All patients (n = 558) who met the criteria were followed up for 1-year. All biochemical parameters and complications of liver cirrhosis were also recorded.

After a 1-year follow-up, all patients with H. pylori infection (n = 270) were treated with two regimens according to the clinical status, patient's general condition, and possible adverse effects related to drugs. The first one; pantoprazole 40 mg, clarithromycin 500 mg, and amoxicillin 1000 mg, twice daily, were used for 14 days. The second one; pantoprazole 40 mg and amoxicillin 1 g twice daily, and levofloxacin 500 mg once daily, were used also for 2 weeks. Treatment with pantoprazole (dose as mentioned above) was given to all patients for 1 more month, to complete the eradication therapy (15). All patients have been notified of the side effects of the drugs used. Therapeutic compliance was checked by tablet counting and all the patients were given an emergency telephone number, though no complications were reported leading to treatment discontinuation during the study period. To evaluate the effect of H. pylori eradication, all parameters were assessed again in patients with successful H. pylori eradication (n = 212) after a 3-months follow-up.

Patients who had not responded to any of the two above mentioned H. pylori therapeutic protocols (n = 58, 21.5% of patients) have received another H. pylori regimen used in our outpatient clinics (15).

All patients (n = 205; as seven patients were lost during the follow-up period) who achieved eradication were followed up for one further year in comparison with H. pylori-negative patients (n = 276; as 12 patients were lost during the follow-up period). All biochemical parameters and complications of liver cirrhosis were also recorded again.

The baseline data was captured within 1 week from the time of enrollment and the after-treatment data was captured 3 months after finishing therapy. The end-of-study data was captured in the last week of the 1 year after therapy follow up period. At the end of the study, clinical examination, radiological and biochemical findings of the patients did not show any problems affecting the follow-up parameters in the subjects' health that were not recorded at the start of the study.

Liver cirrhosis was assessed by clinical evaluation, unequivocal biochemical results, abdominal ultrasonography (US), elastography, histopathological evaluation of liver biopsy, or endoscopic results suggesting portal hypertension. The severity of liver cirrhosis was scored according to the Child-Pugh classification and MELD scoring system (16).

A color Doppler ultrasonography using a 3.5–5 MHz convex probe (SonoAce X6 Ultrasound System; Medison Electronics, Seoul, Korea) was done to all patients. Once, PVT was suspected by routine Doppler US examination, the final diagnosis was approved by portal angiography that also enabled a more precise differentiation between complete and partial obstructive thrombosis and their extension (17). Treatment and follow-up of all patients with PVT were carried out according to EASL clinical practice guidelines (18).

Diagnosis of SBP was based on positive ascitic fluid bacterial culture and/or ascitic fluid absolute polymorphonuclear leukocyte (PMN) count of at least 250 cells/mm³ without an intra-abdominal source of infection (19). According to EASL guidelines, patients with suspected SBP received an appropriate empirical antibiotic therapy, and then antibiotic was shifted depending on the results of culture and sensitivity as well as secondary antibiotic prophylaxis in patients with cirrhosis with a prior history of SBP was recommended (20).

In all cirrhotic individuals, US imaging and alpha-fetoprotein (AFP) were utilized every 6 months for surveillance purposes. Two imaging systems, both showing a focal lesion >2 cm in diameter with highlights of arterial hypervascularization, or a solitary radiologic study with these highlights joined with a serum AFP level of >400 ng/ml may be considered as noninvasive diagnostic tools especially for earlier stages of HCC (21). Surveillance and management of patients with HCC were carried out according to EASL guidelines (22).

HRS was diagnosed according to the International Club of Ascites-Acute Kidney Injury (ICA-AKI) criteria (23).

Overt HE is diagnosed depending on excluding other etiologies of altered mental status and by clinical findings (24). The West Haven criteria are considered the cornerstone for classifying the severity of overt HE for most patients (25). The minimal hepatic encephalopathy (MHE) is diagnosed based on specific psychometric testing (26). The management of patients with HE was recommended according to the recent EASL guidelines (24). In this study, HE may be considered as overt HE and/or MHE.

