Associations of Helicobacter pylori infection with NAFLD, MAFLD, MASLD, and Chinese MAFLD

Abstract

Background:

The association of Helicobacter pylori (H. pylori) infection with non-alcoholic fatty liver disease (NAFLD) remains controversial. Additionally, its associations with metabolic dysfunction-associated fatty liver disease (MAFLD), metabolic dysfunction-associated steatotic liver disease (MASLD), and Chinese MAFLD using the most recent diagnostic criteria were unclear.

Objective:

To analyze the associations of H. pylori infection with NAFLD, MAFLD, MASLD, and Chinese MAFLD.

Design:

Cross-sectional study.

Methods:

This study screened 1172 inpatients who underwent both H. pylori test and liver computed tomography or ultrasound examination between June 2020 and May 2024. Multivariate Logistic regression analyses were performed to evaluate the associations of H. pylori infection with the severity of hepatic steatosis and risk of hepatic fibrosis. Adjusted odds ratios (aORs) with 95% confidence intervals (CIs) were calculated after adjusting for gender, hypertension, hyperlipidemia, body mass index (BMI), fasting plasma glucose (FPG), and high-sensitivity C-reactive protein (HsCRP) in NAFLD analyses; age, gender, drinking, hypertension, diabetes, hyperlipidemia, BMI, and HsCRP in MAFLD analyses; and gender, drinking, hypertension, hyperlipidemia, BMI, FPG, and HsCRP in MASLD and Chinese MAFLD analyses.

Results:

Overall, 875, 982, 869, and 869 patients were included in NAFLD, MAFLD, MASLD, and Chinese MAFLD analyses, respectively. In NAFLD, MAFLD, MASLD, and Chinese MAFLD analyses, 257, 302, 257, and 257 patients had H. pylori infection, respectively. H. pylori infection was independently associated with severe hepatic steatosis in NAFLD (aOR = 3.956; 95% CI = 1.171–13.359, p = 0.027), MAFLD (aOR = 3.730; 95% CI = 1.083–12.850, p = 0.037), and MASLD (aOR = 3.962; 95% CI = 1.158–13.557, p = 0.028) analyses, but not Chinese MAFLD analyses. However, H. pylori infection was not independently associated with the risk of hepatic fibrosis.

Conclusion:

H. pylori infection may increase the severity of NAFLD, MAFLD, and MASLD, but not the risk of liver fibrosis.

Introduction

Helicobacter pylori (H. pylori) is a kind of helical shape gram-negative bacterium that colonizes human gastric epithelium and generates many bacterial virulence factors, such as cytotoxin-associated gene A and vacuolating cytotoxin A. ¹ It can be widely spread via oral-oral and fecal-oral routes in the population. The prevalence of H. pylori infection varies across regions. Despite a downward trend in the global prevalence rate of H. pylori infection, it is still as high as 43.1%. ² As we all know, H. pylori infection has been confirmed to be closely associated with many gastric diseases, primarily including chronic gastritis, peptic ulcer, gastric mucosa-associated lymphoid tissue lymphoma, and even gastric cancer. ³ It may also play a certain role in the occurrence and development of diverse extra-gastric diseases, such as reflux esophagitis, inflammatory bowel disease, cholecystitis, diabetes mellitus, coronary heart disease, Alzheimer’s disease, multiple sclerosis, Parkinson’s syndrome, and asthma.^{4 –6}

Non-alcoholic fatty liver disease (NAFLD) is defined by the presence of steatosis in >5% of hepatocytes, in the absence of other causes of hepatic steatosis, such as significant alcohol intake, long-term use of a steatogenic medication, or monogenic hereditary disorders.^7,8 It has become the most common chronic liver disease, and its global prevalence has increased from 24.4% in 1991–2006 to 36.0% in 2016–2020, contributing to an enormous economic and clinical burden. ⁹ Accordingly, it is of great significance to identify the risk factors for NAFLD. Multiple mechanisms are involved in the pathogenesis of NAFLD, such as insulin resistance (IR), changes of gene polymorphism, abnormal immune microenvironment of the liver, and changes in the composition and structure of gut microbiota. ¹⁰ H. pylori infection is associated with inflammation and immune system responses, which may cause IR, further increasing the risk of NAFLD. ¹¹ Since Cindoruk et al. ¹² initially identified H. pylori 16s rDNA in liver samples of NAFLD patients in 2008, the association between H. pylori infection and NAFLD has been successively explored. However, the conclusions remain inconsistent.^{13 –16} A previous meta-analysis by our group showed a weak association between H. pylori infection and NAFLD, but emphasized some limitations from the current evidence. ¹⁷ Therefore, it was necessary to further clarify their association.

