One hundred and forty-four subjects suffering from upper gastrointestinal tract symptoms were enrolled in this study. Ten subjects were considered negative control cases [non-ulcer dyspepsia (NUD) patients with normal findings]. The patients were diagnosed by specialist physicians and recruited accordingly. Demographic information—including age, sex, marital status, literacy status, residential status, and socioeconomic status—was obtained by referring to medical records and conducting personal interviews.

Throughout the study period, 73 women and 71 men aged between 18 and 80 years (mean age 51.09 ± 15.39 years) presented with dyspepsia and were referred to the Esophago-Gastroduodenoscopy Unit at Benghazi Medical Center (B.M.C.) for upper endoscopy between March 2020 and February 2022. Based on the endoscopic examination, patients were grouped into six categories: gastritis, peptic ulcer disease (PUD), gastric tumor cancer (GC), mucosa-associated lymphoid tissue (MALT) lymphoma, other gastric diseases (including gastroenteropathy, gastric angiodysplasia, and grade 2 esophagitis), and normal oesophago-gastro-duodenoscopy (OGD) findings. Normal OGD patients were defined as those who had no endoscopic lesions of ulcers and/or malignancies.

The protocol of this study was approved by the Ethics and Research Committees of the hospital, and all patients gave informed consent to the study according to the Declaration of Helsinki (WHO, 1993).

To collect samples from the targeted population and avoid any misinterpretation of the results, the following conditions were applied: patients had to be adults suffering from various dyspeptic symptoms and must not have received nonsteroidal anti-inflammatory drugs (NSAIDs), antibiotics, H₂ receptor antagonists, or any proton pump inhibitors for at least one month prior to the study. Patients were followed up by their physicians, and all upper endoscopy abnormalities were recorded. Clinical specimens were collected, stored, and transported according to established protocols.

Four antral biopsies were collected during upper endoscopy: one biopsy for the rapid urease test (RUT), taken from a position adjacent to normal gastric epithelium within 5 cm of the pylorus to avoid gastric atrophy or gastrointestinal metaplasia, with a thickness of about 1.8 to 3.3 mm; the other three biopsies, each about 5 mm thick, were used for molecular investigations targeting 16S rRNA, GLM, HPU, vacA, cagA, and iceA genes. The three biopsy specimens for molecular analysis were preserved immediately at −80˚C.

According to the manufacturer, the QIAamp Tissue DNA Extraction Kit was used to extract genomic DNA from frozen biopsy specimens. Briefly, 25 mg of the crushed biopsy specimen was transferred into a microcentrifuge tube, followed by the addition of 180 μL of lysis buffer ATL. Then, 20 μL of proteinase K solution was added, the mixture was vortexed, and then incubated at 56˚C until the tissue was completely lysed. Lysates were spun at 8000 rpm for 15 seconds, and then 200 μL of AL buffer was added, vortexed, and incubated at 70˚C for 10 minutes. Ethanol (96% - 100%) 200 μL was added to the sample, vortexed for 15 seconds, and spun at 8000 rpm for 15 seconds. The lysates were then applied to the QIAamp Mini spin column and spun at 8000 rpm for 1 minute. Five hundred microliters of wash buffer AW1 were added to the QIAamp Mini spin column and spun at 8000 rpm for 1 minute. The column was transferred to a clean collection tube, and then 500 μL of wash buffer AW2 was added to the column and spun at 14,000 rpm for 3 minutes. The column was transferred to a new Eppendorf tube, and the DNA sample was eluted by adding 200 μL of buffer AE directly onto the membrane of the column. The column was left for 5 minutes at room temperature and then spun at 8000 rpm for one minute. Genomic DNA samples were stored at −20˚C for further investigations.