It is generally recommended that all patients with cirrhosis at the time of diagnosis undergo elective esophagogastroduodenoscopy (EGD) screening for varices and periodically thereafter if small or no varices are identified. The New Italian Endoscopic Club has categorized the severity of portal hypertensive gastropathy (PHG) depending on the presence of 4 elementary lesions: mosaic-like pattern, red point lesions, cherry red spots, and black-brown spots, that are typically localized in the gastric mucosa related to the corpus or fundus of the stomach (27). Gastric antral vascular ectasia (GAVE) is characterized by red spots or patches in either a linear or diffuse array in the gastric antrum (28). For EGD, an Olympus GIF-Q200 (Olympus Optical Co. Ltd., Tokyo, Japan) was used.

In all patients, the fecal antigen test was used to establish the diagnosis of H. pylori infection as well as to prove its eradication. This test was done one month after antibiotic treatment completion and 2 weeks after stopping therapy with pantoprazole (29). This test was also repeated at the end of therapy to identify any reinfection. Many types of research affirmed that the monoclonal antibodies utilization may have the highest sensitivity and specificity before and after eradication treatment (30, 31), and may also increase the diagnostic value in post-treatment testing and decrease the inter-test variability problem (32).

All patients completed a standardized, self-validated questionnaire from which we collected data about their employment condition, marital status, crowding index, income, alcohol consumption, medical history; especially diabetes, malignancy and hypertension, and smoking habits, as well as previous therapeutic history. BMI was calculated as weight (kg) divided by height squared (m²). Crowding index is the number of individuals/rooms excluding both the kitchen and bathroom, which correlated inversely with socioeconomic status (33).

A professional nutritional team started counseling to the patients with cirrhosis and malnutrition for proper protein and calorie intake. They instructed them to ingest the recommended daily protein intake of 1.5 g/kg of actual body weight. Vitamins and micronutrients were administered to treat clinically suspected or confirmed deficiencies. Patients with cirrhotic ascites following sodium restriction had 5 g of salt added daily to their diet to improve its palatability based on EASL guidelines of recommended daily sodium intake of 80 mmol/ day in these cases (34).

Following overnight fasting, fresh venous blood samples (7 mL) were collected from all patients [5 mL with no anticoagulants for serum testing and 1 mL on EDTA for complete blood count (CBC)]. The serum samples were then divided into aliquots and frozen at −20°C until subsequent evaluations. Three ml of venous blood were collected into plain tubes. Samples were then transported immediately to the laboratory, separated within 15 min of collection and analyzed immediately for serum ammonia.

Complete liver function tests and serum creatinine were done on a Dimension Xpand Plus Chemistry Analyzer using respective kits (by Siemens Technology, Princeton, New Jersey). Serum C-reactive protein (CRP) was assessed on COBAS C111 using its standard kits (Roche Diagnostics, Basel, Switzerland). Interleukin-6 (IL-6) and tumor necrosis factor-α (TNF-α) were measured by ELISA utilizing kits supplied by Dia Source (Rue du Bosquet, Louvain-la-Neuve, Belgium. Serum nitric oxide (NO) was evaluated using ELISA kits provided by MyBiosource (San Diego, CA 92195–3308, USA), with its detection range 1.56 nmol/ml−100 nmol/ml (nmol/ml = μmol/L). Serum VEGF was assessed by ELISA kits provided by Invitrogen Corporation (Flynn Road, Camarillo, CA, USA) with its detection range (0–1.5 ng/ml). Estimated glomerular filtration rate (eGFR) was assessed according to the following equation = 186 x (Creatinine/88.4)^−1.154 × (Age)^−0.203 × (0.742 if female) x (1.210 if black) (35). Serum ammonia was measured using a Colorimetric kit supplied by Elabscience (14780 Memorial Drive, Suite 216, Houston, Texas 77079, USA). Assessment of H. pylori antigens in stool was done by a sandwich ELISA technique using specific monoclonal antibodies for H. pylori from Immundiagnostik AG (Stubenwald-Allee 8a, 64625 Bensheim, Germany). Based on their absorbances (OD), the findings were categorized as follows: positive (OD > 0.170), borderline (OD = 0.130–0.170), and negative (OD < 0.130).

The study protocols and all procedures were reviewed and approved by the Mansoura Institutional Review Board. Written consents were obtained from all patients. The work was done in accordance with the Helsinki Declaration's guidelines.

