Hrev_master Healthcare in Low-resource Settings 2023; volume 11(s2):11379 Helicobacter Pylori infection and non-alcoholic fatty liver disease. Is there a relationship? Neveen Rashad Mostafa,1 Abeer A.M. Ali,2 Mona Gamalludin Alkaphoury,3 Roy Rillera Marzo4,5 1Department of Experimental and Clinical Internal Medicine, Medical Research Institute, Alexandria University, Egypt; 2Department of Chemical Pathology, Medical Research Institute, Alexandria University, Egypt; 3Department of Diagnostic Radiology, Ain Shams University, Egypt; 4Department of Community Medicine, International Medical School, Management and Science University, Shah Alam, Malaysia; 5Global Public Health, Jeffrey Cheah School of Medicine and Health Sciences, Monash University Malaysia, Kuala Lumpur, Malaysia Abstract The most prevalent infection that causes chronic gastritis, gas- tric ulcers, and gastric cancer is Helicobacter pylori infection. Recent research has implicated H. pylori in the pathogenesis of non-gastrointestinal diseases such as cardiovascular, autoimmune, and metabolic disorders. In addition, since H. pylori is believed to be implicated in insulin resistance, numerous studies have been conducted to determine the relationship between H. pylori infec- tion and nonalcoholic fatty liver diseases (NAFLD), but the results have been contested. The purpose of this study is to determine the relationship between H. Pylori infection and nonalcoholic fatty liver diseases. One hundred patients were examined via urea breath test for the presence of H. pylori infection and vibration-controlled transient elastography for the diagnosis of non-alcoholic fatty liver disease. After adjusting for other variables, age, body mass index (BMI), and H. pylori infection were associated with elastography 248dB/m. Infection with H. pylori contributes to the development of NAFLD, and its eradication may influence prognosis. Introduction Helicobacter pylori infection is a prevalent condition world- wide, particularly in developing countries. It is considered the most common cause of gastric mucosa causing chronic gastritis, gastric ulcers, and gastric cancer.1 Recently, H. Pylori was found to be involved in the pathogenesis of other non-gastric diseases, and involved in the pathogenesis of insulin resistance and several metabolic and autoimmune diseases that affects the liver.2 Nonalcoholic fatty liver disease (NAFLD) is a group of metabolic diseases caused mainly by insulin resistance with hereditary susceptibility. It is considered a manifestation of metabolic syndrome in the liver, in the absence of alcohol con- sumption, and includes nonalcoholic fatty liver, nonalcoholic steatohepatitis, liver fibrosis, and cirrhosis.3 NAFLD is a common disease condition affecting 25% of the population worldwide, with higher prevalence rates observed in the Middle East (32%), and in Latin America (31%).4 Hepatic lipid homeostasis is controlled by signaling/transcrip- tional pathways mediated by hormones, transcription factors, and nuclear receptors. Triglyceride accumulation mostly is considered the first step in the development of NAFLD and results from a dis- turbed balance between TG production and utilization and unreg- ulated insulin signaling at the level of the adipose tissue.5 In obese and diabetic patients with insulin resistance there is increased lipolysis with increased formation of nonesterified fatty acids directed to the liver where they are taken up by hepatocytes.6 CD36 also facilitates their uptake and accumulation in other cell types (macrophages, adipocytes, enterocytes, and myocytes). CD36 has been shown to rise in animal models with hepatic steatosis. In humans, morbidly obese patients with NAFLD showed a correlation between messenger RNA levels of CD36 and liver fat content.7 It has been shown that there are other two factors contributing to fat accumulation in the liver, these factors are dietary fat and de novo lipogenesis. Two enzymes catalyze hepatic Correspondence: Neveen Rashad Mostafa, Department of Experimental and Clinical Internal Medicine, Medical Research Institute, Alexandria University, Egypt. E-mail: doctor.aj.2000@gmail.com Key words: Helicobacter Pylori, lipid, non-alcoholic fatty liver. Contributions: NRM, data collection, manuscript drafting and statisti- cal analysis; AAMA, biochemical assay; MGAK, elastography; RRM, manuscript editing, language revision. Conflict of interest: the authors declare no potential conflict of inter- est, and all authors confirm accuracy. Ethics approval and consent to participate: the study was approved by the Medical Research Institute Hospital’s Local Ethics