NAFLD has been renamed as metabolic dysfunction-associated fatty liver disease (MAFLD) in 2020 and metabolic dysfunction-associated steatotic liver disease (MASLD) in 2023.^18,19 More recently, the Chinese MAFLD definition has also been proposed in 2024. ²⁰ Notably, their diagnostic criteria are a bit different. Unlike NAFLD, all of the three new definitions (MAFLD, MASLD, and Chinese MAFLD) included metabolic-related indicators, such as body mass index (BMI), serum lipid, and blood glucose. Besides, it was unnecessary to diagnose MAFLD by excluding other causes of liver disease or significant alcohol intake. Until now, no study has investigated the associations of H. pylori infection with MAFLD, MASLD, and Chinese MAFLD.

This cross-sectional study aimed to analyze the associations of H. pylori infection with NAFLD, MAFLD, MASLD, and Chinese MAFLD.

Methods

The reporting of this study conforms to the Strengthening the Reporting of Observational Studies in Epidemiology (STROBE) statement. ²¹

Study design

We consecutively reviewed the medical records of 1172 patients who were admitted to the Department of Gastroenterology of the General Hospital of Northern Theater Command and underwent both H. pylori detection and liver computed tomography (CT) or ultrasound examination between June 1, 2020 and May 31, 2024. Exclusion criteria were as follows: (1) age <18 years; (2) repeat admissions; (3) undefined H. pylori infection status; (4) use of proton pump inhibitors, H₂ blockers within the prior 2 weeks or bismuth, antibiotics within the prior 4 weeks; (5) history of cancer; (6) history of gastric surgery; and (7) severe mental or neurological disorders. In the analyses regarding association of H. pylori infection with NAFLD, there were other exclusion criteria, as follows: (1) history of significant alcohol intake; (2) history of chronic liver disease, such as chronic hepatitis B, chronic hepatitis C, and autoimmune liver disease; and (3) use of steatogenic medications, such as methotrexate and corticosteroids, among others. In the analyses regarding the association of H. pylori infection with MAFLD, the patients were further excluded, if all key indicators for diagnosing MAFLD [i.e., BMI, diabetes, fasting plasma glucose (FPG), glycosylated hemoglobin (HbA1c), hypertension, blood pressure, triglyceride (TG), high-density lipoprotein cholesterol (HDL-C), history of specific lipid-lowering drug treatment, and high-sensitivity C-reactive protein (HsCRP)] were missing. In the analyses regarding association of H. pylori infection with MASLD, there were other exclusion criteria, as follows: (1) history of significant alcohol intake; (2) history of chronic liver disease; (3) use of steatogenic medications; and (4) absence of all key indicators for diagnosing MASLD (i.e., BMI, diabetes, FPG, HbA1c, hypertension, blood pressure, TG, HDL-C, and history of specific lipid-lowering drug treatment). In the analyses regarding association of H. pylori infection with Chinese MAFLD, there were other exclusion criteria, as follows: (1) history of significant alcohol intake; (2) history of chronic liver disease; (3) use of steatogenic medications; and (4) absence of all key indicators for diagnosing Chinese MAFLD (i.e., BMI, diabetes, FPG, HbA1c, hypertension, blood pressure, TG, HDL-C, and history of specific lipid-lowering drug treatment).

Data collection

We collected the baseline data (i.e., age, gender, height, weight, smoking, drinking, systolic blood pressure, and diastolic blood pressure), underlying diseases (i.e., hypertension, diabetes, coronary heart disease, and hyperlipidemia), laboratory parameters [i.e., white blood cell count (WBC), red blood cell count, hemoglobin, platelet count (PLT), total bilirubin, direct bilirubin, alanine aminotransferase (ALT), aspartate aminotransferase (AST), alkaline phosphatase, gamma-glutamyltransferase (GGT), serum albumin (Scr), blood urea nitrogen, serum creatinine, FPG, HbA1c, homocysteine, total cholesterol, TG, HDL-C, low density lipoprotein cholesterol, and HsCRP], fatty liver related indicators (i.e., presence and absence of fatty liver, severity of hepatic steatosis, and risk of hepatic fibrosis), and H. pylori infection status (i.e., negative or positive). BMI was calculated according to the following formula: BMI = weight (kg)/height² (m²). ²²

Diagnosis and group

Diagnosis of NAFLD

According to the guidelines published in 2016 by the European Association for the Study of the Liver (EASL), European Association for the Study of Diabetes (EASD), and European Association for the Study of Obesity (EASO), ⁷ all of the following conditions should be met to establish the diagnosis of NAFLD in our study: (1) hepatic steatosis detected on liver CT or ultrasound; (2) no significant alcohol intake (daily alcohol intake <30 g for men and <20 g for women); and (3) no coexisting causes of chronic liver disease.

Diagnosis of MAFLD

According to the international expert consensus published in 2020, ¹⁸ except for hepatic steatosis detected on liver CT or ultrasound, one of the following criteria should also be met to establish the diagnosis of MAFLD in our study: (1) overweight/obesity (defined as BMI ⩾23 kg/m²); (2) type 2 diabetes; and (3) lean/normal weight (defined as BMI <23 kg/m²) with at least two of the five following criteria: 1) blood pressure ⩾130/85 mmHg or specific drug treatment; 2) TG ⩾1.70 mmol/L or specific drug treatment; 3) HDL-C <1.0 mmol/L for men and <1.3 mmol/L for women or specific drug treatment; 4) prediabetes (i.e., FPG level 5.6–6.9 mmol/L or HbA1c 5.7%–6.4%); and 5) plasma HsCRP level >2 mg/L.

