Population- and Gender-Based Investigation for Prevalence of Helicobacter pylori in Dhamar, Yemen

Among 35 species of genus Helicobacter, H. pylori is the most common causative agent of human gastritis, peptic ulcer, and gastric cancer. The infection can spread through direct human-to-human contact, fecal–oral route, and contaminated water. The study was designed to investigate the rate of prevalence of H. pylori in the population of Dhamar, Yemen. In this one-year study, 460 including 250 male and 210 female stool specimens were collected between January to December 2020 in Dhamar Governorate, Yemen. Of the total 460, 215 rural (male: n = 120 and female: n = 95) and 245 urban (male: n = 130 and female: n = 115) specimens were investigated for identification of H. pylori by serological test using Helicobacter pylori stool antigen (HpSA) test. In addition, for comparing an improved recovery of H. pylori, conventional culture-based isolation was also carried out using three selective media. Modified Campy-blood Agar (MCA), Belo Horizonte Agar (BHA), and Egg yolk Emulsion (EYE) medium supplemented with antimicrobial agents including vancomycin (10 mg/L), cefsulodin (5 mg/L), trimethoprim (5 mg/L), and amphotericin B (5 mg/L) and isolates were phenotypically characterized. The HpSA test results revealed that of the total 460 specimens, 89 (19.3%) were positive for H. pylori with relatively low in male (n = 43; 17.2%) as compared to the female (n = 46; 21.9%) specimens. After 3–10 days of incubation, H. pylori was recovered at a variable rate on each selective (MCA: 16.5%; BHA: 15.0%; EYE: 13.0%) media. However, culture-based assay results showed less recovery (n = 81; 17.6%) with no significant difference among all selective media tested and between genders (male: n = 39; 15.6%; female: n = 42; 20.0%). The infection rate was comparatively higher in rural (n = 45; 20.9%) as compared to urban (n = 36; 14.7%) population. Overall, the study data showed the prevalence of infection in both genders of all age groups. The present study showed a relatively high rate of infection of H. pylori in the Dhamar population. The serological identification and culture-based methods are important for rapid detection, aid in treatment, and developing policies for the control and eradication of H. pylori infection and to prevent the disease in different age groups in Yemen.


Introduction
Te species of genus Helicobacter are microaerophilic, motile, and Gram-negative curved rods bacterium that belongs to the family Helicobacteraceae. Currently, the genus consists of over 30 species that have been classifed into two major lineages: gastric and nongastric or enterohepatic [1]. Te gastric lineage represents only one-third of all Helicobacter species that colonize in the stomach of humans and a wide range of animal species e.g., cattle, sheep, cats, dogs, monkeys, cheetahs, rhesus, ferrets, dolphins, and whales. Among eight Helicobacter species of gastric group, H. pylori (∼2-3.5 μm × 0.5 1.0 μm) is the representative species of medical importance and considered as a causative agent of active chronic gastritis, peptic and duodenal ulcer, gastric cancer, and mucosaassociated lymphoid malignancies in humans [2,3]. In the last decades, an increasing number of studies have performed an analysis of the gastric mucosa to identify the role of H. pylori in peptic ulcer, carcinogenesis, and in some forms of gastric lymphoma [4]. It has been observed that H. pylori colonizes under the coating layer of the gastric pits close to gastric epithelial cells and in the gastric mucin covering [1,4]. However, the enterohepatic or nongastric Helicobacter species have been more frequently identifed in the human intestinal tract and liver, other mammals (e.g., rodents, rats, mice, and hamsters), and birds. In many cases, they have been linked with infammation or malignant transformation in immunocompetent hosts and more severe clinical diseases in immunocompromised humans and animals [1,5].
Te prevalence of H. pylori infection has been considered as one of the most common infections in both developing and developed countries with an estimated 4.4 billion cases reported in 2015 [3]. Clinically, over 80% of cases are asymptomatic [6]. Te prevalence of H. pylori infection ranged between 7.3 and 92.0%, depending on diferent factors such as geographic location, age, and socio-economic status [7,8]. Te prevalence in Asia, South America, and South and East Europe is ˃50%, whereas only one-third of population has been reportedly infected in North American and North European countries [9]. Previous epidemiological studies have reported more H. pylori infection in developing countries (up to 90%) in some regions such as Bhutan and Myanmar [10,11]. Te socio-economic and hygiene status have been considered as main risk factors for infection where high prevalence rates have been reported in crowded areas, especially in rural areas with low socio-economic and unhygienic conditions [10,12].
Several diagnostic methods have been developed and applied for the identifcation and characterization of H. pylori. Invasive diagnostic tests including endoscopic imaging, histology (microscopy), rapid urease test, and culture methods are mostly used to obtain biopsy materials to aid in the treatment of infection [13,14]. However, the most common noninvasive diagnostic assays, including stool antigen tests (SATs), serology-based tests, urea breath tests, and molecular assays have been widely applied [15].
Previous epidemiological studies of H. pylori in Yemen population have not been well-reported [7,[16][17][18]. Terefore, the present population-and gender-based study was designed to detect and identify the rate of prevalence of H. pylori infection in urban and rural male and female patients in the city of Dhamar, Yemen. Moreover, we also assessed and compared the rate of recovery of H. pylori on three selective media, especially when cell concentration is low in the specimen.

