Helicobacter pylori Isolate from Endoscopy-Examined Patients in Bahir Dar, Northwest Ethiopia

Abstract

Background: Helicobacter pylori infection is widely prevalent across the globe and is a major etiological agent of various gastric pathologies. This bacterium colonizes the human stomach, where it induces a range of mucosal abnormalities observable upon clinical examination. Accordingly, the present study aimed to assess the prevalence and clinical implications of H. pylori infection among patients undergoing endoscopic evaluation. Method: A cross-sectional study was conducted from January to May 2019 at endoscopy service-providing health institutions. Sociodemographic and clinical data were collected. Gastric biopsies were collected during endoscopic procedures and immediately inoculated into brain–heart infusion broth and plated out. Then, phenotypic bacterial identification was done. The collected data have been analyzed using SPSS version 23. A logistic regression model was used for association determination. Result: Among the 135 individuals enrolled in the study, 59.3% are male, and 40.7% are female, with a mean age of 45 years. H. pylori is isolated in 17.8% of participants (24/135). Notably, the majority of these isolates 71% (17/24) are from male participants, while 29% (7/24) are from females. A statistically significant association is identified between H. pylori infection and both high salt intake [AOR = 3.3; 95% CI: 1.5–10.8; p = 0.04] and the presence of duodenal ulcers [AOR = 3.8; 95% CI: 1.2–11.9; p = 0.02]. The highest prevalence of H. pylori is observed among patients diagnosed with atrophic pangastritis. Conclusions: The prevalence of H. pylori among the study participants is comparatively low. However, a significant association was observed between H. pylori infection and both high dietary salt intake and the presence of duodenal ulcers.

1. Introduction

Helicobacter pylori is a Gram-negative, spiral- or curved-shaped bacterium characterized by the presence of four to seven polar flagella [1]. It thrives under microaerophilic conditions, requiring reduced oxygen levels for survival. The organism was first successfully isolated from gastric biopsies in 1982 by Australian scientists Robin Warren and Barry Marshall [2]. Taxonomically, H. pylori belongs to the domain Bacteria, phylum Proteobacteria, class Epsilonproteobacteria, order Campylobacterales, family Helicobacteraceae, and genus Helicobacter [3].

This pathogen is widely recognized as a major etiological agent in a spectrum of gastric-associated diseases, including chronic gastritis, duodenal and gastric ulcers, gastric carcinoma, and mucosa-associated lymphoid tissue (MALT) lymphoma [4,5]. Due to its strong association with gastric malignancies, the World Health Organization (WHO) and the International Agency for Research on Cancer (IARC) have classified H. pylori as a Group I carcinogen, denoting it as a definite human cancer-causing agent [6,7,8].

Due to suboptimal environmental sanitation, developing countries exhibit a significantly higher prevalence of H. pylori infection [9]. Transmission typically occurs via oral ingestion, with initial exposure most commonly taking place during early childhood [10]. The familial clustering of H. pylori infections suggests a household-based transmission dynamic, wherein older individuals are more vulnerable to both persistent infection and its associated pathological outcomes, including H. pylori-induced gastric carcinoma [3].

A variety of diagnostic approaches are available for patients presenting with gastric-related complaints. Among these, bacterial detection, particularly of H. pylori, and endoscopic examination play pivotal roles. Endoscopy not only enables direct visualization of mucosal abnormalities but also facilitates grading of disease severity and targeted biopsy collection for histopathological, molecular, or microbiological analyses. These methods are essential for accurate diagnosis and tailored treatment. The integration of endoscopy and microbiological testing enhances diagnostic precision, especially in cases of chronic gastritis, ulcers, or suspected malignancy [11,12].