Committee, which follows the Helsinki Declaration terms. Informed consent was taken from all participants. Consent for publication: the manuscript does not contain any indi- vidual person’s data in any form. Availability of data and materials: data sharing is not applicable as no dataset was generated or analyzed during the current study. Received for publication: 11 April 2023. Accepted for publication: 10 June 2023. This work is licensed under a Creative Commons Attribution 4.0 License (by-nc 4.0). ©Copyright: the Author(s), 2023 Licensee PAGEPress, Italy Healthcare in Low-resource Settings 2023; 11(s2):11379 doi:10.4081/hls.2023.11379 Publisher's note: all claims expressed in this article are solely those of the authors and do not necessarily represent those of their affili- ated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article or claim that may be made by its manufacturer is not guaranteed or endorsed by the publisher. [page 12] [Healthcare in Low-resource Settings 2023; 11(s2):11379] No n- co mm er cia l u se on ly fatty acid synthesis which is acetyl-CoA carboxylase and fatty acid synthase and are controlled by insulin and by glucose through liver X receptors which directly induce acetyl-CoA carboxylase and fatty acid synthase. De novo lipogenesis is markedly increased in NAFLD mainly due to the coexistent hyperinsulinemia and increased intake of simple sugars.6 The mechanism claimed to be involved in the development of NAFLD is the “second hit hypothesis” which is the oxidative stress, as the “first hit” usually is the fat accumulation in hepato- cytes, and the “second hit” is the oxidation that causes hepatic injury.8 Recently, the “multiple hits” hypothesis is more reliable and accepted, as proposed by Takaki et al.,9 and Buzzetti et al.,10 they found that multiple factors work together on genetically pre- disposed subjects that lead to the development of NAFLD; these factors include insulin resistance, adipose tissue hormones, nutri- tion, and gut microbiota. Khosravi et al.,11 found that H. Pylori infection was related to gut microbiota by using germ-free and specific pathogen-free mice and found that there is a strong relation between normal gut micro- biota and infection with H. Pylori which alters the metabolism and induce gut inflammation considering H. Pylori as one of the mech- anisms that cause NAFLD through gut microbiota dysbiosis. This was confirmed by Sumida et al.,12 as they found that invasion of H. Pylori into intestinal mucosa may increase gut permeability and alter gut microbiota and subsequently increase the passage of bac- terial endotoxin through the portal vein to the liver and initiate inflammation and considered as one of the multiple hits causing NAFLD. In this study, we are aiming at finding the relationship between H. Pylori infection and the development of NAFLD. Materials and Methods Study design and population This study was conducted on 100 patients, 50 of whom were diagnosed with H. Pylori infection while the other 50 were not. The patients were examined for the presence of fatty liver in the absence of alcohol consumption. They were selected from either outpatient clinics or inpatient wards of the Medical Research Institute for any medical reason during the period from June 2022 to December 2022, in Alexandria, Egypt. Participants with DM, hypertension, alcohol consumption, and chronic liver disease were excluded from the study. Informed consent was obtained from all patients in a case-control study and approved by the Medical Research Institute Ethics Committee. Clinical data The clinical data collected included: i) thorough clinical exami- nation including weight and height; ii) routine laboratory investiga- tions include liver function tests, renal function tests, lipid profiles, electrolytes, complete blood pictures, and fasting blood sugar;13 iii) the urea breath test was done for the diagnosis of H. pylori infection;14 iv) NFLD was diagnosed using vibration-con- trolled transient elastography, the cut-off for steatosis is >248 dB/m.15 Statistical analysis Data were fed to the computer and analyzed using IBM SPSS software package version 20.0. (Armonk, NY: IBM Corp). Categorical data were represented as numbers and percentages. The chi-square test was applied to investigate the association between the categorical variables. For continuous data, they were tested for normality by the Shapiro-Wilk test. Quantitative data were expressed as a range (minimum and maximum), mean, stan- dard deviation, and median. Student t-test was used to compare two groups for normally distributed quantitative variables. On the other hand, the Mann-Whitney