Diagnosis of MASLD

According to the guidelines published in 2024 by the EASL-EASD-EASO, ²³ all of the following conditions should be met to establish the diagnosis of MASLD in our study: (1) hepatic steatosis detected by liver CT or ultrasound; (2) no significant alcohol intake (history of alcohol consumption <20 g/day in women and <30 g/day in men); (3) no other cause of steatosis; and (4) meeting at least one of five cardiometabolic criteria: 1) BMI ⩾23 kg/m²; 2) FPG ⩾5.6 mmol/L or HbA1c ⩾5.7% or type 2 diabetes or treatment for type 2 diabetes; 3) blood pressure ⩾130/85 mmHg or specific antihypertensive drug treatment; 4) TG ⩾1.70 mmol/L or lipid-lowering treatment; and 5) HDL-C ⩽1.0 mmol/L for men and ⩽1.3 mmol/L for women or lipid-lowering treatment.

Diagnosis of Chinese MAFLD

According to the guidelines published in 2024 by the Chinese Society of Hepatology of the Chinese Medical Association, ²⁰ all of the following conditions should be met to establish the diagnosis of Chinese MAFLD in our study: (1) hepatic steatosis detected by liver CT or ultrasound; (2) no significant alcohol intake; (3) no other cause of steatosis; and (4) meeting at least one of five risk factors for metabolic cardiovascular disease: 1) BMI ⩾24 kg/m²; 2) FPG ⩾6.1 mmol/L or HbA1c ⩾5.7% or type 2 diabetes; 3) blood pressure ⩾130/85 mmHg or specific antihypertensive drug treatment; 4) TG ⩾1.70 mmol/L or lipid-lowering treatment; and 5) HDL-C ⩽1.0 mmol/L for men and ⩽1.3 mmol/L for women or lipid-lowering treatment.

Diagnosis of H. pylori infection

H. pylori infection would be diagnosed if one of the following criteria was met: (1) positive ¹³C/¹⁴C urea breath test; (2) positive fecal antigen test; and (3) positive serum antibody test without previous eradication therapy.^14,24 Patients would be assigned to the H. pylori positive group if they were diagnosed with H. pylori infection; otherwise, patients would be assigned to the H. pylori negative group.

Severity of hepatic steatosis

Severity of hepatic steatosis was evaluated by hepatic attenuation divided by splenic attenuation (CT_L/S). The attenuation value of the liver (CT_L) was measured by averaging the Hounsfield units (HU) of three circular regions of interest (ROIs) with a size of 1.5 ± 0.05 cm² in the liver. The attenuation value of the spleen (CT_S) was measured by averaging HU values of two circular ROIs with a size of 1.5 ± 0.05 cm² in the spleen, avoiding vessels, hepatic ducts, calcifications, and artifacts. CT_L/S was calculated according to the following formula: CT_L/S = CT_L/CT_S. According to the CT_L/S, the severity of hepatic steatosis was divided into mild (0.7 < CT_L/S ⩽ 1), moderate (0.5 < CT_L/S ⩽ 0.7), and severe (CT_L/S ⩽ 0.5) ²⁵ (Figure 1).

Figure 1.

CT images of severity of hepatic steatosis. (a) A CT image of mild hepatic steatosis showing that CT_L (46.5 HU) was higher than CT_S (49.9 HU), and the CT_L/S was 0.9; (b) A CT image of moderate hepatic steatosis showing that CT_L (28.0 HU) was lower than CT_S (50.6 HU), and the CT_L/S was 0.6; (c) A CT image of severe hepatic steatosis showing that CT_L (3.5 HU) was remarkably lower than CT_S (42.8HU), and the CT_L/S was 0.1.

CT, computed tomography; CT_L, the attenuation value of the liver; CT_L/S, hepatic attenuation divided by splenic attenuation; CT_S, the attenuation value of the spleen; HU, hounsfield units.

Risk of hepatic fibrosis

Risk of hepatic fibrosis was evaluated by the fibrosis-4 (FIB-4) index. FIB-4 index was calculated using the following formula: age (years) × AST (U/L)/[(PLT (10⁹/L) × ALT (U/L)]^(1/2). The risk of liver fibrosis was stratified as low (FIB-4 index <1.3), intermediate (1.3⩽ FIB-4 index ⩽2.67), and high (FIB-4 index >2.67) for age <65 years; and that was stratified as low (FIB-4 index <2.0), intermediate (2.0 ⩽ FIB-4 index ⩽2.67), and high (FIB-4 index >2.67) for age ⩾65 years.^{8,23,26 –28}

