|
| Age (by year) | Mean | 51.00 | 46.90 | – |
| SD | 12.083 | 11.731 | ||
| P value | .188 | |||
| Gender | Male | 9 (30%) | 10 (33.3%) | 19 (31.7%) |
| Female | 21 (70%) | 20 (66.7%) | 41 (68.3%) | |
| P value | >.99 | |||
|
BMI weight (kg)/height (m 2) |
Mean | 26.133 | 26.813 | – |
| SD | 5.8077 | 4.6809 | ||
| P value | .619 | |||
| Blood group | O | 12 (40%) | 6 (20%) | 18 (30%) |
| B | 3 (10%) | 5 (16.7%) | 8 (13.3%) | |
| A | 4 (13.3%) | 5 (16.7%) | 9 (15%) | |
| AB | 1 (3.3%) | 1 (3.3%) | 2 (3.3%) | |
| Unknown | 10 (33.3%) | 13 (43.3%) | 23 (28.3%) | |
| P value | –a | |||
| Rh | Positive | 18 (60%) | 14 (46.7%) | 32 (53.3) |
| Negative | 2 (6.7%) | 3 (10%) | 5 (8.3%) | |
| Unknown | 10 (33.3%) | 13 (43.3%) | 23 (38.3%) | |
| P value | –a | |||
| Education level | Cycle | 21 (70%) | 18 (60%) | 39 (65%) |
| Diploma | 3 (10%) | 9 (30%) | 12 (20%) | |
| Associate degree | 2 (6.7%) | 1 (3.3%) | 3 (50%) | |
| Bachelor | 1 (3.3%) | 0 (0) | 1 (1.7%) | |
| Master's degree | 0 (0) | 0 (0) | 0 (0) | |
| Doctorate | 1 (3.3%) | 1 (3.3%) | 2 (3.3%) | |
| Other | 2 (6.7%) | 1 (3.3%) | 3 (5%) | |
| P value | –a | |||
| Job | Housekeeper | 21 (70%) | 20 (66.7%) | 41 (61.8%) |
| Unemployed | 0 (0) | 0 (0) | 0(0) | |
| Employee | 1 (3.3%) | 1 (3.3%) | 2 (3.3%) | |
| Free | 8 (26.7%) | 9 (30%) | 17 (28.3%) | |
| P value | –a | |||
| Marital status | Single | 2 (6.7%) | 3 (10%) | 5 (8.3%) |
| Married | 28 (93.3%) | 27 (90%) | 55 (91.7%) | |
| P value | >.99 | |||
| Nationality | Non-Iranian | 2 (6.7%) | 1 (3.3%) | 3 (5%) |
| Iranian | 28 (93.3%) | 29 (96.7%) | 57 (95%) | |
| P value | >.99 | |||
aBlank space: Due to the lack of necessary assumptions for performing the chi-square test, there is no P-value.
Primary outcome (urea breath test)
Given that the UBT values were not normally distributed, the Mann–Whitney test was used. Table 2 presents the descriptive statistics (median, quartiles, minimum, and maximum) for UBT values. The median score in the intervention group decreased from 77 at baseline to 11 after treatment, whereas the control group remained stable (median = 55). The next variable analyzed was the change in secondary UBT values after treatment compared to baseline in the intervention and control groups. Comparison of patients’ improvement in three states of improvement, no change, and worsening based on the normal range of UBT showed that only one patient in the control group showed worsening, 5 patients in each group exhibited no change, and 10 patients in the intervention group versus 4 in the control group demonstrated improvement. Although no significant difference was found (Table 3). When “no change” was combined with “worsening,” the improvement rate was 66.7% in the intervention group and 40% in the control group (difference 26.7%; P = .241) based on Pre protocol analysis.
Table 2.