Specimen Collection.
A total of 460 human stool specimens were collected between January to December 2020 from General Dhamar Hospital and diferent medical diagnostic laboratories located in Dhamar Governorate, Yemen. Te stool specimens were collected from 250 males (rural: 120; urban: 130) and 210 females (rural: 95; urban: 115). Te age of the participants ranged from <1 to 80 years.

H. Pylori Antigen
Screening. Te principle of the H. pylori stool antigen (HpSA) test is a noninvasive diagnostic module based on immunochromatography (ICA) assay for H. pylori infection where One-StepH. pylori antigen test card (LumiQuick, Santa Clara, USA) was used. Briefy, approx. 200 mg of stool sample was transferred into a vial containing 2 mL phosphate bufer saline (PBS), the solution was shaken for 30 seconds and then 120-150 μL of homogenous suspension was loaded in the well of One-StepH. pylori antigen test card strip (LumiQuick, USA). Te results were recorded after 10-15 min based on a red line developed at the C&T region, whereas a red line developed at the C band was recorded as negative as described by the manufacturer.

Cultivation and Species Identifcation.
Te putative H. pylori culture isolates recovered from each selective growth media were identifed based on colony morphology, Gram staining reaction, and microscopic characteristics, including S-shaped or curved thin rods. Similarly, a panel of biochemical tests including catalase, oxidase, urease, indole, and growth in 1% glycine and 3.5% NaCl were performed. Single colonies were subcultured onto blood agar, incubated at 37°C for 48-72 h and stored at −20°C in cysteine storage medium containing 20% glycerol for further analysis.

Antimicrobial Susceptibility
Testing. Antimicrobial susceptibility test was performed using disc difusion (Kirby-Bauer) method [21]. Two types of antibiotics, nalidixic acid (30 µg) and cephalothin (30 µg) were used as described by the manufacturer (HiMedia, Düsseldorf, Germany). Te H. pylori cell suspension was prepared in 2 mL sterile normal saline at an inoculum concentration equivalent to McFarland 3, where 10 µL was spread plated on Columbia agar media. Te plates were incubated under microaerophilic conditions at 37°C for 48-72 h using an anaerobic candle jar. Te zone of inhibition was measured to classify them as sensitive or resistant according to the CLSI (2012) and CASFM (2017) guidelines and interpretative criteria.

Questionnaire. All patients investigated in this study
were enrolled based on their clinical symptoms and history of treatment including antibiotics or other medications. A computer-assisted questionnaire including demographic and socio-economic information (age, gender, birthplace, education, and household population), clinical symptoms (chronic gastritis, gastric ulcer and/or duodenal ulcer, and stomach pain), any antibiotic and/or other treatment, and other factors (smoking and alcohol) was completed.