Statistical analyses

Continuous variables were expressed as mean ± standard deviation and median (range), and compared by the nonparametric Mann–Whitney U test or the independent-sample t-test. Categorical variables were expressed as frequency (percentage), and compared by the chi-square test or Fisher’s exact tests. Severity of hepatic steatosis and risk of hepatic fibrosis were compared between H. pylori negative and positive groups. Logistic regression analyses were performed to evaluate the associations of H. pylori infection with the severity of hepatic steatosis and risk of hepatic fibrosis. Crude odds ratios (cORs) with 95% confidence intervals (CIs) were calculated in univariate analyses. Adjusted odds ratios (aORs) with 95% CIs were calculated after adjusting for the factors that are statistically significant in the univariate regression analyses and potentially affect the occurrence of NAFLD/MAFLD/MASLD/Chinese MAFLD. All statistical analyses were performed using SPSS 27.0 software (IBM Corp, Armonk, NY, USA), and a two-tailed p-value <0.05 was considered statistically significant.

Results

NAFLD analyses

A total of 875 patients were included (Figure 2). Their mean age was 55.63 ± 13.22 years old, and 447 patients (51.1%) were female. Among them, 24.8% (217/875) had hypertension, 11.4% (100/875) diabetes, 9.7% (85/875) coronary heart disease, 30.9% (258/836) hyperlipidemia, and 25.9% (227/875) NAFLD. The prevalence of H. pylori infection was 29.4% (257/875). In NAFLD patients, 71.2% (99/139), 18.0% (25/139), and 10.8% (15/139) had mild, moderate, and severe hepatic steatosis, respectively; and 71.6% (159/222), 26.1% (58/222), and 2.3% (5/222) had low-, intermediate-, and high-risk hepatic fibrosis, respectively (Supplemental Table 1).

Figure 2.

Flowchart of patients’ enrollment.

CT, computed tomography; H. pylori, Helicobacter pylori; MAFLD, metabolic-associated fatty liver disease; MASLD, metabolic dysfunction-associated steatotic liver disease; NAFLD, non-alcoholic fatty liver disease.

Compared with the H. pylori negative group, the H. pylori positive group had significantly higher levels of Scr (p = 0.020), FPG (p = 0.021), and HsCRP (p = 0.044), but a lower proportion of female (p = 0.016). The prevalence of NAFLD (p = 0.956) and the proportions of mild (p = 0.724), moderate (p = 0.402), and severe (p = 0.121) hepatic steatosis and low- (p = 0.299), intermediate- (p = 0.210), and high-risk (p = 0.628) hepatic fibrosis were not significantly different between H. pylori positive and negative groups (Supplemental Table 2).

Univariate logistic regression analyses showed that H. pylori infection was not significantly associated with NAFLD (cOR = 1.009, p = 0.956), mild (cOR = 1.261, p = 0.310), moderate (cOR = 0.937, p = 0.887), and severe (cOR = 2.755, p = 0.053) hepatic steatosis, and low- (cOR = 1.074, p = 0.711), intermediate- (cOR = 0.696, p = 0.268), and high-risk (cOR = 1.607, p = 0.605) hepatic fibrosis. After adjusting for gender, hypertension, hyperlipidemia, BMI, FPG, and HsCRP (Supplemental Table 9), multivariate logistic regression analyses showed that H. pylori infection was independently associated with higher odds of severe hepatic steatosis (aOR = 3.956, p = 0.027), but not NAFLD (aOR = 0.917, p = 0.708), mild (aOR = 1.113, p = 0.739) and moderate (aOR = 0.703, p = 0.570) hepatic steatosis, and low- (aOR = 1.026, p = 0.923), intermediate- (aOR = 0.613, p = 0.255), and high-risk (aOR = 2.486, p = 0.552) hepatic fibrosis (Table 1).

Table 1.

Logistic regression analyses of association between H. pylori infection and NAFLD.

Comparisons	H. pylori negative	H. pylori positive	Univariate analyses		Multivariate analyses*
			Crude OR (95% CI)	p-Value	Adjusted OR (95% CI)	p-Value
NAFLD versus absence of NAFLD	160 versus 458	67 versus 190	1.009 (0.725–1.406)	0.956	0.917 (0.583–1.442)	0.708
Degree of hepatic steatosis by CTL/S
Mild hepatic steatosis versus absence of NAFLD	65 versus 458	34 versus 190	1.261 (0.806–1.973)	0.310	1.113 (0.595–2.081)	0.739
Moderate hepatic steatosis versus absence of NAFLD	18 versus 458	7 versus 190	0.937 (0.385–2.281)	0.887	0.703 (0.209–2.369)	0.570
Severe hepatic steatosis versus absence of NAFLD	7 versus 458	8 versus 190	2.755 (0.985–7.704)	0.053	3.956 (1.171–13.359)	0.027
Risk of hepatic fibrosis by FIB-4
Low-risk hepatic fibrosis versus absence of NAFLD	110 versus 458	49 versus 190	1.074 (0.737–1.565)	0.711	1.026 (0.617–1.706)	0.923
Intermediate-risk hepatic fibrosis versus absence of NAFLD	45 versus 458	13 versus 190	0.696 (0.367–1.321)	0.268	0.613 (0.264–1.423)	0.255
High-risk hepatic fibrosis versus absence of NAFLD	3 versus 458	2 versus 190	1.607 (0.266–9.694)	0.605	2.486 (0.123–50.113)	0.552

^*Adjusting for gender, hypertension, hyperlipidemia, BMI, FPG, HsCRP.