Comparison of changes in primary and secondary UBT thresholds between placebo and intervention groups.
| Group | NoUBT1 | NoUBT2 | ||
|---|---|---|---|---|
| Intervention | Number of patients | 28 | 14 | |
| Mean | 85.32 | 25.93 | ||
| SD | 82.703 | 34.480 | ||
| Minimum | 0 | 0 | ||
| Maximum | 274 | 85 | ||
| Percentiles | 25 | .00 | .00 | |
| 50 | 77.00 | 11.00 | ||
| 75 | 145.75 | 63.75 | ||
| Control | Number | 27 | 9 | |
| Mean | 80.11 | 52.89 | ||
| SD | 95.914 | 58.420 | ||
| Minimum | 0 | 0 | ||
| Maximum | 317 | 166 | ||
| Percentiles | 25 | .00 | .00 | |
| 50 | 55.00 | 55.00 | ||
| 75 | 132.00 | 90.00 | ||
| P value | .714 | .511 | ||
Table 3.
Comparison of primary and secondary UBT changes based on improvement or non-improvement between placebo and intervention groups.
| Treatment | Total | ||||
|---|---|---|---|---|---|
| Intervention | Control | ||||
| Worsening | Count | 0 | 1 | 1 | |
| % | 0.0% | 10.0% | 4.0% | ||
| No change | Count | 5 | 5 | 10 | |
| % | 33.3% | 50.0% | 40.0% | ||
| Improvement | Count | 10 | 4 | 14 | |
| % | 66.7% | 40.0% | 56.0% | ||
| Total | Count | 15 | 10 | 25 | |
Based on ITT analysis in the intervention group (IgY-enriched egg yolk powder, n = 30), 10 patients achieved H. pylori eradication according to the evaluable post-treatment UBT results. The remaining 20 patients (including those with negative baseline UBT or loss to follow-up) were classified as treatment failures. Thus, the ITT eradication rate was 33.3%.
In the control group (plain egg yolk powder, n = 30), 4 patients achieved eradication, and 26 were considered failures, yielding an ITT eradication rate of 13.3%.
The risk difference was 20.0% (95% confidence interval: −0.6% to 39.4%), and Fisher’s exact test gave a two-sided P-value of .11, indicating no statistically significant difference between groups. These ITT results are consistent with the per-protocol findings and support the conclusion that the addition of IgY did not lead to a statistically significant improvement in eradication rates, although a numerical advantage was observed.
Secondary outcome (clinical symptoms)
An examination of the patients’ clinical symptoms, including anorexia, stomach pain, burping and bloating, and nausea in the intervention and control groups, revealed that despite a reduction in the severity of symptoms in the intervention group compared with the control group, no statistically significant differences were observed in symptoms, except for anorexia (P = .048). Given the exploratory nature of secondary outcome analyses, no adjustment for multiple comparisons was applied. Findings for secondary outcomes should be interpreted cautiously, and the significant result for anorexia (P = .048) is considered hypothesis-generating. We performed a post-hoc Bonferroni correction for the four clinical symptoms (adjusted α = 0.0125). After correction, the P-value for anorexia (.048) did not reach statistical significance. The trend of clinical symptom scores illustrated that the symptoms improvement in the intervention group was faster than in the control group. Even nausea scores in the control group during the sixth visit showed an upward trend, unlike the intervention group. However, no significant difference was observed (Figs 2 and 3). No patient in the intervention group discontinued treatment due to adverse events. In the control group, two patients experienced mild nausea (grade 1) that resolved spontaneously without discontinuation of study medication. No allergic reactions to egg proteins were reported in either group.
Figure 2.
Distribution of participants across five severity grades (Grade 0 to Grade 4) at each of six study visits, with left and right panels showing the intervention and control groups, respectively.
Figure 3.
Estimated marginal means for clinical symptom scores in the intervention and control groups across six study visits, with lower scores indicating less severe symptoms and error bars representing 95% confidence intervals.