Polymerase chain reaction (PCR) was performed on extracted DNA from biopsies using primers specific for H. pylori 16S rRNA under the following conditions: initial denaturation at 95˚C for 5 minutes, followed by 37 cycles of denaturation at 95˚C for 60 seconds, annealing at 60˚C for 60 seconds, and extension at 72˚C for 60 seconds, with a final extension time of 72˚C for 5 minutes. The PCR amplification was performed using a thermocycler system (T3 thermocycler, Whatman Biometra, USA). Each 25 μL PCR reaction mixture contained 12.5 μL of PCR master mix (Promega, GoTaq® Green Master Mix, USA), 1.5 μL of each primer (Metabion, Planegg, Germany), 3 μL of template DNA, and 6.5 μL of PCR-grade water. For each PCR experiment, appropriate positive and negative controls were included. The H. pylori strain J99 and nuclease-free water were used as positive and negative controls, respectively.

To detect the amplified product, 5 μL of amplicons were visualized by electrophoresis through a 2% agarose gel (Promega, USA) at 100 V for 60 minutes in 1 × TBE buffer and stained with ethidium bromide (500 ng/mL) (Sigma-Aldrich, USA) using the gel photo documentation system (Bio-Rad Laboratories, Inc., American developer). The bands were identified by comparing the band sizes with molecular weight markers (Promega, USA). Samples were considered positive when the visible band was the same size as the positive control DNA. The primer for the 110 bp product of the 16S rRNA sequence was represented by the forward primer sequence: 5'-CTG GAG AGA CTA AGC CCT CC-3' and the reverse one: 5'-ATT ACT GAC GCT GAT TGT GC-3' (Metabion International AG, Germany).

Virulence genes, including GLM, HPU, vacA (subtypes: s1, s2, m1, and m2), cagA, and iceA, were detected using PCR-specific primers ( Table 1 ) with the same amplification conditions and assay protocol as described earlier.

Data were analyzed using chi-square tests to determine the association between H. pylori positivity and epidemiological risk factors, virulence factors, and gastroduodenal disease diagnosis. Briefly, a chi-squared test was used to compare frequencies between various groups, and Fisher’s exact test was used to analyze two-by-two tables of categorical data. The relationship between virulence genes, clinical outcomes, age, and gender was evaluated using two-way ANOVA. P-values less than 0.05 were considered statistically significant.

A total of 144 patients were enrolled in this study, comprising 73 females and 71 males, with a mean age of 51.09 ± 15.39 years (range: 18 - 80 years). The distribution of gastroduodenal diseases diagnosed via esophagogastroduodenoscopy (EGD) is detailed in Table 2 . Gastritis was the most prevalent condition, observed in 66 patients (45.83%), followed by peptic ulcer disease (PUD) in 26 patients (18.06%), mucosa-associated lymphoid tissue lymphoma (MALT lymphoma) in 7 patients (4.86%), and gastric adenocarcinoma in 3 patients (2.08%). Other conditions—including gastroenteropathy, gastric angiodysplasia, grade 2 esophagitis, and gastroesophageal reflux disease (GERD)—were identified in 20 patients (13.89%). Normal EGD findings were reported in 22 patients (15.28%).

The mean age of male patients was 52.66 ± 13.41 years (range: 18 - 78 years), and that of female patients was 49.58 ± 17.14 years (range: 18 - 80 years). The gender-specific distribution of gastroduodenal diseases is presented in Figure 1 . Gastric adenocarcinoma was observed exclusively in males (3/3, 100%). MALT lymphoma was more prevalent in males (6/7, 85.71%) compared to females (1/7, 14.29%). Conversely, gastritis and normal EGD findings were more common in females, accounting for 60.61% and 63.64% of cases, respectively.

The presence of H. pylori DNA in gastric biopsy specimens was confirmed by PCR amplification of housekeeping genes: 16S rRNA, glmM, and HPU. Among the 144 specimens, 91 (63.19%) were positive for both 16S rRNA and glmM genes, and 90 (62.50%) were positive for the HPU gene. Gel electrophoresis images confirmed the expected amplicon sizes for these genes.

As shown in Table 3 , the detection rates of housekeeping genes were similar between females and males, with no statistically significant differences observed (p > 0.05).