We bold values/text to emphasize the key points.

BMI, body mass index; CI, confidence interval; CT_L/S, hepatic attenuation divided by splenic attenuation; FIB-4, fibrosis-4; FPG, fasting plasma glucose; H. pylori, Helicobacter pylori; HsCRP, high-sensitivity C-reactive protein; NAFLD, non-alcoholic fatty liver disease; OR, odd ratio.

MAFLD analyses

A total of 982 patients were included (Figure 2). Their mean age was 56.08 ± 12.80 years old, and 460 patients (46.8%) were female. Among them, 25.7% (252/982) of patients had hypertension, 12.4% (122/982) diabetes, 9.7% (95/982) coronary heart disease, 30.3% (285/941) hyperlipidemia, and 22.0% (216/982) MAFLD. The prevalence of H. pylori infection was 30.8% (302/982). In MAFLD patients, 71.1% (91/128), 17.2% (22/128), and 11.7% (15/128) had mild, moderate, and severe hepatic steatosis, respectively; and 71.4% (152/213), 25.8% (55/213), and 2.8% (6/213) had low-, intermediate-, and high-risk hepatic fibrosis, respectively (Supplemental Table 3).

Compared with the H. pylori negative group, the H. pylori positive group had significantly higher proportions of smoking (p = 0.018) and drinking (p = 0.040), levels of WBC (p = 0.020), GGT (p = 0.014), Scr (p = 0.032), and FPG (p = 0.028), but lower proportions of female (p = 0.002). The prevalence of MAFLD (p = 0.668) and the proportions of mild (p = 0.685), moderate (p = 0.395), and severe (p = 0.117) hepatic steatosis and low- (p = 0.698), intermediate- (p = 0.438), and high-risk (p = 0.381) hepatic fibrosis were not significantly different between H. pylori positive and negative groups (Supplemental Table 4).

Univariate logistic regression analyses showed that H. pylori infection was not significantly associated with MAFLD (cOR = 1.074, p = 0.668), mild (cOR = 1.182, p = 0.476), moderate (cOR = 0.858, p = 0.752), and severe (cOR = 2.614, p = 0.066) hepatic steatosis, and low- (cOR = 1.088, p = 0.657), intermediate- (cOR = 0.858, p = 0.624), and high-risk (cOR = 2.288, p = 0.313) hepatic fibrosis. After adjusting for age, gender, drinking, hypertension, diabetes, hyperlipidemia, BMI, and HsCRP (Supplemental Table 10), multivariate logistic regression analyses showed that H. pylori infection was independently associated with higher odds of severe hepatic steatosis (aOR = 3.730, p = 0.037), but not MAFLD (aOR = 0.963, p = 0.860), mild (aOR = 0.976, p = 0.937) and moderate (aOR = 0.765, p = 0.636) hepatic steatosis, and low- (aOR = 0.972, p = 0.909), intermediate- (aOR = 0.812, p = 0.572), and high-risk (aOR = 0.208, p = 0.455) hepatic fibrosis (Table 2).

Table 2.

Logistic regression analyses of association between H. pylori infection and MAFLD.

Comparisons	H. pylori negative	H. pylori positive	Univariate analyses		Multivariate analyses*
			Crude OR (95% CI)	p-Value	Adjusted OR (95% CI)	p-Value
MAFLD versus absence of MAFLD	147 versus 533	69 versus 233	1.074 (0.776–1.486)	0.668	0.963 (0.633–1.466)	0.860
Degree of hepatic steatosis by CTL/S
Mild hepatic steatosis versus absence of MAFLD	60 versus 533	31 versus 233	1.182 (0.746–1.872)	0.476	0.976 (0.536–1.777)	0.937
Moderate hepatic steatosis versus absence of MAFLD	16 versus 533	6 versus 233	0.858 (0.331–2.220)	0.752	0.765 (0.253–2.314)	0.636
Severe hepatic steatosis versus absence of MAFLD	7 versus 533	8 versus 233	2.614 (0.937–7.294)	0.066	3.730 (1.083–12.850)	0.037
Risk of hepatic fibrosis by FIB-4
Low-risk hepatic fibrosis versus absence of MAFLD	103 versus 533	49 versus 233	1.088 (0.749–1.581)	0.657	0.972 (0.598–1.580)	0.909
Intermediate-risk hepatic fibrosis versus absence of MAFLD	40 versus 533	15 versus 233	0.858 (0.465–1.584)	0.624	0.812 (0.394–1.673)	0.572
High-risk hepatic fibrosis versus absence of MAFLD	3 versus 533	3 versus 233	2.288 (0.458–11.418)	0.313	0.208 (0.003–12.803)	0.455

^*Adjusting for age, gender, drinking, hypertension, diabetes, hyperlipidemia, BMI, HsCRP.