Discussion
This randomized controlled trial evaluated whether adding anti-H. pylori IgY-enriched egg yolk powder to standard triple therapy improves H. pylori eradication and clinical symptoms compared to plain egg yolk powder. The results show a non-statistically significant trend toward higher eradication rates in the IgY group (33.3% vs. 13.3%; P = .241) and a statistically significant but uncorrected improvement in anorexia (P = .048). These preliminary findings should be interpreted with caution due to the small sample size and multiple comparisons. The results provide several key insights into the intervention’s effects, homogeneity of the study groups, and clinical implications of the findings. The demographic analysis confirmed that the intervention and control groups were well-matched, with no statistically significant differences in variables such as age, sex, BMI, blood type, education level, occupation, marital status, or nationality. This ensured that any observed effects in the study were likely due to the intervention rather than confounding factors. The normal distribution of age and BMI further supports the use of parametric tests for these variables in this study. The UBT results, which served as the primary outcome measure, did not show a statistically significant difference between the intervention and control groups in terms of the mean numerical values. Nevertheless, the median UBT value in the intervention group declined markedly (from 77 to 11), while it remained unchanged in the control group (median 55).
Owing to unforeseen factors during trial execution specifically, 30 of the 60 initially eligible patients had a negative baseline UBT and were excluded from the primary outcome analysis, and an additional 5 patients were lost to follow-up—only 25 patients (n = 15 in intervention, n = 10 in control) had evaluable paired UBT data for the per-protocol analysis. With this effective sample size, the post-hoc power to detect the originally assumed large effect size (Cohen’s h = 0.8) was approximately 25% (calculated using G*Power). Consequently, the non-significant finding for the primary outcome (P = .241) should be interpreted with caution, as the study was underpowered for that analysis. The ITT analysis (all 60 randomized patients, with missing outcomes treated as failures) retained the planned sample size and achieved 85% power under the same effect size assumption, but the actual observed effect size was smaller than anticipated (absolute difference 26.7%, Cohen’s h ≈ 0.55). This discrepancy highlights the need for larger confirmatory trials.
To address sample attrition, we used the ITT analysis in addition to the per-protocol analysis, the results of which are presented in the Table 4. An ITT analysis, considering all 60 randomized participants and classifying dropouts as treatment failures, yielded consistent results, with no statistically significant difference between groups.
Table 4.
Comparison of the primary outcome (UBT) results using per-protocol and ITT analyses.
| Analysis method | Intervention group (recovery rate) | Control group (recovery rate) | Absolute difference | Odds ratio |
|---|---|---|---|---|
| ITT (missing = failure) | 33.3% (10/30) | 13.3% (4/30) | 20.0% | 3.25 |
| Per-protocol | 66.7% (10/15) | 40.0% (4/10) | 26.7% | 3.00 |
Further classification of outcomes into improvement, no change, and worsening revealed that the intervention group had a higher percentage of improvement (33.3%) compared to the control group (13.3%), resulting in a 20% difference between groups (P-value = .241).
A recent review of H. pylori eradication studies across Iran reported that standard triple therapy (PPI, amoxicillin, clarithromycin) has largely fallen to unacceptable eradication rates, often below 70%–80%, due to rising clarithromycin resistance. In contrast, bismuth based quadruple regimens consistently achieved higher per protocol eradication rates, ranging from 70% to 96%, depending on the region and specific drug combination (e.g. 89.6% in Ardabil, 90% in Sari, and up to 96.3% in Isfahan with furazolidone). Our study found that adding anti H. pylori IgY to standard triple therapy yielded a per protocol eradication rate of 66.7% and an ITT rate of 33.3%, the latter being substantially lower than the bismuth-based regimens reported in the literature. However, the marked numerical reduction in median UBT values (from 77 to 11) and the complete absence of adverse events in the IgY group contrast favorably with the frequent gastrointestinal side effects of bismuth quadruple therapy [30].