Statistical Analyses.
Data was analysed using SPSS statistical software, version 25. Confdence intervals were calculated by the normal approximation method and α-level was set to 0.05. Te Chi-square test was used to evaluate diferences between groups and p < 0.05 was considered as signifcant.

Comparison and Assessment of Culture-Based Assays for
Recovery and Detection. Similar to HpSA test results, H. pylori was recovered, between 3-10 days, from all three selective growth media with relatively low frequency (n = 81; 17.6%) in male (n = 39; 15.6%; rural: 22 and urban: 17) and female (n = 42; 20%; rural: 23 and urban: 19) specimens. Similar to serological test results, comparatively a high frequency of H. pylori positive cultures was recovered from rural males (n = 22; 49%) and females (n = 23; 51%) than in urban males (n = 17; 47%) and females (n = 19; 53%) with no signifcant (p > 0.05) diference (Table 1). Of three selective growth media, H. pylori cultures were isolated at a variable frequency on MCA (16.5%), BHA (15.0%), and EYE (13.0%) media with no signifcant (p > 0.05) diference. Te cells were slow growing with small and translucent colony morphology. Microscopically, the Gramnegative colonies appeared as spiral-shaped rods. A slow cell growth was observed on blood agar base at 37°C under anaerobic condition. H. pylori cells grown on MCA media were small, round, and appeared translucent due to the blood agar background. Whereas golden colony morphology was observed on BHA due to 2,3,5-triphenyltetrazolium chloride as compared to EYE media where small red colony morphology was observed due to egg yolk agar base containing oxidationreduction indicator (tetrazolium red) that changed yellow to red. H. pylori colonies on EYE media were distinctively visible, and confuent growth was very obvious against the yellow background. Based on the colony morphology observed on three selective agar media, BHA had more visible and distinctive (translucent golden) colonies followed by EYE and MCA media.
All culture isolates recovered from three selective culture media showed positive biochemical reactions to catalase, oxidase, urease, and TSI with Pb acetate paper tests and negative to indole. Moreover, isolates were positive for H 2 S production and hippurate hydrolysis test. Te culture showed clear growth in 1% glycine and Brucella broth with 3.5% sodium chloride. All culture isolates (n � 81) showed resistance to nalidixic acid (30 µg) and sensitivity to cephalothin (30 µg).

Population-Based Assessment of H. Pylori Infection.
A total of 460, including 250 (54.3%) male (rural: 120; urban: 130) and 210 (45.7%) female (rural: 95; urban: 115), participants involved and completed the questionnaires for this study. All patients were further investigated based on their clinical symptoms such as stomach pain, heartburn and various degree of nausea, vomiting, loss of appetite, and sometimes headache. Te patients were not treated with antibiotics or other medication at least two months prior to visiting university's internal diseases clinic.
Overall, prevalence of H. pylori in Dhamar population was ranged from 15.74% to 22.96% (95% CI). According to antigen and cultural-based results, Table 1 shows the gender group-based rate of prevalence of H. pylori for all specimens investigated in the study. Although the frequency of H. pylori in females was relatively high, no gender-associated signifcant (p > 0.05) correlation with H. pylori infection was observed in both serological and culture-based assay results.
Moreover, the rate of prevalence among various participants' age groups ranged from <1 to 80 years was assessed. Te highest prevalence rate (28.6%) was observed among adolescents. In contrast, H. pylori infection among children <11 year was signifcantly (p < 0.05) lower than other age groups ( Table 2). Te detailed distribution of positive cases is graphically depicted in Figure 1 based on a 10-year-age interval. Regarding the residence area, H. pylori prevalence among participants from rural was higher than urban areas. However, this diference was not statistically signifcant (χ 2 = 1.05, p > 0.05).
Te data analysis was further performed to assess the seasonal occurrence of H. pylori infection where the distribution of H. pylori cases was monitored for a full year ( Figure 2). Te highest prevalence in December (28.9%) while the lowest rate in October (13.2%) with no signifcant (p > 0.05) seasonal diference was recorded. Based on the questionnaire data results, the source of drinking water and household population did not show signifcant (p > 0.05) diference. Moreover, 78% of participants showed asymptomatic positive cases of H. pylori infection.