We bold values/text to emphasize the key points.

BMI, body mass index; CI, confidence interval; CT_L/S, hepatic attenuation divided by splenic attenuation; FIB-4, fibrosis-4; H. pylori, Helicobacter pylori; HsCRP, high-sensitivity C-reactive protein; MAFLD, metabolic-associated fatty liver disease; OR, odd ratio.

MASLD analyses

A total of 869 patients were included (Figure 2). Their mean age was 55.67 ± 13.24 years old, and 443 patients (51.0%) were female. Among them, 25.0% (217/869) of patients had hypertension, 11.5% (100/869) diabetes, 9.8% (85/869) coronary heart disease, 31.0% (258/831) hyperlipidemia, and 25.2% (219/869) MASLD. The prevalence of H. pylori infection was 29.6% (257/869). In MASLD patients, 71.1% (96/135), 17.8% (24/135), and 11.1% (15/135) had mild, moderate, and severe hepatic steatosis, respectively; and 72.1% (155/215), 25.6% (55/215), and 2.3% (5/215) had low-, intermediate-, and high-risk hepatic fibrosis, respectively (Supplemental Table 5).

Compared with the H. pylori negative group, the H. pylori positive group had significantly higher levels of Scr (p = 0.023), FPG (p = 0.027), and HsCRP (p = 0.038), but lower proportions of female (p = 0.017). The prevalence of MASLD (p = 0.702) and the proportions of mild (p = 0.739), moderate (p = 0.423), and severe (p = 0.146) hepatic steatosis and low- (p = 0.342), intermediate- (p = 0.249), and high-risk (p = 0.635) hepatic fibrosis were not significantly different between H. pylori positive and negative groups (Supplemental Table 6).

Univariate logistic regression analyses showed that H. pylori infection was not significantly associated with MASLD (cOR = 1.067, p = 0.702), mild (cOR = 1.328, p = 0.218), moderate (cOR = 0.997, p = 0.995), and severe (cOR = 2.767, p = 0.052) hepatic steatosis, and low- (cOR = 1.119, p = 0.560), intermediate- (cOR = 0.749, p = 0.380), and high-risk (cOR = 1.614, p = 0.602) hepatic fibrosis. After adjusting for gender, drinking, hypertension, hyperlipidemia, BMI, FPG, and HsCRP (Supplemental Table 11), multivariate logistic regression analyses showed that H. pylori infection was independently associated with higher odds of severe hepatic steatosis (aOR = 3.962, p = 0.028), but not MASLD (aOR = 0.962, p = 0.868), mild (aOR = 1.145, p = 0.674) and moderate (aOR = 0.757, p = 0.655) hepatic steatosis, and low- (aOR = 1.093, p = 0.736), intermediate- (aOR = 0.586, p = 0.219), and high-risk (aOR = 2.124, p = 0.622) hepatic fibrosis (Table 3).

Table 3.

Logistic regression analyses of association between H. pylori infection and MASLD.

Comparisons	H. pylori negative	H. pylori positive	Univariate analyses		Multivariate analyses*
			Crude OR (95% CI)	p-Value	Adjusted OR (95% CI)	p-Value
MASLD versus absence of MASLD	152 versus 460	67 versus 190	1.067 (0.765–1.490)	0.702	0.962 (0.610–1.518)	0.868
Degree of hepatic steatosis by CTL/S
Mild hepatic steatosis versus absence of MASLD	62 versus 460	34 versus 190	1.328 (0.846–2.085)	0.218	1.145 (0.609–2.155)	0.674
Moderate hepatic steatosis versus absence of MASLD	17 versus 460	7 versus 190	0.997 (0.407–2.443)	0.995	0.757 (0.223–2.566)	0.655
Severe hepatic steatosis versus absence of MASLD	7 versus 460	8 versus 190	2.767 (0.989–7.738)	0.052	3.962 (1.158–13.557)	0.028
Risk of hepatic fibrosis by FIB-4
Low-risk hepatic fibrosis versus absence of MASLD	106 versus 460	49 versus 190	1.119 (0.767–1.634)	0.560	1.093 (0.652–1.832)	0.736
Intermediate-risk hepatic fibrosis versus absence of MASLD	42 versus 460	13 versus 190	0.749 (0.393–1.428)	0.380	0.586 (0.250–1.375)	0.219
High-risk hepatic fibrosis versus absence of MASLD	3 versus 460	2 versus 190	1.614 (0.268–9.737)	0.602	2.124 (0.106–42.543)	0.622

^*Adjusting for gender, drinking, hypertension, hyperlipidemia, BMI, FPG, HsCRP.

We bold values/text to emphasize the key points.

BMI, body mass index; CI, confidence interval; CT_L/S, hepatic attenuation divided by splenic attenuation; FIB-4, fibrosis-4; FPG, fasting plasma glucose; H. pylori, Helicobacter pylori; HsCRP, high-sensitivity C-reactive protein; MASLD, metabolic dysfunction-associated steatotic liver disease; OR, odds ratio.