test was used to compare two groups for not normally distributed quantitative variables. And Receiver operating characteristic curve (ROC) was used to deter- mine the diagnostic performance of the markers, an area of more than 50% gives an acceptable performance, and an area of about 100% is the best performance for the test. The significance of the obtained results was judged at the 5% level. Results The effect of demographic data on NAFLD was tested, and we found that fatty liver is more common in females but not statisti- cally significant. p=0.680, while age was significantly related to fatty liver where the mean age in patients with steatosis was 52.5±12.4 years, and the mean age of patients without steatosis was 38.2±14.3 (p<0.001). BMI also was a risk factor for steatosis, where the mean BMI in Patients with NAFLD was 32±3.9 kg/m2, and in patients without NAFLD was 28.8±4.3kg/m2, p<0.001 (Table 1). Various biochemical markers were evaluated for their relation- ship to NAFLD, including liver enzymes such as alanine amino- transferase (ALT), Aspartate aminotransferase (AST), total choles- terol (TC), low-density lipoprotein (LDL), high-density lipopro- tein (HDL), and triglyceride level (TG). However, none of these markers showed a significant relationship with NAFLD (Table 2). Article Table 1. Relation between elastography and demographic data. Total n = 100 Elastography Test of Sig. p ≤248 dB/m >248 dB/m Gender Male (%) 38 (38.0) 18 (36.0) 20 (40.0 χ2= 0.170 0.680 Female (%) 62 (62.0) 32 (64.0) 30 (60.0) Age (years) Mean±SD 45.4±15.1 38.2±14.3 52.5±12.4 U=566.0* <0.001* Median (Min-Max) 46 (19-68) 35 (19-66) 57 (29-68) BMI (kg/m2) Mean±SD 30.4±4.37 28.8±4.3 32±3.9 t=3.963* <0.001* Median (Min-Max) 30.5 (18.5-38) 29.0 (18.5-37.0) 32 (26-38) SD, standard deviation; χ2, chi-square test; t, student t-test; U, Mann-Whitney test. *Statistically significant at p≤0.05. [Healthcare in Low-resource Settings 2023; 11(s2):11379] [page 13] No n- co mm er cia l u se on ly The relation between NAFLD and H. Pylori infection was examined, revealing a prevalence of H. Pylori infection in patients with NAFLD was 68% and in patients without NAFLD 32%. Additionally, we found that the mean elastography value in H. Pylori +ve patients was 250.4±33.59 dB/m, while the mean in H. Pylori-ve patients was 229.3±33.83 dB/m, p=0.002 (Table 3). Furthermore, H. Pylori was found to have a significant prog- nostic performance of H. Pylori infection with elastography values ≥248 dB/m, (95% CI 0.582-0.794, p=0.001, sensitivity 68% and specificity 68%; Table 4, Figure 1). Assessment of various biochemical markers, demographic data, and H. Pylori infection in patients with and without NAFLD in a univariate analysis showed that age, BMI, and H. Pylori infec- tion were associated with elastography ≥ 248 dB/m after adjust- ment with other variables. Multivariate analysis showed that these factors were independent risk factors for NAFLD (Table 5). Discussion Non-alcoholic fatty liver disease is a common metabolic health problem that has become a public health concern. The peak of fatty liver incidence is between 40-50 years of age in males and 60-69 years in females, with minimal reduction in older (>70 yrs) cohorts.16 In a retrospective cohort study conducted on 351 patients with NAFLD diagnosed by biopsy, the patients were divided into (≥60 yrs), (≥50 to <60 yrs), and a younger (<50 yrs) group. The study found that NAFLD was more prevalent in the mid- dle-aged and the elderly.17 In our study, we found that the mean age of patients with NAFLD was 52.5±12.4 years and in patients with- out NAFLD it was 38.2±14.3 years. Hence, confirming that the incidence increases with age and it is an independent risk factor for NAFLD. This is probably due to an increase in all risk factors for fatty liver in older age groups such as hypertension, diabetes, hyperlipidemia, and obesity. According to previous longitudinal studies, NAFLD is more common in males as compared to females.18 However, a study specifically investigating NAFLD in females found that the inci- dence is higher in menopausal females (7.5%) and postmenopausal females (6.1%) than in premenopausal females (3.5%). The study also reported that postmenopausal women had an increased risk of NAFLD at univariate but not at multivariate analysis after adjust- ment for age, metabolic syndrome, and BMI.19 In our study, we found that the incidence of NAFLD is more common in females but was not statistically significant, this may be due to the increased age of patients with NAFLD in our study, and the fact that most of the females studied were postmenopausal. Female sex hormones are known to protect against dysmetabolism and pro- mote the division of fatty acids into ketone bodies rather than into very low-density lipoprotein-triacylglycerol. The senescence of the Article Table 2. Relation between elastography and different parameters. Total n=100 Elastography Test of Sig. p ≤248 dB/m >248 dB/m ALT Mean±SD 28.7±23.1 24.2±12.8 33.3±29.6 U=1014.0 0.103 Median (Min-Max) 23 (10-153) 20 (11-72) 23 (10-153) AST Mean±SD 25.6±16.5 22.9±8.49 28.3±21.5 U=1046.0 0.159 Median (Min-Max) 22 (11-125) 22 (13-49) 26 (11-125) Total cholesterol Mean±SD. 183.7±30 181.6±26.0 185.8±33.6 t 0.692 0.490 Median (Min.