Discussion
Since H. pylori infection has been related to gastritis, peptic ulcers, and gastric cancer [22], therefore, epidemiological surveillance of H. pylori helps in establishing public health counter measures that could reduce and control the transmission and acquisition of infection [23]. A variable (ranged between 30 and 80%) rate of prevalence of H. pylori infection between developed and developing countries has been reported [24]. As compared to 6 to 12% in developed countries (Japan, Germany, Netherlands, and USA), high infection rate (26 to 66%) has been reported in developing (Chile, Venezuela, and Nigeria) countries [25]. Te rate of frequency (19.3%), in our study, is similar to the previous surveillance studies (ranged 12 to 18%) conducted in countries (Lebanon, Egypt, Saudi Arabia, and Iran) located in the same geographical area [26][27][28][29]. In contrast, high Canadian Journal of Gastroenterology and Hepatology 5 prevalence rates (ranged from 40 to 82%) were also reported in the same area [30][31][32][33]. Tis serological variable data could be due to numerous contributing factors such as socioeconomic status, household population, hygienic lifestyle, living in crowded families, and family history of GI cancer that might impact on the prevalence rate of H. pylori. Moreover, the detection methods are also one of the major contributing factors [11,34,35]. Although, no single test can be considered as the gold standard [36]. Generally, there are two (enzyme immunoassay (EIA) and immunochromatography assay (ICA)) types of SATs for the diagnosis of H. pylori infection using either mono-or polyclonal antibodies. Although both tests are highly sensitive and specifc, the EIA-based tests appear to be more accurate than the ICA-based tests, but the ICA-based tests do not require special equipment, easy to use and useful for rapid diagnosis of H. pylori infection [37]. H. pylori stool antigen (HpSA) test is a rapid assay applied for rapid screening and diagnostics since it is accurate, sensitive, economical, and simple. However, the results may be infuenced by some factors such as the amount of antigen in stool, the nature of biochemical reaction in the kit, and the heterogeneity of antigens [34]. Urea breath test (UBT) and stool antigen test (SAT) have been widely applied for the detection of H. pylori infection. Since the sensitivity and specifcity of SAT ranged between >80% and >90% [38], Canadian Agency for Drugs and Technologies in Health extensively reviewed SAT test for H. pylori detection and approved due to its diagnostic accuracy, clinical, and cost efectiveness [37]. In recent meta-analysis study, the pooled sensitivity and specifcity SATs test results between 92 and 94% [39]. Similarly, Schulz et al. [40] evaluated nine diferent strategies for screening of H. pylori-infected asymptomatic immigrants and refugees to Australia. Stool antigen screening and repeat testing were found the most costefective approach for reducing the future burden of  peptic ulcer (PU) and gastric cancer in the population. Previous studies have evaluated indirect methods, including antibody-based tests (serology and urine test), UBT, and SAT that have been used for population-based screening for detecting H. pylori infection.
Similar to previous research studies, our study results have showed no signifcant correlation associated with gender-based (male and female) distribution of H. pylori infection [14,41]. Te data indicates that gender has a negligible role in the H. pylori infection. Also, rate of prevalence in rural and urban area results did not show signifcant diference. In contrary, based on age group data analysis, a signifcant diference in the prevalence of H. pylori infection where low (7.7%) frequency in age group between 1 and 9 years as compared to age group between 10 and 19 years with a high (28.6%) frequency was recorded. Our results are incongruence with previous studies where high infection rate was reported after childhood as compared to other studies where H. pylori infection was reported during childhood and adolescence [42][43][44][45][46]. On the other hand, other studies reported high prevalence in children regardless the diferences in the associated risk factors [27,47]. Interestingly, increased rates of infection, as well as spontaneous recovery was observed primarily in children under 5 years [48]. In a recent study, Park et al. [25] reported that H. pylori infection in children and adolescents was not signifcantly associated with age and gender.
We also assessed seasonal association with H. pylori prevalence. Te statistical analysis showed no signifcant (p > 0.05) diference between seasons. Tese fndings indicate the independency of H. pylori prevalence regardless the season. However, previous study showed an increase of infection frequency from 70% in the dry months to 96% in the rainy months among youngsters only, while incidence pattern in adults showed no statistical association with season [49]. Similarly, H. pylori infection frequency was increased in February (40%) and declined afterwards [7]. Te participants of this study used tap water pipe as drinking water source, therefore, showed no signifcant diferences between the populations. On the contrary, other previous studies found a signifcant correlation between the source of drinking water and H. pylori infection [14,50].
A comparative analysis among three selective media was performed to assessthe enhanced and improved recovery of H. pylori from both urban and rural male and female patients. Generally, detection of H. pylori from stool samples showed low (18%) recovery rate [51]. However, Dore et al. [52] successfully recovered H. pylori (26.3%) from stool of known H. pylori infected patients after treating stool specimens with cholestyramine before plating on culture medium. Other studies recovered H. pylori between 15 and 39.1% of stool specimens [53,54]. According to Alsulaimany et al. [38], the cultural method showed sensitivity ranged between 63 and 90% and specifcity between 93 and 100%. Tese variations are mostly linked to factors such as specimens and/or culture media and incubation conditions as well as the stage of infection [35,51]. Indeed, the density of bacteria in the stomach is an important determinant of successful recovery of H. pylori from stool. Additionally, the use of cholestyramine in the initial treatment of stool samples improves the yield of H. pylori in cultural methods, which may be related to the successful isolation of the metabolically active form of H. pylori [35].
In addition, we also investigated antimicrobial resistance of these culture isolates, where all H. pylori strains showed resistance to nalidixic acid and sensitivity to cephalothin with no statistically signifcant diference between male and female rural and urban specimens. Similar results were reported by Meng et al. [55], where variable drug resistance to metronidazole, clarithromycin, amoxicillin, levofoxacin, and furazolidone with no statistically signifcant diference between diferent sex, age, and disease category was reported. Tus, the antibiotic resistance of H. pylori could be considered a risk factor for the development of gastric cancer [56]. However, it has been recently reported that rabeprazole-bismuth-tetracycline-tinidazole regimen is a highly efective treatment for H. pylori infection where men have higher eradication results than women [57]. Further research is warranted to investigate the mechanism of action of these compounds that variably impact on the treatment of H. pylori infection in each gender.