Statistical analysis was performed using SPSS software (version 20; SPSS Inc., Chicago, IL, USA). Categorical and continuous parameter differences were expressed using Pearson's chi-square test and Mann–Whitney U-test respectively. Our results are expressed as numbers (%) and mean ± SD. Univariate and multivariate logistic regression analyses were performed to identify independent indicators for the presence of various cirrhotic complications. The Bonferroni correction for multiple comparisons was used where appropriate. P-values < 0.05 were considered statistically significant.

There was a significant increase in serum ammonia, CRP, IL-6, TNF-α, NO, VEGF, AFP, ascitic PMNs, and Child–Pugh score as well as there was a significant decrease in WBCs, platelet count, AST, serum albumin, eGFR, and ascitic fluid protein (all P < 0.05) as highlighted in Table 2.

There was a significant increase in total bilirubin, serum ammonia, CRP, VEGF, NO, AFP, and ascitic PMNs as well as there was a significant decrease in WBCs, platelet count, ALT, AST, serum albumin, prothrombin time, serum creatinine, and e GFR (all P < 0.05) as listed in Table 2.

There was a significant increase WBCs, platelet count, ALT, AST, serum albumin, prothrombin time, serum creatinine, eGFR, serum ammonia, CRP, IL-6, TNF-α, NO, AFP, VEGF levels, Child–Pugh score, and ascitic PMNs as well as there was a significant decrease in ascitic fluid protein in H. pylori-positive vs. negative patients (all P < 0.05), as observed in Table 2.

There was a significant increase in the incidence of esophageal varices (EV), gastric varices (GV), PHG, GAVE, HCC, SBP and its recurrence, HE, PVT, and HRS in H. pylori-positive vs. negative patients (all P < 0.05), as highlighted in Table 2.

According to ascitic fluid analysis, among the 47 patients who developed SBP in both groups, 18 (38%) had positive ascitic fluid cultures; the isolated organism was only Escherichia coli (E. coli). The rest of the SBP patients had culture-negative SBP (n = 29). Ascitic fluid with positive cultures was significantly increased in H. pylori-positive vs. negative patients (P = 0.027) as demonstrated in Table 2.

There was a significant decrease in WBCs count, serum albumin, creatinine, ammonia, CRP, IL-6, TNF-α, NO, AFP, and VEGF levels as well as there was a significant increase in hemoglobin level (all P < 0.001), as reported in Table 3.

There was no significant changes regarding all variables as well as there was no statistically significant difference in the incidence of EV, GV, PHG, GAVE, HCC, SBP and their recurrence, HE, PVT, and HRS in H. pylori-positive patients with eradication vs. H. pylori-negative patients (all P > 0.05), as observed in Table 4.

According to ascitic fluid analysis, among the 33 patients who develop SBP in both groups, 11 (33%) had positive ascitic fluid cultures; the isolated organism was only Escherichia coli (E. coli). The rest of the SBP patients had culture-negative SBP (n = 22). No significant differences were found between the two groups (P = 0.873) as shown in Table 4.

All patients with PVT in both groups had been subjected to measurements of activated partial thromboplastin time, fibrinogen, protein C, protein S, antithrombin III, serum homocysteine, D-dimer, antinuclear antibody, anticardiolipin IgG antibodies, anti-double-strand DNA after 1 year of the follow-up and after 1 year of H. pylori eradication. No significant difference between the two groups was evident in both conditions. The details of diagnostic biochemical criteria were described in Tables S1, S2.

Univariate analysis revealed that the presence of H. pylori infection was associated independently with the development of SBP, PVT, and HCC (all P < 0.05) while multivariate regression analysis model reported that the presence of H. pylori infection was associated independently with the development of only PVT and HCC, after adjustment for age, BMI, inflammatory markers (CRP, IL-6, and TNF-α), vascular mediators (VEGF and NO), Child-Pugh score and MELD score as shown in Table 5.

As regards to PVT, the IR was 0.115 (95% CI, 0.078–0.163) before therapy, and changed to 0.072 (95% CI, 0.0385–0.124) after therapy. The IRD was 0.043 (95% CI, −0.016–0.102) with P = 0.042. The IRR was 1.59 (95% CI, 0.81–3.31).

As regards to HCC, the IR was 0.059 (95% CI, 0.034–0.096) before therapy, and changed to 0.036 (95% CI, 0.015–0.074) after therapy. The IRD was 0.023 (95% CI, −0.018–0.064) with P = 0.037. The IRR was 1.64 (95% CI, 0.64–4.72).

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.