When compared with the recent Iranian trial by Zamanifard and colleagues (2025) in Arak, our findings show notable similarities and differences. In their study, standard 14-day OAC triple therapy achieved 54% eradication, while adding bismuth sub citrate dramatically increased eradication to 94% (P < .001). In our trial, OAC plus IgY gave a per protocol eradication of 66.7% versus 40.0% for OAC alone, a 26.7% absolute improvement that did not reach significance (P = .241), likely due to small sample size. Thus, the efficacy of IgY adjunctive therapy (66.7%) was substantially lower than that of bismuth quadruple therapy (94%). However, the bismuth regimen was associated with frequent adverse effects (severe bitter taste, nausea, abdominal discomfort) leading to 8% treatment discontinuation, whereas no adverse events occurred in our IgY group. The divergent performance of standard OAC between studies (40% in our control vs. 54% in Zamanifard) probably reflects geographic variability in antibiotic resistance and diagnostic methods (UBT vs. stool antigen). Collectively, while bismuth quadruple therapy remains the most effective option, its side effect burden is considerable. IgY based adjunctive therapy, although not superior in this underpowered study, offers an excellent safety profile and merits further investigation as a well-tolerated alternative or complementary approach [31].
The study by Nan Hao et al in China in 2024 on the treatment of patients for 4 weeks with polyclonal IgY against VacA, HpaA, CagA, and UreB showed that the eradication rate of H. pylori in the intervention group was 50.04%, which was significantly higher compared to the control group (20.85%). On the other hand, in this study, a comparison of the improvement in clinical symptoms of patients in the two intervention and control groups showed that the improvement in the intervention group (61.76%) was greater than that of the control group (30.30%) [24].
Another study by Guo et al. on the treatment of 75 patients for 2 weeks with IgY against UreB, BabA2, and FlaA reported an improvement in clinical symptoms of 87.3%. In this study, the difference in the urea breath test before and after treatment with IgY showed a significant decrease (25.9 ± 14.1 before treatment vs. 11.4 ± 9.78 after treatment, P < .001), and the eradication rate of H. pylori reached 30.6% [25].
Nonetheless, a consistent trend toward faster symptom resolution was observed for all assessed symptoms in the intervention group (Fig. 3). Moreover, symptoms such as nausea and bloating tended to worsen during later follow-up visits in the control group, whereas they continued to improve in the IgY group. These observations, although not statistically significant for most symptoms, suggest a potential clinical benefit.
An ITT analysis, considering all 60 randomized participants and classifying dropouts as treatment failures, yielded consistent results, with no statistically significant difference between groups.
Additionally, side effects were reported in only two patients, both of whom were in the control group. This suggests that the intervention was well tolerated and had no side effects, reinforcing its potential feasibility for wider clinical application.
This trial did not find a statistically significant difference in H. pylori eradication rates between the IgY and control groups. The observed difference of 20 percentage points, while clinically interesting, was not statistically significant, likely due to the small sample size (post-hoc power ~25%). Therefore, these results should be considered exploratory and hypothesis-generating rather than conclusive.
Future studies with larger sample sizes, optimized IgY dosages, and longer follow-up periods are needed to establish the clinical utility of IgY in H. pylori management.
Conclusion
These results contribute to the growing body of evidence on alternative treatments for H. pylori infection and highlight the importance of considering both objective measures (UBT) and subjective symptoms when evaluating therapeutic outcomes. In this underpowered trial, adding anti-H. pylori IgY-enriched egg yolk powder to standard triple therapy did not significantly improve eradication rates. These findings are preliminary and require confirmation in adequately powered, multicenter trials with longer follow-up. IgY was well tolerated with no attributable adverse events. However, due to the small sample size, further studies in patients are needed.
Supplementary Material
Clinical trial registration: The registration number (NCT06973889) corresponds to a publicly accessible registry entry and matches the reported protocol.
Contributor Information
Zahra Esmaeili, Department of Microbiology and Virology, School of Medicine, Mashhad University of Medical Sciences, Mashhad, 9177948564, Iran.