Chinese MAFLD analyses

A total of 869 patients were included (Figure 2). Their mean age was 55.67 ± 13.24 years old, and 443 patients (51.0%) were female. Among them, 25.0% (217/869) of patients had hypertension, 11.5% (100/869) diabetes, 9.8% (85/869) coronary heart disease, 31.0% (258/831) hyperlipidemia, and 24.7% (215/869) Chinese MAFLD. The prevalence of H. pylori infection was 29.6% (257/869). In Chinese MAFLD patients, 71.4% (95/133), 18.0% (24/133), and 10.5% (14/133) had mild, moderate, and severe hepatic steatosis, respectively; and 72.0% (152/211), 25.6% (54/211), and 2.4% (5/211) had low-, intermediate-, and high-risk hepatic fibrosis, respectively (Supplemental Table 7).

Compared with the H. pylori negative group, patients in the H. pylori positive group had significantly higher levels of Scr (p = 0.023), FPG (p = 0.027), and HsCRP (p = 0.038), but lower proportions of female (p = 0.017). The prevalence of Chinese MAFLD (p = 0.677) and the proportions of mild (p = 0.909), moderate (p = 0.435), and severe (p = 0.252) hepatic steatosis and low- (p = 0.381), intermediate- (p = 0.282), and high-risk (p = 0.636) hepatic fibrosis were not significantly different between H. pylori positive and negative groups (Supplemental Table 8).

Univariate logistic regression analyses showed that H. pylori infection was not significantly associated with Chinese MAFLD (cOR = 1.074, p = 0.677), mild (cOR = 1.351, p = 0.192), moderate (cOR = 0.998, p = 0.997), and severe (cOR = 2.424, p = 0.102) hepatic steatosis, and low- (cOR = 1.119, p = 0.564), intermediate- (cOR = 0.769, p = 0.425), and high-risk (cOR = 1.616, p = 0.601) hepatic fibrosis. After adjusting for gender, drinking, hypertension, hyperlipidemia, BMI, FPG, and HsCRP (Supplemental Table 12), multivariate logistic regression analyses still showed that H. pylori infection was not independently associated with Chinese MAFLD (aOR = 0.967, p = 0.886), mild (aOR = 1.195, p = 0.584), moderate (aOR = 0.760, p = 0.659), and severe (aOR = 3.232, p = 0.073) hepatic steatosis, and low- (aOR = 1.083, p = 0.766), intermediate- (aOR = 0.604, p = 0.249), and high-risk (aOR = 2.129, p = 0.621) hepatic fibrosis (Table 4).

Table 4.

Logistic regression analyses of association between H. pylori infection and Chinese MAFLD.

Comparisons	H. pylori negative	H. pylori positive	Univariate analyses		Multivariate analyses*
			Crude OR (95% CI)	p-Value	Adjusted OR (95% CI)	p-Value
Chinese MAFLD versus absence of Chinese MAFLD	149 versus 463	66 versus 191	1.074 (0.768–1.501)	0.677	0.967 (0.607–1.538)	0.886
Degree of hepatic steatosis by CTL/S
Mild hepatic steatosis versus absence of Chinese MAFLD	61 versus 463	34 versus 191	1.351 (0.860–2.123)	0.192	1.195 (0.632–2.258)	0.584
Moderate hepatic steatosis versus absence of Chinese MAFLD	17 versus 463	7 versus 191	0.998 (0.407–2.446)	0.997	0.760 (0.224–2.575)	0.659
Severe hepatic steatosis versus absence of Chinese MAFLD	7 versus 463	7 versus 191	2.424 (0.839–7.005)	0.102	3.232 (0.896–11.659)	0.073
Risk of hepatic fibrosis by FIB-4
Low-risk hepatic fibrosis versus absence of Chinese MAFLD	104 versus 463	43 versus 191	1.119 (0.764–1.638)	0.564	1.083 (0.640–1.834)	0.766
Intermediate-risk hepatic fibrosis versus absence of Chinese MAFLD	41 versus 463	13 versus 191	0.769 (0.403–1.467)	0.425	0.604 (0.256–1.423)	0.249
High-risk hepatic fibrosis versus absence of Chinese MAFLD	3 versus 463	2 versus 191	1.616 (0.268–9.748)	0.601	2.129 (0.106–42.657)	0.621

^*Adjusting for gender, drinking, hypertension, hyperlipidemia, BMI, FPG, HsCRP.

BMI, body mass index; CI, confidence interval; CT_L/S, hepatic attenuation divided by splenic attenuation; FIB-4, fibrosis-4; FPG, fasting plasma glucose; H. pylori, Helicobacter pylori; HsCRP, high-sensitivity C-reactive protein; MAFLD, metabolic associated fatty liver disease; OR, odd ratio.