The rationale for focusing on H. pylori in Ethiopia lies in the growing burden of gastric diseases and the availability of advanced clinical diagnostic tools such as endoscopic and histopathological investigations in major medical centers. These tools allow for detailed assessment of gastric mucosal changes and disease severity. However, there remains a critical gap in microbiological or culture-based identification of H. pylori in local research. While clinical and pathological evaluations are routinely performed, bacterial isolations are rarely incorporated. This limits our understanding of local need for integrated studies that include culture-based diagnostics to support evidence-based management and national guidelines. Despite a rising incidence of gastric-related complaints, culture-based isolation of H. pylori and its correlation with endoscopic findings remain scarcely documented in Ethiopia, particularly within the study area. This study is predicated on the hypothesis that there is a significant association between H. pylori infection rates and specific endoscopic abnormalities, offering insights into the bacterium’s potential role in the development of distinct gastric and duodenal pathologies. Accordingly, the primary objective of this investigation is to determine the prevalence of H. pylori isolates in gastro–duodenal regions, evaluate their association with endoscopic findings, and identify related clinical and demographic variables among patients undergoing endoscopic examination for gastric disorders.

Operational Definition

Gastric-associated disease: Clients with gastritis, duodenal ulcer, atrophic pangastritis, non-atrophic pangastritis, antral gastritis, gastric ulcer, and a combined category encompassing any form of gastritis and ulceration.

High salt intake: A daily salt intake exceeding 12,000 mg or the consumption of excessively salty meals such as foods heavily seasoned with salt can lead to a distinctly salty taste during eating. For instance, adding 2 full teaspoons of salt to 500 mL of watt (Ethiopian traditional food) or an equivalent amount in food preparation may result in noticeably high saltiness during consumption.

2. Materials and Methods

2.1. Study Area

The study has been carried out at selected healthcare institutions in Bahir Dar City that offer endoscopy services. These included the Kidane Mihret Specialty Higher Clinic, Eyasta Specialty Higher Clinic, and Gamby Teaching General Hospital. Bahir Dar is situated approximately 565 km northwest of Addis Ababa, the capital of Ethiopia. Geographically, the city lies at a latitude of 11.6° N and a longitude of 37.3614° E, with an elevation of 1784 m above sea level.

2.2. Study Design and Period

From January to May of 2019, an institutional-based cross-sectional study was conducted on patients with gastric-associated diseases who had endoscopic examinations.

2.3. Sample Size Determination and Sampling Technique

The study conducted in Tehran (Iran) reported a H. pylori prevalence of 90% among patients with various gastric disorders, with a 95% confidence level. The sample size for this study has been calculated using the single population proportion formula, ensuring statistical rigor. Although two earlier studies were conducted in Ethiopia, they are now considered outdated and insufficient as reference points for current epidemiological patterns. The findings in Tehran (Iran) were more consistent with the high prevalence typically observed in developing countries, including those with similar socioeconomic and healthcare conditions. Therefore, the 90% prevalence estimate from Tehran study finding. was adopted in this study as a more relevant and representative baseline for sample size determination and comparative analysis [13].

N = (Za_/2)²* P (1 − P)/W², (1.96)² 0.9 (1 − 0.9)/(0.05)² = 138

(1)

N = number of study participants

W = margin of error

P = proportion of previous study

A total of 135 study subjects participated in this study. Convenient sampling technique is used, and all patients who fulfill the inclusion criteria at endoscopy sites have been enrolled in the study.

2.4. Inclusion and Exclusion Criteria

All clients presented to selected health institutions for endoscopic confirmation of gastric-associated diseases during the study period were included in this study. Clients who had a history of antibiotics therapy within 15 days and who had taken anti-acid therapy at sample collection day were excluded from the study.

2.5. Sociodemographic and Clinical Data Collection

Sociodemographic data are collected through structured interviews conducted with the study participants. Additional clinical information was obtained by consulting the attending physicians during endoscopic examinations and reviewing the patients’ medical records.

2.6. Specimen Collection, Transport, and Processing

Biopsy specimen collection has been adhered, at a minimum, to the guidelines of the Sydney System protocol. During endoscopic examinations of patients with suspected gastric disorders, two to four gastric biopsy samples were obtained from different anatomical regions of the stomach using sterile endoscopic forceps. Biopsy samples were obtained from both the antrum and corpus regions. The endoscopic examination also encompassed evaluation of the patients’ duodenal features [14]. Based on a comprehensive assessment integrating endoscopic and histopathological findings, the final diagnoses were classified into the following categories: normal mucosa, atrophic pangastritis, non-atrophic pangastritis, antral gastritis, gastric ulcer, and a combined category encompassing any form of gastritis and ulceration. Clinical decisions during specimen collection were made with careful consideration of potential risks that could compromise patient safety or lead to adverse outcomes. To optimize patient comfort and improve the efficacy of the endoscopic procedure, a topical anesthetic of 4–5 mL of lidocaine spray via mouthwash was administered, along with 1 mL (5 mg) of intravenous diazepam for mild sedation.