-Max.) 181 (105-254) 180 (137-245) 182 (105-254) LDL Mean±SD 100.7±22.5 98.5±22 102.9±23.1 t=0.985 0.327 Median (Min-Max) 95 (59-157) 93 (68-155) 100 (59-157) HDL Mean±SD 49.96±8.80 50.5±8.84 49.4±8.83 t=0.589 0.557 Median (Min-Max) 49 (29-74) 53 (29-67) 46 (38-74) Triglyceride Mean±SD 114.8±46.49 112.4±44.52 117.2±48.72 U=1250.0 1.000 Median (Min-Max) 96.5 (51-246) 98 (51-246) 94 (60-225) H. pylori (%) 50 16 34 χ2=12.96* <0.001* SD, standard deviation; t, student t-test; U, Mann-Whitney test; χ2, chi-square test. *Statistically significant at p≤0.05 Figure 1. ROC curve for Elastography to prognoses positive H. Pylori patients. [page 14] [Healthcare in Low-resource Settings 2023; 11(s2):11379] No n- co mm er cia l u se on ly ovaries also increases the formation of hepatic steatosis and pro- gression to fibrosis.20 Obesity has been linked to fatty liver disease in all stages, start- ing from simple steatosis to steatohepatitis and fibrosis. Obesity causes the accumulation of fat inside liver cells through increasing insulin resistance and leads to progression to non-alcoholic steato- hepatitis and its related cirrhosis.21 In a cross-sectional study con- ducted on 3202 individuals to investigate the association of BMI with fatty liver found that a dose-response analysis with adjust- ment of other factors like age, gender, hypertension, total choles- terol, triglycerides, glucose, high-density lipoprotein, low-density lipoprotein, uric acid, homocysteine, creatinine, aspartate amino- transferase, and alanine transaminase showed that overweight and obesity were significantly related to fatty liver risk (p=0.004 or lower). They reported that high BMI (overweight/obesity) is an independent, dose-dependent risk factor for fatty liver.22 In a study conducted on the Sudanese population to assess the risk factors for non-alcoholic fatty liver disease, they found that increasing age and obesity were the most prominent predisposing factors in devel- oping NAFLD23 In our study, we found that the mean BMI in patients with NAFLD was 32±3.9 kg/m2, and in patients without NFLD it was 28.8±4.3 kg/m2, and increased BMI was highly sig- nificantly related to fatty liver p<0.001 and was an independent risk factor for NAFLD p≤0.009 NAFLD occurs when there is an imbalance between the rate of uptake of fatty acids and triglycerides from circulation, increased lipogenesis, and a decreased ability to oxidize fatty acids and export very low-density lipoprotein-TG. Therefore, changes in liver and serum lipid parameters can be a predictor of disease development.24 Kantartzis et al.25 in their study on 16 patients with fatty liver and 24 control subjects found that fatty liver was associated with decreased levels of high-density lipoprotein 2 (HDL2) which is potent antiatherogenic. Moreover, in patients with NAFLD, abnor- mal serum ALT and AST are usually present when the disease progress to steatohepatitis or hepatic fibrosis.26 However, in a study done by Ma et al.27 they found the prevalence of normal ALT in patients with NAFLD reached over 90%. In our study, we found that in patients with NAFLD, total cholesterol, LDL, and TG, as well as ALT and AST were higher than in patients without NAFLD. However, none of these were statistically significant. This may be attributed to the low number of patients with dyslipi- demia involved in the study and the early stage of NAFLD in those patients. Several studies have been conducted to demonstrate the rela- tionship between H. pylori infection and NAFLD. This is because the main pathogenic mechanism in NAFLD is insulin resistance which makes hepatocytes more susceptible to oxidative stress and lipid peroxidation. At the same time, H. Pylori was implicated in the development of insulin resistance through increasing pro- inflammatory cytokines and reactive oxygen species production. Numerous studies aimed to find out whether there is a relationship between them or not.28 In a study done by Polyzos et al.29 on 28 patients with biopsy-proven NAFLD