Discussion

Previous studies have investigated the association between H. pylori infection and NAFLD, but their conclusions are controversial. Jiang et al. ¹⁴ and Xu et al. ²⁹ found an independent association between H. pylori infection and NAFLD. Similarly, Wang et al. ³⁰ and Chen et al. ³¹ found that H. pylori infection increased the risk of NAFLD only in female patients and only in the diabetic population, respectively. Córdova-Gallardo et al. ³² further supported the association of H. pylori infection with liver fibrosis in patients with obesity and MASLD undergoing bariatric surgery. Conversely, other studies did not demonstrate that H. pylori infection was an independent risk factor for NAFLD.^{16,33 –35} The difference among studies may be explained by the heterogeneity and potential drawbacks in study design. First, Chen et al. ³¹ did not conduct multivariate regression analyses in the overall population. Second, some studies did not analyze the relationships between H. pylori infection and severity of hepatic steatosis or risk of hepatic fibrosis.^14,16,29,33 Third, by the diagnostic methods employed in the study by Han et al., ³⁵ the current status of H. pylori infection could not be determined. Finally, some studies would have explored the relationship between H. pylori infection and MASLD, but they still employed the diagnostic criteria for NAFLD, rather than MASLD.^36,37

Our current study demonstrated that H. pylori infection positively correlated with increased risk of severe hepatic steatosis in NAFLD, MAFLD, and MASLD analyses after adjusting for confounding factors, which is partially consistent with the results of our previous meta-analysis ¹⁷ that H. pylori infection is associated with NAFLD, especially moderate and severe NAFLD. But we found that H. pylori infection was not associated with Chinese MAFLD, which might be related to stricter metabolic indicators in the diagnostic criteria for Chinese MAFLD as well as a relatively small sample size. Compared with previous studies, our current study has some strengths. First, we have for the first time explored the associations of H. pylori infection with MAFLD, MASLD, and Chinese MAFLD by employing the most recent diagnostic criteria according to their relevant practice guidelines. Second, we have employed diverse diagnostic methods for H. pylori infection, which can indicate the current status of H. pylori infection. Third, we have explored the effects of H. pylori infection on NAFLD, MAFLD, MASLD, and Chinese MAFLD in terms of severity of hepatic steatosis and risk of hepatic fibrosis.

IR and altered gut microbiota are both crucial components in the “multiple hit” hypothesis about the pathogenesis of NAFLD. ¹⁰ Notably, H. pylori infection can also cause IR and change gut microbiota. First, H. pylori infection can secrete a wide spectrum of inflammatory factors, such as Interleukin (IL)-6, IL-1β, IL-18, IL-10, tumor necrosis factor-α, and transforming growth factor-β, which lead to IR by suppressing the insulin pathway through a variety of mechanisms.^{38 –43} Second, H. pylori infection increases the level of fetuin-A, ⁴⁴ an endogenous ligand of toll-like receptor 4, which facilitates lipid-induced IR. ⁴⁵ Third, H. pylori infection causes alterations in some adipokines, such as a decrease in adiponectin as an insulin sensitizer, and an increase in leptin as a regulator for releasing various inflammatory factors, leading to IR.^{46 –48} Last, H. pylori infection can alter gut microbiota and increase gut permeability, facilitating the passage of bacterial endotoxins and bacterial lipopolysaccharide via the portal vein to the liver.^49,50 Collectively, IR and gut microbiota alteration may contribute to the association of H. pylori infection with NAFLD.

Our current study also has some limitations. First, this is a cross-sectional study, which can only establish possible associations of H. pylori infection with NAFLD, MAFLD, and MASLD, but not cause-effect associations. Second, liver biopsy, which is the gold standard for diagnosing hepatic steatosis and fibrosis, and hepatic elastography (FibroScan), which is one of the most common alternative tools to evaluate fatty liver, have not been employed yet. Third, considering that CT findings are more objective than ultrasound, only CT was used to assess the severity of hepatic steatosis. However, CT is more sensitive for moderate to severe hepatic steatosis than mild hepatic steatosis. ⁵¹ In addition, not all included patients underwent CT examination. Fourth, the medications that may influence the pathophysiology of NAFLD and hepatic steatosis, such as pioglitazone, GLP-1 receptor agonists, SGLT2 inhibitors, metformin, or statins, have not been comprehensively evaluated. Fifth, we used serological tests alone to diagnose H. pylori infection, which may result in false-positive results, in a minority of patients. Finally, the number of patients with severe hepatic steatosis and a high risk of hepatic fibrosis was small, which may be caused by limited accuracy of our diagnostic methods. This hampers robust subgroup analyses. Future prospective studies with larger cohorts and standardized imaging-based fat quantification are needed to clarify causal pathways and therapeutic implications.

Conclusion

In conclusion, H. pylori infection may increase the risk of severe NAFLD, severe MAFLD, and severe MASLD using their contemporary diagnostic criteria. Thus, patients who are diagnosed with severe NAFLD, severe MAFLD, and severe MASLD should be advised to screen for H. pylori infection. More large-scale prospective studies are needed in the future to verify the relationships of H. pylori infection with NAFLD, MAFLD, MASLD, and Chinese MAFLD.