Immediately following collection, biopsy specimens were inoculated into brain–heart infusion (BHI) broth and transported to the Medical Microbiology Laboratory at the College of Medical and Health Sciences, Bahir Dar University. The inoculated broths were incubated under microaerophilic conditions at 37 °C for 48 h, after which they were sub-cultured onto charcoal cefoperazone deoxycholate agar (CCDA) and CAMPY agar supplemented with 5% sheep blood.

For broths showing no initial growth, a secondary subculture was performed after 10 days of incubation. Both the culture plates and inoculated broth bottles were maintained in microaerophilic environments (comprising 5% O₂, 10% CO₂, and 85% N₂) using candle jars. To sustain these conditions, anaerobic catalysts and gas-generating kits (OXOID, Basingstoke, UK) were employed.

Subcultures were inspected for bacterial growth every 48 h for the first 3 days post-inoculation. In cases where no growth was observed, cultures continued to be examined every 24 h for up to 10 days to ensure accurate detection (Figure 1).

2.7. Bacterial Identification

The selective media that permit the growth of H. pylori and Campylobacter species were used. Preliminary bacterial colonies were examined using a series of identification tests: Gram staining, oxidase, catalase, and urease tests (Figure 1).

2.8. Quality Control

The data collection questionnaire was meticulously designed to ensure clarity and ease of comprehension. During data collection, each participant’s questionnaire was thoroughly reviewed to confirm completeness. Adherence to standardized operating procedures (SOPs) was rigorously maintained throughout the study to uphold the integrity and quality of the investigation from initiation to completion. To verify sterilization efficacy, Geobacillus stearothermophilus (ATCC 7953) spores were subjected to autoclaving followed by incubation to monitor for any residual growth. For sterility assurance, 5% of each batch of prepared media underwent overnight incubation at 35–37 °C. The performance of the culture media was validated using Campylobacter jejuni (ATCC 33560), chosen due to its comparable growth characteristics to H. pylori strains.

2.9. Data Processing and Analysis

Upon verification for completeness, the collected data were entered into Epi Data software version 3.1 and subsequently imported into SPSS version 23 for statistical analysis. Analytical procedures included multivariable analysis, binary logistic regression, Fisher’s exact test, and the chi-square test. The results were presented using frequencies, tables, and graphical figures to elucidate data patterns and explore associations with relevant variables. Statistical significance was determined at a threshold of p sis.

3. Results

3.1. Sociodemographic Characteristics of Study Subjects

This study involved 135 patients presenting with gastric-related conditions who underwent endoscopic examination during the study period. Of these participants, 59.3% (80/135) were male, while 40.7% (55/135) were female. The mean age was 45 years, ranging from 18 to 88 years. The majority of participants (81.5%) belonged to a low-income bracket, with 17% and 1.5% classified as medium- and high-income, respectively (Table S1).

3.2. H. pylori Isolates from Gastric Biopsies

The prevalence of H. pylori isolated from gastric-associated patient biopsies was 17.8% (24/135) in the current study. Among those positive study participants, 71% (17/24) were males, and 29% (7/24) were females, with a high proportion of positivity in the age group of 30 to 60 years old (54.2%, 13/24). Rural residents had a higher proportion of H. pylori infection (75% of the total of 24), while urban residents had only 25% (6 of the total of 24). The majority of culture-positive study participants (75%, 18/24) were married. Furthermore, 100% (24/24) of the culture-positive results came from study participants with low-income levels (Table S2).

3.3. Factors Associated with H. pylori Infection

In a statistical analysis of different predictor variables for H. pylori-induced gastric-associated diseases, only high salt intake showed a significant association [AOR = 3.3, 95% CI = (1.5, 10.8), p-value = 0.042]. As a result, patients who consume a lot of salt are three times more likely to develop H. pylori-induced gastric-associated disease.