Conclusion
Te study reports on population-and gender-based rate of H. pylori prevalence and infection in Dhamar governorate, Yemen. Overall, H. pylori infection was detected at a relatively low level in the area and in both genders. It is not conclusive whether gender is a risk factor for the increased prevalence seen among females. Similarly, residents of rural areas may become more infected due to low socio-economic status and level of education, as well as suboptimal sanitary conditions and increased susceptibility as a result of a genetic tendency. Temporal impact on H. pylori infection was not observed as no statistical diferences across seasons were found. Personal hygiene and proper food cooking and processing are recommended to reduce the spread of H. pylori infections. Further epidemiological molecular characterization is warranted to understand the transmission pathway in order to develop and improve strategies to control infection in the community.

Data Availability
Data are available on request.

Ethical Approval
Tis study obtained approval from Knowledge University, Erbil, Iraq. Te study was structured in accordance with the Helsinki Declaration 2000 and reviewed and approved by the Ethics Committee of Tamar University, Yemen.

Consent
Te study objectives and procedures including samples and data collection were explained to all participants (adolescents, adult, or who acts on behalf of minors), and their Canadian Journal of Gastroenterology and Hepatology consent was obtained prior to collecting samples and completing the survey questionnaire.

Conflicts of Interest
Te authors declare that they have no conficts of interest to report.

Authors' Contributions
DAA, AAH, and IUHK conceived and designed the study. SMM, HIM, and HIM collected specimens and performed laboratory analysis. AAH, IUHK, and DAA analysed data and drafted the manuscript. All authors reviewed, edited, and approved the fnal version for submission.