and 25 healthy controls, they Article Table 3. Relation between H. pylori and elastography. Total n=100 H. pylori Test of Sig. p Negative (n=50) Positive (n=50) Mean±SD 239.8±35.18 229.3±33.83 250.4±33.59 3.133* 0.002* Median (Min-Max) 248.5 (150-296) 230 (150-296) 259 (170-294) SD, standard deviation; t, student t-test; U. *Statistically significant at p≤0.05. Table 4. Prognostic performance for Elastography to prognoses positive H. Pylori patients (n = 50) from negative H. Pylori patients (n = 50). AUC p 95% C.I Cut off Sensitivity Specificity PPV NPV Elastography 0.688 0.001* 0.582-0.794 >248 68.0 68.0 68.0 68.0 AUC, area under a curve; CI, confidence intervals; NPV, negative predictive value; PPV, positive predictive value. *Statistically significant at p≤0.05. Table 5. Univariate and multivariate Logistic regression analysis for the parameters affecting Elastography >248dB/m. Univariate Multivariate p OR (LL–UL 95%C.I) p OR (LL–UL 95%C.I) Male 0.680 1.185 (0.528-2.660) Age (years) <0.001* 1.075 (1.041-1.110) <0.001* 1.082 (1.039-1.127) BMI (kg/m2) 0.001* 1.227 (1.091-1.380) 0.009* 1.225 (1.052-1.427) ALT 0.077 1.023 (0.998-1.049) AST 0.151 1.028 (0.990-1.067) Total cholesterol 0.487 1.005 (0.991-1.018) LDL 0.324 1.009 (0.991-1.027) HDL 0.553 0.986 (0.943-1.032) Triglyceride 0.607 1.002 (0.994-1.011) H. pylori <0.001* 4.516 (1.949-10.463) 0.001* 5.632 (1.967-16.130) OR, odd’s ratio; CI, confidence interval; LL, lower limit; UL, upper limit. *Statistically significant at p≤0.05. [Healthcare in Low-resource Settings 2023; 11(s2):11379] [page 15] No n- co mm er cia l u se on ly found that H. Pylori diagnosed by serology were found in 82% of NAFLD patients and 56% of healthy controls. Meta-analysis of data from cross-sectional and case-control studies involving 91,958 individuals concluded that H. Pylori infection was also associated with increased NAFLD incidence.30 In addition, another study found a remarkable effect of H. Pylori infection on NAFLD after ruling out many confounding factors like age, dyslipidemia, diabetes, hypertension, and liver enzymes, H. Pylori infection was found to be an independent risk factor for NAFLD (95% CI 1.02- 1.79, OR 1.35, p=0.036).31 Yan et al.32 conducted a wide-scale study on 1185 patients. Abdominal color Doppler ultrasound was used to assess nonalcoholic fatty liver disease and13 C-urea breath test was used to diagnose H. Pylori infection, NAFLD was found in 44.6% (n=529), distributed in 362 males and 167 females. The study concluded that H. pylori is a significant and independent risk factor for NAFLD (95% CI 1.02-1.79, p=0.036, OR=1.35). On the other hand, a similar study was conducted using abdominal color Doppler ultrasonography as well as transient elastography, fat attenuation parameter, and liver stiffness for diagnosis of NAFLD,13 C-urea breath was the method for diagnosis of H. pylori infection. The study found no association between H. pylori infection and NAFLD or elevated liver steatosis, but it could be a risk of increased liver stiffness in males.33 Other studies intended to demonstrate the effect of H. Pylori eradication on hepatic fat contents. One of these studies performed by Jamali et al.34 on 100 patients diagnosed H. Pylori positive and given slandered treatment and re-tested again to confirm eradica- tion, found no effect of eradication on hepatic fat content checked by NAFLD liver fat score. In our study, H. Pylori infection was significantly associated with NAFLD, where it existed in 68% of patients with elastogra- phy > 248 dB/m (95% CI 0.582-0.794, p=0.001, sensitivity 68% and specificity 68%). Univariate analysis showed that age, BMI, and H. Pylori infection were associated with NAFLD after adjust- ment with ALT, AST, total cholesterol, LDL, HDL, and TG, Multivariate analysis showed that they were still independent risk factors for NAFLD. Conclusions Increasing age, weight, and H. Pylori infection are indepen- dent risk factors for the development of NAFLD. Therefore, weight reduction and treatment of H. Pylori infection may help to reduce the incidence of fatty liver. References 1. Narayanan M, Reddy K, Marsicano E. Peptic ulcer disease and Helicobacter pylori infection. Mo Med 2018;115:219-24. 2. Waluga M, Kukla M, Zorniak M. From the stomach to other organs: Helicobacter pylori and the liver. World J Hepatol 2015;18:2136-46. 3. Kim D, Kim WR. Non-obese. Clin Gastroenterol Hepatol 2017;15:474-85. 4. Younossi Z, Anstee M, Marietti M. Global burden of NAFLD and NASH: trends, predictions, risk factors and prevention. Nat Rev Gastroenterol Hepatol 2018;15:11-20. 5. 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