3.4. H. pylori Isolates and Clinical Syndrome

Almost all patients who were involved in the study had symptoms of dyspepsia in 96.3% (130/135) and burning sensations in 97.0% (131/135). All H. pylori isolates were found in patients with peptic ulcer disease symptoms, such as dyspepsia and burning sensations.

3.5. H. pylori Isolates and Endoscopy Findings

Endoscopic examination of patients with duodenal ulcers revealed 50.4% (68/135) and 25% (17/68) positive H. pylori cultures (Figure 2). This phenomenon also showed a significant association with H. pylori isolation from antrum mucosal biopsies of duodenal ulcerated patients [AOR = 3.8, 95% CI = (1.2, 11.9), p-value = 0.02]. Therefore, patients with H. pylori infection are four times more likely to develop duodenal ulcers than patients with normal mucosa on their duodenum (

Table 1. Gastric-integrated endoscopic and histopathological findings and H. pylori isolates among patients attending endoscopy examination.

Stomach Endoscopy Findings	H. pylori Isolate
	Positive N (%)	Negative N (%)
Normal mucosa	4 (17.4)	19 (81.6)
Atrophic pan gastritis	9 (30.0)	21 (70.0)
Non-atrophic pangastritis	1 (3.6)	27 (96.4)
Antral gastritis	3 (11.1)	24 (88.9)
Gastric ulcer	0 (0)	8 (100)
Any gastritis and ulcer	7 (36.8)	12 (63.2)
Total	24 (17.8)	111 (82.2)

and

Table 2. Binary and multiple variable analysis of associated factors and H. pylori findings.

	Positive N (%)	Negative N (%)	OR 95%CI	p-Value	AOR 95%CI	p-Value
High salt intake
Yes	16 (31.4)	35 (68.6)	4.3 (1.69–11.1)	0.001	3.3 (1.5–10.8)	0.042
No	8 (9.5)	76 (90.5)	1		1
Khat chewing
Yes	2 (40)	3 (60)	3.3 (5.2–20.8)	0.22 *	NA	NA
No	22 (16.9)	108 (83.1)	1
Coffee drinking habit
Yes	11 (31.4)	24 (68.6)	3.1 (1.22–7.1)	0.014	2 (0.6–5.3)	0.22
No	13 (13.0)	87 (87.0)	1		1
Alcoholism
Yes	2 (12.5)	14 (87.5)	0.6 (0.1–0.9)	0.55	NA	NA
No	22 (18.5)	97 (81.5)	1
Sex
Male	17 (21.2)	63 (78.8)	1.9 (1.71–4.8)	0.20	0.4 (0.15–0.21)	0.095
Female	7 (12.7)	48 (87.3)	1		1
Residence
Urban	6 (18.8)	26 (81.2)	1.1 (0.4–3.0)	0.87	NA	NA
Rural	18 (17.5)	85 (82.5)	1

Variables H. pylori. * = Fisher’s exact test, NA = Not applicable.

).

The overall proportion of abnormal gastric mucosa was 83.0% (112/135). Among those, 22.2% (30/135) were atrophic pangastritis, and 20.7% (28/135) were non-atrophic pangastritis. Patients with pan-atrophic gastritis and any gastritis with ulcerated stomach, on the other hand, had a higher positivity for H. pylori isolates (92.5%) than other endoscopic findings (29.2%, 7/24). Among patients who tested positive for H. pylori, 83.3% (20/24) exhibited abnormal gastric mucosa upon endoscopic examination, with varying degrees of severity ranging from mild gastritis to ulcerative lesions. Interestingly, even among H. pylori-negative individuals, approximately 82% (92/111) demonstrated endoscopic features suggestive of H. pylori infection, such as mucosal erythema, edema, erosions, or nodularity/ulcerations.

4. Discussion

The experiment design used in the current study is new and innovative, enabling better bacterial isolation and bypassing biopsy grinding. The reason we did it is because H. pylori is microaerophilic, and while grinding, it may contact environmental excess oxygen, which is toxic for bacteria (methodology).