Sara Kamal Shahsavar, Department of Microbiology and Virology, School of Medicine, Mashhad University of Medical Sciences, Mashhad, 9177948564, Iran.
Elham Khosravi, Department of Microbiology and Virology, School of Medicine, Mashhad University of Medical Sciences, Mashhad, 9177948564, Iran.
Hamid Ariannejad, Department of Biotechnology, Ferdowsi University of Mashhad, Mashhad, 9177948974, Iran.
Mohammadreza Nassiri, Department of Agriculture, Ferdowsi University of Mashhad, Mashhad, 9177948978, Iran.
Ali Beheshti Namdar, Department of Gastroenterology and Hepatology, Faculty of Medicine, Mashhad University of Medical Sciences, Mashhad, 91778 99191, Iran.
Mitra Ahadi, Department of Gastroenterology and Hepatology, Faculty of Medicine, Mashhad University of Medical Sciences, Mashhad, 91778 99191, Iran.
Ramin Sadeghi, Department of Nuclear Medicine, Faculty of Medicine, Mashhad University of Medical Sciences, Mashhad, 9177948564, Iran.
Vahid Reza Dabbagh, Department of Nuclear Medicine, Faculty of Medicine, Mashhad University of Medical Sciences, Mashhad, 9177948564, Iran.
Faezeh Sabet Birjandi, Department of Microbiology and Virology, School of Medicine, Mashhad University of Medical Sciences, Mashhad, 9177948564, Iran.
Majid Khadem Rezaiyan, Department of Community Medicine, Faculty of Medicine, Mashhad University of Medical Sciences, Mashhad, 913791-3316, Iran.
Kiarash Ghazvini, Department of Microbiology and Virology, School of Medicine, Mashhad University of Medical Sciences, Mashhad, 9177948564, Iran.
Acknowledgments
Not applicable.
Author contributions
Zahra Esmaeili (Investigation [lead], Methodology [lead], Writing—original draft [equal], Writing—review & editing [equal]), Sara Kamal shahsavar (Data curation [equal], Methodology [equal]), Elham Khosravi (Investigation [equal], Methodology [equal]), Hamid Ariannejad (Data curation [equal], Funding acquisition [equal]), Mohammadreza Nassiri (Investigation [equal], Methodology [equal], Visualization [equal]), Ali Beheshti Namdar (Investigation [equal], Methodology [equal]), Mitra Ahadi (Validation [equal], Visualization [equal]), Ramin Sadeghi (Resources [equal], Writing—original draft [equal]), Vahid Reza Dabbagh (Resources [equal], Software [equal]), Faezeh Sabet Birjandi (Software [equal]), Majid Khadem Rezaiyan (Formal analysis [equal], Software [equal]), and Kiarash Ghazvini (Conceptualization [equal], Funding acquisition [equal], Project administration [lead])
Conflicts of interest
The authors declare that they have no competing conflicts or interests.
Funding
This project was financially supported by the Vice-Chancellor for Research of the Mashhad University of Medical Science (MUMS).
Data availability
The data supporting the findings of this study are available upon request from the corresponding author.
Ethics and consent statement
The authors declare that this research (NCT06973889) was approved by Mashhad university of medical sciences ethics committee. All procedures involving human participants were conducted in accordance with the ethical standards of Iran and the Declaration of Helsinki (1964), as revised in 2013. The authors confirm that informed consent was obtained from all patients before the start of the study. The study protocol was approved by the Mashhad university of medical sciences ethics committee (Approval No. IR.MUMS.REC.1401.381; 7 February 2023).
Animal research statement
Not applicable.
Permission to reproduce material from other sources
Not applicable.
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Associated Data
This section collects any data citations, data availability statements, or supplementary materials included in this article.
Supplementary Materials
Data Availability Statement
The data supporting the findings of this study are available upon request from the corresponding author.