The prevalence of H. pylori housekeeping genes varied significantly across different age groups ( Figure 2(A) and Figure 2(B) ). The highest detection rate was in the 39 - 59 years group (48/65, 73.85%), followed by the 60 - 80 years group (30/49, 61.22%) and the 18 - 38 years group (13/30, 43.33%). Statistical analysis indicated a significant association between age and the presence of housekeeping genes (χ² = 8.34- 8.50, p < 0.05). As shown in Figure 3 , the agarose gel electrophoresis results demonstrate successful amplification of key H. pylori housekeeping genes. The genus-specific 16S rRNA gene (110 bp), as shown in Figure 3(A) , the glmM gene (294 bp), as indicated in Figure 3(B) , and the HPU gene (411 bp), as shown in Figure 3(C) were consistently detected in patient biopsy samples, indicating the presence of H. pylori across the majority of tested samples.

A significant association was detected between housekeeping genes and clinical outcomes (p ≤ 0.0001). The highest detection rates were observed in patients with MALT lymphoma (7/7, 100%), PUD (20/26, 76.92%), and gastritis (50/66, 75.76%), as indicated in Table 4 .

PCR analysis detected one or more virulence genes (vacA, cagA, and iceA1) in 87 out of 91 patients (95.60%) who were positive for housekeeping genes. Four patients (4.40%) were negative for all tested virulence genes, as indicated in Table 5 .

Among the 77 vacA-positive patients, the s1 allele was detected in 41 patients (53.25%), as shown in Figure 4(A) , and the s2 allele in 10 patients (12.99%) ( Figure 4(B) ). The m1 allele was found in 33 patients (42.86%), and the m2 allele in 29 patients (37.66%). The most common allelic combinations were s1/m2 (16 patients, 20.78%) and s1/m1 (13 patients, 16.88%), as shown in Table 6 .

The distribution of vacA genotypes among different clinical outcomes is presented in Table 7 . Although variations were observed in genotype frequencies among disease categories, no statistically significant association was found between vacA genotypes and clinical outcomes (p > 0.05).

A significant association was observed between vacA genotypes and the detection of other virulence genes (cagA and iceA1) (χ² = 62.1, p ≤ 0.0001) ( Table 8 ). For instance, the s1/m1 genotype was predominantly associated with cagA positivity (12/13 patients, 92.31%).

The cagA gene was detected in 53 of 91 H. pylori strains (58.24%). The distribution of cagA positivity among different clinical outcomes is presented in Table 9 . The highest prevalence of cagA-positive strains was observed in patients with MALT lymphoma (6/7, 85.71%) and gastric adenocarcinoma (2/2, 100%). However, no significant differences were found in the frequency of cagA-positive strains between clinical groups (p = 0.31). The agarose gel electrophoresis of PCR amplified products for H. pylori cagA gene is shown in Figure 5 .

The iceA1 gene was identified in 27 out of 91 H. pylori strains (29.67%). Its distribution among clinical outcomes is shown in Table 10 . No significant differences were observed in the frequency of iceA1-positive strains between clinical groups (p = 0.92). PCR amplification of the H. pylori iceA gene yielded a product size of 558 bp in multiple patient samples, as confirmed by agarose gel electrophoresis. Positive amplification was observed in lanes 2, 3, 4, 5, 6, 8, 9, 10, 12, 13, and 14, while the negative control (lane 7, no-template control) and a negative sample (lane 11) showed no amplification. These results indicate the presence of the iceA gene in most tested samples, supporting its prevalence among the isolates, as indicated in Figure 6 .

A significant association was detected between cagA and iceA1 genes (p = 0.01), as shown in Table 11 . Among the 53 cagA-positive patients, 10 (18.87%) were also positive for iceA1.

An analysis of the relationship between virulence genes and clinical outcomes revealed a significant association (F = 12.5, p ≤ 0.0001), as indicated in Figure 7 . Patients with more severe gastroduodenal diseases, such as PUD, MALT lymphoma, and gastric adenocarcinoma, exhibited higher frequencies of certain virulence genes, particularly cagA and specific vacA genotypes.

We could not detect a significant association between the presence of virulence genes and patient gender (F = 1.3, p = 0.3), as shown in Figure 8(A) . However, a significant association was observed with age groups (F = 13.5, p = 0.005) ( Figure 8(B) ). Younger patients (18 - 38 years) had different distributions of virulence genes compared to older patients.