This study holds significant medical importance due to its potential to improve local healthcare outcomes. H. pylori is a major cause of gastric-associated diseases, including gastric cancer, which are underdiagnosed in alignment with pathological findings and endoscopic investigation due to the difficulty of isolating bacteria because of the fastidious nature of the organism. Furthermore, understanding the epidemiology and burden of H. pylori can lead to better diagnostics, preventive measures, and overall management of gastrointestinal diseases, thereby contributing to public health improvements. The overall prevalence of H. pylori isolates is 17.8% (24/135) in this culture-based analysis of gastric biopsies taken from patients undergoing endoscopic examinations. The global prevalence of Helicobacter pylori infection remains substantially high, with estimates indicating that approximately 44% of the world’s population is affected. This figure is markedly higher than the prevalence observed in the current study. However, it is important to note that global prevalence rates vary considerably depending on geographic region, age group, socioeconomic status, and the diagnostic methods employed [15]. Unlike the referenced global systematic analysis, which primarily relied on serological methods known for their higher sensitivity but lower specificity, our study utilized culture-based isolation techniques, which, although more specific, may yield lower detection rates due to the demanding nature of culturing H. pylori. This methodological difference likely contributes to the lower prevalence reported in our findings and underscores the importance of standardizing diagnostic approaches when comparing epidemiological data across regions. But this finding is in agreement with our previous investigation that was conducted in northwestern Ethiopia, aiming at the antimicrobial resistance profile of H. pylori [16]. However, it is lower than the studies conducted in Ethiopia at Butajira Hospital [17] and Jigjiga University student clinics [18], with proportions of 52.4% and 71%, respectively. Both studies employed only serological tests, which detect antibodies against H. pylori antigens. The higher proportion of H. pylori infections in those studies could be due to past infections or might not be active infections.

The prevalence of H. pylori isolates observed in the current study is substantially lower compared to findings reported in several studies conducted in other regions of the world. For instance, study in Tehran. documented a 90% prevalence of H. pylori among patients with gastric disorders, and an other study also reported a prevalence of 90.5%, both significantly exceeding the rates found in our study. Furthermore, a study conducted in southwestern Iran by M. Amin et al. reported a prevalence of 38.7%, which, although lower than the aforementioned figures, remains considerably higher than that of our findings [13,18,19,20]. These discrepancies may reflect geographic variation, diagnostic methodologies, study populations, and healthcare access, underscoring the importance of generating updated, locally representative data to guide clinical and public health interventions. This could also be due to the fact that the study subjects in our study were only patients who were eligible for endoscopic investigations.

In this study, the majority of study participants have dyspepsia (96.3%) and burning sensations (97%). These findings are in agreement with a study that revealed more than 70% of H. pylori-positive patients have dyspepsia. Higher H. pylori infection rates are observed in the age group of 30–60 years, with a prevalence of 9.6%. Similar findings and the association of this age group with H. pylori infection were indicated by Sasidharan et al. [21]. Moreover, from the study conducted in Bangladesh, the 41–50 age group had the highest prevalence of H. pylori infection at 25.5%. Endoscopic results showed that 101 dyspeptic patients (97.1%) had gastritis, with most H. pylori infections occurring in patients histopathologically diagnosed with duodenal ulcers, which is completely in agreement with our study [22]. Notably, approximately 82% of individuals who tested negative for H. pylori still exhibited endoscopic features typically associated with infection, including mucosal erythema, edema, erosions, nodularity, or ulcerations. This observation reveals a notable discordance between endoscopic impression and microbiological confirmation, which may stem from factors such as low bacterial density, focal or patchy colonization, previous antibiotic exposure, or the inherent limitations of culture-based diagnostic methods. These findings reinforce the importance of employing a multimodal diagnostic strategy integrating endoscopy with histopathology and laboratory techniques with the best diagnostic accuracy to enhance the sensitivity and accuracy of H. pylori detection.

Although logistic regression did not show a significant association, a higher frequency of H. pylori infection was observed in male patients than female ones. Unlike our report, Sasidharan and colleagues showed a significant association between sex and H. pylori isolation, with a higher prevalence of H. pylori infection observed among males (12.6%) compared to females (5.2%) [21]. This difference could be due to the fact that males have different predisposing practices, such as a high alcohol consumption habit, than females do. However, an earlier study reported that H. pylori infection did not correlate with gender differences [23].

In contrast to our study findings, a study from South Chile showed that a higher proportion of H. pylori was found among female participants (42% vs. 40.2%), although the difference was not statistically significant [24].

According to the logistic regression analysis of our data, a significant association of H. pylori infection with the presence and severity of gastritis was observed. In total, 83% of patients with H. pylori infection exhibited symptoms of gastritis. In addition, 96% of patients with dyspepsia had an H. pylori infection. Similarly, a previous study from Jordan revealed that 82% of dyspeptic patients with gastritis had H. pylori infection [25].

Patients with gastric-associated diseases who had a history of high dietary salt intake were more likely to develop gastric-associated diseases than patients who ate less or no salt. It concurred with the study by Geddy and his colleagues [26]. This could be due to the combined effect of salt and H. pylori adherence on gastric mucosa, which causes gastric-associated diseases. Salt intake might also affect the growth of H. pylori and genetic expressions [27,28]. This study also explored the potential association between Helicobacter pylori isolation and various dietary and lifestyle habits, including coffee consumption, alcohol intake, and khat (Catha edulis) chewing. However, none of these factors demonstrated a statistically significant association with H. pylori isolation in the study population. While such habits have been hypothesized in other studies to influence gastric mucosal integrity or alter gastric acidity potentially affecting H. pylori colonization, the current findings suggest that these behaviors may not play a direct or measurable role in the bacterial isolation outcome. Further research with larger sample sizes and controlled confounders is warranted to clarify these relationships.

Regarding educational status, a higher proportion of H. pylori isolates was observed in patients with a university educational level (20%) and patients who cannot read and write (18.3%) than in other groups. Patients who cannot read or write may be more vulnerable to H. pylori infection due to poor personal and environmental sanitation, whereas patients with a university education may have a better understanding of the importance of visiting endoscopy physicians and being examined on a regular basis. Our findings of higher H. pylori isolates in educated people agreed with previous research from Chile and other Latin American countries [29].

In the current study, the prevalence of H. pylori infection in different age groups is generally very low. All positive findings were from patients with an age of >20 years old, though only four patients were examined for endoscopic and H. pylori cultures. Unlike our findings, a review of the global scenario of H. pylori by Mohammed and Alvi from Saudi Arabia indicated that the prevalence in Ethiopia at ages 2 up to 4 and 6 years was 48% and 80%, respectively [30]. This could be due to the methodological difference that this study included patients who presented to endoscopic examination at the margin of chronic gastric-associated diseases.

As noted, this study found no statistically significant association between H. pylori infection and key lifestyle or physiological factors, including alcohol consumption, smoking habits, and body mass index, while previous evidence by Kibru and colleagues [16] showed a significant association. Another study done at Jigjiga University students’ clinic also showed a significant association between the presence of H. pylori infection and the clinical manifestation of gastritis, the habit of alcohol drinking, and khat chewing [17].

5. Limitations

The current study could not employ a CO₂ incubator, which better supports the growth of H. pylori than a candle jar does, due to the unavailability of the equipment. This study considered only participants sent for endoscopic examinations, while gastric complaints or patients in outpatient departments were being seen at growing rates. Another limitation was the inability to get previously done culture-based studies on H. pylori infection for better comparison.

6. Conclusions and Recommendations

In conclusion, the present study showed that the prevalence of H. pylori infection was lower compared to other studies done previously. Results confirmed that the severity of gastritis and high salt intake practices were significantly associated with the existence of H. pylori isolates in endoscopic specimens. Endoscopic sites should incorporate culture-based H. pylori isolation in laboratory testing as part of the differential diagnosis, as gastric-associated diseases can arise from various factors beyond infection. If it could be difficult to implement, the stakeholders should influence the referral laboratories to capacitate H. pylori identification. Furthermore, we strongly recommend further studies be conducted.